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September 2009 Operating Instructions PALM RoboSoftware 4.3 SP1

1. dont care Lamp don t care w dontcare Baseport don t care w Sideport don t care On the left there is a list of the microscope parts for which you can make prior settings There are two groups of fields in the middle and one on the right in which you can specify the setting you 170 want for the respective part which is to be select ed automatically when PALM RoboSoftware is launched or closed Every field contains a drop down list containing all the possible settings for the respective microscope part In addition each in cludes an entry don t care If you select this en try the setting that is currently active at the time PALM RoboSoftware is launched or closed will be the one that is retained If all the current settings are to be retained when PALM RoboSoftware is launched or closed e Click on the Don t care button under Startup or Shutdown All entries in the fields for Startup or Shutdown will be set to Don t care If certain settings for the individual parts of the mi croscope are to be made automatically when PALM RoboSoftware is launched or closed e Open the respective drop down list Reflector Condenser Filter Shutter Reflected Transmit ted Lamp Baseport Sideport and select the desired setting of course the selection options for launching and closing of PALM RoboSoftware are the same Info The entries in the lists are
2. Common Reflected Light HW Settings Objective bjecti 5 10 20 40 63 100 3 2 8 8 9 8 UI ocus C Auto change Fi q sani Cut Delta LPC 2 ulting iteration cles Focus Light ra AF active z Cut z focus delta 3 pm Set Workpos 3200K 10000 um 100 m 0 O O 6 Copyright Info mors PR anmo 909 2 00 E HERE 5 For Help press F1 Slide2 ES 10f4 53 Idle TrapXY2 6595 13165 Menu bar toolbars and tools 1 Menus 9 Color Palette 2 Tool Bar 10 Speed Tools 3 Camera Tools camera and display settings 11 Laser Tools 4 Microscope Tools settings for the microscope 12 Start cutting laser for fluorescence and hardware 13 Cutting laser status display 5 Status Bar 14 Switch on off trapping laser 6 Cut Tools E trapping laser status display 7 Selection of a well for a collection device 15 Setting for arrow keys and joystick 8 Graphic Tools When the program has been launched the main The icon bars the tools and the Status Bar can be window of PALM RoboSoftware as depicted above toggled on and off as shown in chapter 2 2 will appear showing the camera image the Menu Bar various icon bars and tools as well as the Status Bar 415109 2614 101 e 09 09 15 PALM RoboSoftware layout Info The possible options refer to chapter 1 1 which are available are dependent upon the configuration of your system This can be adapted to your persona
3. To switch the display of a Laser Marker on or off Laser View Laser Marker Cut Laser Trap 1 Lazer Trap 2 Laser e Inthe Laser menu click on menu item View Laser Marker A submenu opens containing a list of all available Laser Markers e Click on the name of the Laser Marker you want to show or hide To change the appearance of a Laser Marker Laser Layout Laser Marker ego Cut Laser 1 i e Trap 1 Laser ws Trap 2 Laser e Inthe Laser menu click on menu item Layout Laser Marker A submenu opens containing a list of all Laser Markers e Click on the name of the Laser Markers whose appearance you want to change This opens the Laser Marker window Laser Marker LaserMarker Cut marker Triangle filled EX green dle Height 4 gt width 415109 2614 101 e 09 09 In the Laser Marker field you can select the La ser Marker whose appearance you want to change Cut marker Cut marker Laserbdarker Trap 2 marker If you want to change the appearance of more than one Laser Marker you can change from one to the other here e Select the required shape for the Laser Marker from the Type list e Select the required style for the Laser Marker from the Style list e Setthe required height and width for the Laser Marker using the Height and Width arrow buttons e Setthe required line thickness of the border for the Las
4. When the automatic cutting laser functions are started the stage is automatically moved along the defined cut lines or from dot to dot You can set the speed to suit the various methods of moving the stage refer to chapter 5 3 In order to prevent the stage from being moved in advertently you can freeze its movement in any position ie disable all functions that would cause the stage to move The program distinguishes accordingly between the various movement modes and the Freeze Mode The active mode is displayed on the Status Bar You can and must disable Stage Mode and Freeze Mode yourself To use the other movement methods you must be in Cursor Mode the modes are automatically entered as soon as you select the appropriate functions and automatically exited at the end of the movement task You can immediately interrupt the movement of the stage for example in case of an emergency The speed with which the stage is moved can be programmed separately for each movement type 415109 2614 101 e 09 09 Movement types 5 1 Movement types 5 1 1 Positioning the stage with the mouse Stage Mode In Stage Mode you control the movement of the stage synchronal with the mouse Each mouse ac tion is translated into a movement of the stage If you are using an objective that inverts the im age a mouse movement to the right in Stage Mode will appear on the monitor as a movement to the left If you select Invert Stag
5. First set the required height then define whether the setting for the height is to apply during a mo vement or for the working height 143 Settings e Clickon the left or right arrow button to move the collection device up or down The actual value is indicated in the field under the arrow buttons Info You can also enter a value in the field under the arrow buttons and click on the Goto but ton beside this field e Click on the Set button by moving height or by working height depending on which height you want to define Info You can use the Goto buttons to move the collection device to the moving height or working height set To set the settings back to their factory default values e Click on the Set to default button 144 12 4 4 Referencing PALM RoboMover or PALM CapMover II If PALM RoboMover or PALM CapMover II should be moved other than under program control e g by hand as a consequence of a collision an error message appears e g Error 18 In such a case you must reference PALM RoboMover or PALM CapMover II to send the exact position to PALM RoboSoftware Reference PALM RoboMover or 15 Reference PALM CapMover II e Click on the Reference PALM RoboMover or Reference PALM CapMover II icon button 415109 2614 101 e 09 09 13 MicroTweezers Option PALM MicroTweezers System is an optical tweezers with which can you trap and move objects in sus pension under the
6. If you are in Freeze Mode and move outside the Scroll Rectangle with the mouse while drawing the stage does not follow the movement How ever you can continue to draw even beyond the border of the visible image You can check the result by exiting Freeze Mode again and moving the stage so that your element is dis played in its entirety on the monitor However this practice of drawing outside the border in Freeze Mode is not to be recommended be cause it means that you have no control over your drawing Ifyou are in Cursor Mode and you move outside the Scroll Rectangle with the mouse while drawing the stage follows the movement auto matically for all tools except the Freehand and the Line Tool refer to chapter 7 3 5 The Free hand Tool and chapter 7 3 6 The Line Tool Select a low scrolling speed for the stage so that the movement of the image on the monitor can be easily followed with the naked eye 415109 2614 101 e 09 09 7 3 4 The Reference Point Tool Reference Point Tool Reference Point With the Reference Point Tool you define reference points on your sample For example you need them for the Serial Sections function refer to chapter 8 7 e Select the Reference Point Tool in the Graphic Toolbar e Click on that point in your image at which you want to set a reference point 7 3 5 The Freehand Tool Freehand Tool Use the Freehand Tool to draw irregularly shaped elements e Select
7. 17 4 Displaying log file Open the menu for the Incubation icon but ton Click on the View Log file menu entry 187 188 415109 2614 101 e 09 09 Field of View Analysis 18 Field of View Analysis 2 Thresholds interactive Object Definition A A penne pena Ego eae o Pixel Click O Outline Click Tolerance asl JOE Show graphics plane Single image Remove unsegmented channels Invert result Create binary image Automatic thresholds Do Auto Threshold Histogram Color Model RJ sJe RGB HLS Interactive The Field of View Analysis function can be used Info to automatically generate elements using PALM The Field of View Analysis function is only RoboSoftware For this purpose a script is used available in the PALM RoboSoftware that searches your microscope image for struc Pro Mode Please contact Carl Zeiss tures and automatically marks them as elements MicroImaging GmbH if you wish to upgrade You can either use the default script supplied with your system PALM RoboSoftware or integrate and choose from up to ten custom scripts The analysis always covers the image section currently visible in the PALM RoboSoftware win dow 415109 2614 101 e 09 09 189 Field of View Analysis How to integrate custom scripts Adjustments PALMA obo e Inthe Adjustments menu click on the PALM Robo option The Preferences and Configuration window opens
8. 415109 2614 101 e 09 09 8 Working with the Element List Window A Element List File Edit Motion Laser Collection Device E Ea A RENQ CloseCut AutolPC v Slide Slide2 Slide3 Summary Show Types all olor r Type O Reference Freehand N 1 2 3 Line 4 Line 5 6 7 8 Rectangle Circle Dot Dot Freehand liver Freehand Freehand Rectangle Rectangle Rectangle Rectangle Laser function CloseCut CloseCut CloseCut AutoLPC CloseCut CloseCut LPC LPC CloseCut CloseCut CloseCut CloseCut CloseCut CloseCut CloseCut E Objective 5x Fluar 5x 0 25 5x Fluar 5x 0 25 5x Fluar 5x 0 25 5x Fluar 5x 0 25 40x LD Plan Neofluar 40x 0 6 Korr 5x Fluar 5x 0 25 5x Fluar 5x 0 25 5x Fluar 5x 0 25 5x Fluar 5x 0 25 5x Fluar 5x 0 25 5x Fluar 5x 0 25 20x LD Plan Neofluar 20 0 4 Korr 20x LD Plan Neofluar 20x 0 4 Korr 20x LD Plan Neofluar 20x 0 4 Korr 20x LD Plan Neofluar 20 0 4 Korr Ruler 5x Fluar 5x 0 25 18 Grp30 Group 5x Fluar 5x 0 25 Well 1C 2C 1D 2D RT E TE IS E E TT ETE NECE LD Plan Neofluar 20x 0 4 Area un Grp 218431 194210 108821 176680 112231 96350 103096 58530 49193 49193 49193 49193 3E K cut shot Comment Reference Grp30 example Morph4 2x2 Col Morph 2x2 Col Morph4 2x2 Col Morph4 2x2 Col H x w 277 2 x 85 8 um 655 9 x 616 3 um 667 7 x 436 9 um 511 5 x 557 8
9. 95 Defining wells for the next laser operation 8 6 2 Setting automatic distribution of the elements to wells e Select the elements for which you want to define wells for the next laser operation e Click on the Calculation entry in the Element List window on the Collection Device menu The Distribution Calculator window opens Select an operating mode in the left half of the window The related settings appear once you have made the selection Operational Configuration Operating Mode n elements per field hi spread elements evento sequence 1 14 THES area per field sequence area area delta area xdelta e Select the required Operating Mode in the Operational Configuration area The significance of the Operating Modes and the related settings are described in detail in the fol lowing Fields that reflect the distribution of the elements to the wells are displayed in the right half of the window The significance of these fields is de scribed in chapter 3 6 3 The window has three buttons If you click on the Cancel button the window will be closed your settings discarded If you click on the Apply button your settings will be applied i e entered in the Well col umn in the Element List window The Distribution Calculator window remains open Ifyou click on OK the settings will be applied The window will close 96 Operating Mode n elements per fie
10. e Click on Enter Select Data in the File menu in either the main window or the Element List window The Please select or enter data window opens i Please select or enter data Slide1 Slidez Collection Device Data Source Data Label Liver Type Description Specimen Data Label Description Holder Label Holder Type Experiment Data Label Description Blue labeled fields are mandatory You must enter all blue fields to enable a position For working e If you are using a slideholder that can accom modate more than one slide select the tab for the slide for which you wish to load elements In the Source Data Specimen Data and Experiment Data sections of the selection lists in the Label fields you will find all the entries that you have written to the database refer to chapter 7 2 2 e Open these selection lists one at a time start ing at the top and select the desired entry e Close this window by clicking on OK Your elements are displayed on the monitor and in the Element List window 415109 2614 101 e 09 09 9 4 Exporting and importing elements With the Export function you create a file that can be opened in any text editor Info The functions Export Elements and Import Elements are intended primarily for exchang ing data with other programs If you are working in File Mode the Save Elements function is recommended for saving elements w
11. R Pointer Tool e Select the Pointer Tool in the Graphic Toolbar e Click once with the mouse on the element or move the cursor with the mouse button depressed to pull up a rectangle that surrounds the element you require entirely or partly Y Ctrl key Pointer Tool If your elements are overlapping you might not be able to select a single element with the described methods without selecting adjacent elements also e Inthis case keep the Ctrl key pressed and click on the desired element until it is selected with each click the next of the stacked elements will be selected To select multiple elements R Shift key Pointer Tool e Select the Pointer Tool in the Graphic Toolbar e Clickon the first element you want to select e Press and hold down key Shift click on all other elements you want to select as well 27 Deleting elements and restoring deleted elements To stop selection of elements Edit DeSelect All Ctrl e Click on the menu item DeSelect All in the Edit menu or click in the microscope image on any dot out side the selection rectangle Info If elements outside of the image section dis played on the monitor are selected it is possi ble that you do not see the selection rectangle as it is larger than the monitor In this case you cannot deselect the elements with one mouse click Info If you want to deselect only single of multiple selected elements first you have to deselect
12. The following section provides instructions for working with a movable trapping laser beam If you have purchased a system with two movable trapping laser beams you will use the same meth ods for working with both beams The moving trap also operates with the trapping laser beam switched off This means that you do not have to switch the trapping laser on to move the beam to a different site There are three ways to move the trapping laser beam with the mouse see Chapter 13 11 2 with the arrow keys see Chapter 13 11 3 with the Joystick see Chapter 13 11 4 13 11 1 Setting speed of the moving trap You can set the speed separately for every type of trapping laser beam movement TrapXY Speed of movement in Trap Mode TrapXY Speed when moving with arrow keys or joystick If you have installed a system with two movable trapping laser beams the speed values set apply to both beams For the speed setting you can select between the units steps sec and Percent You can save the speed settings together with oth er parameters in a settings file refer to chapter 3 7 You have the following options for setting the movement speed of the trapping laser beam with the Speed Tools tool with keyboard or menu commands You can set the speeds of both types of move ments with the Speed Tools tool Speed changes with keyboard or menu commands always refer to the type of movement that is currently s
13. e Click on the Pickup button in the Pixeldata section e Move the mouse pointer to the point on the microscope image for which you want to display the color values and brightness and click on it The fields Red Green and Blue show the brightness values for the three primary colors and their ratio to one another while the Brightness field shows the brightness of the selected point in the image To display the lowest and highest brightness value for the entire image e Click on the Refresh button in the Imagedata section 37 38 415109 2614 101 e 09 09 4 Calibrating the system Note The system is calibrated by the manufacturer for the objectives with which it is supplied It is therefore not usually necessary to recalibrate Recalibration is only necessary if despite exact positioning of the Laser Marker and correct choice of objective differences occur between the drawn contours and those processed by the laser Carry out a recalibration as described in chapters 4 1 to 4 5 and 4 7 You can reset the cal ibration to the factory default setting at any time refer to chapter 4 6 or 4 8 4 1 Preliminary remarks To achieve the desired results with the laser func tions the following points must be fulfilled the program must be set to the microscope s magnification setting and the movement of the stage and the trap ping laser if you have installed a system with Laser Beam Positioni
14. e Enter the desired value 153 The moving trap To change the speed with menu commands or with keyboard shortcuts Motion Speed slower Chrl S Speed faster Ctrl F e Ifyou want to reduce the speed press key combination Ctrl S or click on the menu item Speed Slower in the Motion menu e If you want to increase the speed press key combination Ctrl F or click on the menu item Speed Faster in the Motion menu Info Changes with keyboard or menu commands al ways refer to the type of movement that is cur rently selected in the Speed Tools tool Therefore activate the tool display before car rying out any speed changes with keyboard shortcuts or menu commands Otherwise you will not be able to see which movement type the changed speed setting will affect If a movement is in progress the selection in Speed Tools is automatically changed to the current movement type and the speed chang es you make are automatically applied to the current motion 154 13 11 2 Moving the trapping laser beam with the mouse TrapXY 1 TrapXY 2 Mode In TrapXY Mode you control the movement of the trapping laser beam with the mouse Each mouse action is translated into a movement of the trap ping laser beam You can also move both trapping laser beams at the same time using the mouse You can perform movements in any direction To switch to TrapXY Mode Laser EEE BJ es MicroBeam Tweezers Trap 1
15. e Select the FoV Script Assignment tab Preferences and Configuration Configuration Elements Images Laser Appearance Microscope Setup Incubation Force Measurement FoW Script Assignment Recorder configuration Field of View user script assignment not assigned not assigned not assigned not assigned not assigned not assigned not assigned not assigned Workgroup PALM Training SL EER 40x not assianed o M not assigned e Open the drop down list for the first field and select the desired script e Select any other scripts you want to use in the subsequent drop down lists e Close this window by clicking on OK 190 How to select the script to be used e Openthe Field of View Analysis menu in the toolbar The menu contains the Default option followed by all custom scripts you have integrated Default refers to the default PALM RoboSoftware script e gt Default Workgroup PALM Training SL EER 40x e Select the script you want The appearance of the icon button depends on your choice of script see Figure below How to perform an analysis 63 Field of View Analysis Default script selected Field of View Analysis script integrated by user selected e Click on the Field of View Analysis icon button The Thresholds interactive window will open Info The exa
16. in the Adjustments menu The Load Settings window opens e Select the settings file you want to load and click on the Open button The window will close The name of the settings file will appear in the Status Bar 3 8 Restoring settings You can restore the factory default settings at any time To restore settings Adjustments Load Factory Defaults e Click on Load Factory Defaults in the Adjustments menu Note If you restore the settings all those settings which you created will be deleted including any change in the position of the Laser Marker 415109 2614 101 e 09 09 3 9 Short term alarm PALM RoboSoftware has a short term alarm that you can set to a fixed pre defined 1 2 3 5 or 10 minutes time or set to a freely selectable time seconds minutes There is also an option to en ter a message text At the end of the specified pe riod a message window is displayed showing any text that you may have entered You may wish to use this short term alarm as a timekeeper in time critical operations or as a re minder to switch off the trapping laser with tem perature sensitive samples or to switch off the fluorescent illumination with samples sensitive to fluorescence before the sample begins to suffer any damage To set the short term alarm Y Alarm Bell e Click on the Alarm Bell icon button on the Tool Bar The Alarm Bell window opens Alarm bell Select a time an
17. positions if you want to produce scans of several samples How to specify the area to be scanned You can specify areas to be scanned for all three Slideholder positions Carry out the steps de scribed below for all desired positions e Using the mouse drag a rectangle over the image of the slide that covers the area you want to scan If you only want to scan a small section from your sample or you do not know exactly where the area of interest is located proceed as follows Navigator Ali cina a e Navigation s can AxioVision Analyze VisDat 415109 2614 101 e 09 09 Set ROl top left Set ROI bottom right e Move the stage such that the top left corner of the area to be scanned is displayed in the middle of the monitor e Click on the Set ROI top left button e Move the stage such that the bottom right cor ner of the area to be scanned is displayed in the middle of the monitor e Click on the Set ROI bottom right button The rectangle selected is displayed on the image of the slide In the Navigator window controls appear for specifying the type of focus correction during the scan and the type of scan see Figure below Slide Navigation ddl Selected slide 2 New Slide Set ROl top left Set ROI bottom right Scan all ROls Y T Focuscorrection None O Suspend scan for focus O Use Auto Focus Draw Elements C Marker elements 55 Making the settings for a scan
18. Microscope motorized activate autofocus 167 adjust light intensity 169 Check Position meaning 168 default settings 170 define Check Position 169 define Work Position 169 Load Position meaning 168 open close shutter 169 set Check Position 169 set focus 167 set Load Position 169 set Work Position 169 switching the lamp on and off 169 Work Position meaning 168 Microscope Tools 167 fluorescence 158 Microscope Window 171 Mode 20 Cursor Mode 45 Stage Mode 45 Trap Mode 154 155 Multi channel fluorescence generate recording 178 navigator 56 save images 179 show images 179 Naming element 34 Navigator continue scan 59 focus correction 56 interrupt scan 59 load scanned image 61 multi channel fluorescence 56 prepare scan 54 save scanned image 61 scan section 60 show elements 62 specify scan type 56 start 54 tile 53 415109 2614 101 e 09 09 O Objective Offset adjust 43 reset 44 Operating mode 96 Overview scan go to section scan 60 P PALM CapMover II 137 catapult elements 140 position collection vessel 143 prepare collector 138 set speed 142 settings 142 PALM RoboMover 137 catapult elements 140 define sequence for the wells 139 operating mode 96 position using joystick 27 position wells 143 prepare collector 138 set speed 142 settings 142 PALMRobo information 23 PALMRobo Instructor 21 Pen 73 Position collection vessel 143 Position marking text eleme
19. Temperature Atmosphere Y Module Temperature Atmosphere Y Module Temperature Channel 1 CO Concentration Module Channel 1 Channel 1 Measured Setpoint Heating CO Channel Measured Setpoint Activate Circulator 1 Measured Setpoint Heating a 3 On 20 0 C Off E OF i Off Status online Status online Status online Temperature Channel 2 02 Concentration Module Channel 2 Channel 2 02 Channel Circulator 2 Measured Setpoint Activate Measured Setpoint Heating Measured Setpoint Heating mooom Mo sce Me e 2000 ER 2 Bgn leo Status online Status online Status online Temperature Channel 3 Channel 3 Air Heating Module Air Heater Selected Channel Measured Setpoint Heating Measured Setpoint Heating Channel 1 DETE gt a 00n go On 0 O REE Status online Status online Channel 2 Temperature Channel 4 Channel 4 Measured Setpoint Heating 20 OOn ME NES Status online Control Sensor T Control Sensor T Measured Status online 186 415109 2614 101 e 09 09 17 3 Starting and stopping logging a z Configuration Start Logging View Log hile e Open the menu for the Incubation icon but ton e Click on the Start Logging menu entry The logging is started The Stop Logging menu entry appears e Click on the Stop Logging menu entry to stop the logging 415109 2614 101 e 09 09
20. To specify which mode PALM RoboSoftware should start up in Adjustments PALMA obo e In the Adjustment menu click on the menu item PALMRobo The Preferences and Configuration window opens The Configuration tab will be displayed Configuration Operating Mode File mode only Database mode with selectable DE Database mode with pre selected DE Database e Inthe Operating Mode section select the mode for PALM RoboSoftware to operate in when launched 25 Basic and Advanced display modes If you have chosen Database Mode with pre se lected DB e Choose the database you wish to work with using either the selection list or the button B Note You can protect your database with a pass word and user ID see InformationCenter manual If you enter this information here then PALM RoboSoftware will be automati cally linked with the selected database ev ery time it is launched This means that every user who starts PALM RoboSoftware will have access to your database To prevent unauthorized access to the data base it is therefore not advisable to enter the User ID and password here in the launch settings These will then be requested each time PALM RoboSoftware is launched e If required enter the relevant User ID and password Note It is not possible to switch between File and Database Mode or to change database from within PALM RoboSoftware You should exit P
21. bar Autoconfig automatic adjustment of exposure time white balance brightness and contrast Auto Live function Automated and continuous adaption of camera exposure times of the live image during e g objective change or move of the sample Info If you change the exposure time manually the Auto Live check box is switched off automati cally Measure automatic adjustment of the expo sure time If you are not satisfied with the result you can set other values using the arrow keys Gain AxioCam ICcl The AxioCam ICc1 supports an analog gain from 0 24 dB AxioCam MRc m Rev 3 The AxioCam MRc m Rev 3 uses a gain factor O 5 as power of 2 for the gain adjustment This way gain values of 1 2 4 8 16 32 are possible The AxioCam MRc m Rev 3 supports an addi tional camera gain Camera Gain 2x White Balance automatic white balance You can perform the automatic white balance using any microscope image It is not necessary for the camera to see a completely empty bright image Advanced Settings use this button to open the Live Image window with further settings e g the beam path settings e Click on the related button to activate the func tion resp enter the required settings Gain Factor 1 Gain Factor 0 32 Gain Factor 2 Gain Factor 3 Exposure Time 400ms To make basic camera settings in the Live Image window Advanced Settings e Click on the Advanced Sett
22. channel fluorescence experiments in conjunction 415109 2614 101 e 09 09 with the time lapse function if used You can dis play a superimposed view of the individual images from multi channel fluorescence experiments E Show with picture viewer To view a multi channel fluorescence image e Click on the Show with picture viewer option in the Multi channel fluorescence menu You also use this menu to open the picture viewer if you want to view a series of recordings from a combination of multi channel fluorescence and extended focus ES Show with picture viewer To view an image recorded with extended focus e Clickon the Show with picture viewer option in the Extended focus menu Use the buttons in the bottom section of the win dow to select the image to be displayed and make settings to improve the image display Selecting the image to be displayed If you are combining a multi channel fluorescence recording with a recording with extended focus for each fluorescence channel the number of images with different focus settings that you specified in the extended focus settings will be recorded see chapter 16 2 For example if you are using two fluorescence channels and seven focus levels in an experiment a total of 14 images will be recorded If you have also used the time lapse function for an experiment a multiple of this number of images will be recorded depending on your setting For exampl
23. combination focus 146 define Reference Position 149 indication of the operating state 151 move objects in z axis 152 movement 152 position using joystick 27 set focus using the mouse 147 settings 145 switch on and off 151 Laser energy cutting laser link Cut and LPC 126 set 125 Laser focus cutting laser link Cut and LPC 126 set 125 199 Laser focus trapping laser set 146 set with the mouse 147 Laser functions cutting laser allocate to an element 94 AutoLPC 122 Center RoboLPC 122 Close amp Cut 121 Close amp Cut AutoLPC 122 Cut 121 JointCut 121 LineAutoLPC 121 LPC 121 Oneclick LPC 135 RoboLPC 122 select 124 set available functions 28 set number of passes 127 three dimensional cutting 127 Laser Marker change appearance 19 meaning 194 standard layout 16 Laser power trapping laser set 146 Laser settings cutting laser 124 energy and focus 125 find suitable values 130 footswitch 135 general 128 set available functions 28 Laser settings trapping laser find suitable values 148 footswitch 150 power and focus 146 Laser Tools trapping laser settings 126 146 147 Line thickness change definition 86 Linking Cut and LPC energy and focus 126 Load elements 109 Load Position return to original position 52 Load Position objective meaning 168 set 169 Load Position stage define 52 targeting 52 200 M Measuring 90 area 90 length of lines 90 Message history 23
24. line is completely closed Then the laser first cuts along the line but leaves a narrow joint The width of the joint can be pre selected in the LPC Param eter window The joint is displayed on the monitor with an LPC dot Finally the sample is catapulted from here Single Beam System PALM MicroTweezers is available as a Single Beam System and as a Double Beam System The Single Beam System configuration is equipped with one trapping laser beam and may optionally include Laser Beam Positioning MonoFlex Mono system Work Position Freely definable focus setting of the used objec tive to set by a menu command motorized micro scope only The setting should be defined so that the sample is displayed sharply on the monitor or in the ocular 415109 2614 101 e 09 09 195 Appendix B Keyboard Shortcuts Keyboard Shortcuts F1 F2 F3 Alt F4 F4 F5 F6 F7 Shift F7 Ctrl F7 F11 F12 Ctrl A Ctrl C Ctrl F 196 Icon Button Menu Command Help gt PALMRobo Help E Incubation Devices gt Incubation Device r InformationCenter File gt Open InformationCenter File gt Exit PALMRobo de Navigator Window View gt Navigator Window E Element List View gt Element List 4 Microscope Window Devices gt Microscope in Stage J Motion gt Stage Mode 4 Trap 1 mode Motion gt Trap 1 mode 2 Trap 2 mode E Motion gt Trap 2 mode Start
25. optional Column onented e e In the Subtype Configuration drop down list select the order in which the wells are to be used Using the Line oriented setting the wells are filled row by row A1 A2 A3 conversely with the Column oriented setting column by column Al B1 C1 Info The Subtype Configuration drop down list is not available for all collector types When PALM RoboMover is operating the co ordinates of the selected cap or well are displayed in Current Field 139 Working with PALM RoboMover or PALM CapMover II 12 2 3 Catapulting elements E Start Laser e Click on the Start Laser icon in the main window for the PALM RoboSoftware The progress during the process is shown in the illustration of the collector in the RoboMover window bb d A I EEE 000000000000 10 00000000080 E 0000000000 FO 00000000000 co o 0cEEEHloloovo HO 00000000000 In the example the next element to be catapulted will be catapulted into the well G9 during the cur rent laser operation itis marked with a red bor der Elements have already been catapulted into the wells G5 to G8 There are already catapulted samples from an earlier laser operation in the wells Al to B9 Note After catapulting check that the elements have been catapulted into the correct wells 140 12 2 4 Viewing the catapulted samples After catapulting you can view the content of the wells using the objec
26. or you can archive all images and elements for the Same project together In the upgraded version of the InformationCenter you can switch at any time between Database Mode and File Mode 415109 2614 101 e 09 09 1 5 About this user manual 1 5 1 Contents This user manual describes all functions that are provided by PALM RoboSoftware refer to chapters 1 1 Functionality of PALM MicroLaser systems and 1 2 PALM RoboSoftware Info Like PALM MicroLaser Systems PALM RoboSoftware has been designed on a modu lar basis During system installation by Carl Zeiss Microlmaging GmbH it will be adapted exactly to your system configuration i e the program will provide those tools and com mands which you require to control your par ticular system Tools and commands which relate to components which have not been in stalled will not appear Each chapter begins with a brief summary of the functions that it covers followed by instructions for using and executing the functions The instructions are organized so that they can be followed step by step However you should al ways read the summary at the start of each chap ter first and familiarize yourself with all the operational tasks of a section before you put them into practice A laser function carried out by mis take or with incorrect settings could destroy your sample Until you gain experience with the pro gram functions work on samples that are no lon ger required
27. position the stage at this point and thus display particular areas of your sample on the monitor very rapidly If your system is equipped with the Definiens software or AxioVision image analysis you can analyze the scanned section directly using a script or a set of rules see chapter 6 11 To facilitate analysis the Live Image window enables you to modify various parameters for the image con trast brightness saturation color gamma value see chapter 3 10 The Navigator window includes the Navigation and Scan tabs The Navigation tab displays an image of your slideholder The Scan tab shows the area to be scanned or the current scan First of all produce what is known as an overview scan Within this overview scan you can select a section if required and then scan it with greater magnification section scan Navigator a A KA Navigation s can AxioVision Analyze VisDat 415109 2614 101 e 09 09 A scan is created by sections the stage is posi tioned then an image of the area visible in the PALM RoboSoftware main window is recorded The stage is then moved so that the immediately adja cent section of your sample is visible Another im age is recorded This process is repeated until your entire sample or the section you specified has been scanned An individual scan section is referred to below as a tile You can save and reload the scan either in its entirety or with the individual tiles
28. storing the images is irrelevant To specify a folder Adjustments PALMA obo e Inthe Adjustment menu click on the menu item PALMRobo The Preferences and Configuration window opens e Click on the Images tab Images Saving Images Folder igstAdministratortMy Documents hay PalmD atas m Filename The name of the currently set folder appears in the Folder field of the Saving Images section e Click on the button rp The Image folder window will open e Select the desired folder 114 To select a folder other than the preset folder File New Image Folder e Click on the menu item New Image Folder in the File menu The Select new image folder window opens Select new image folder Select a folder where images should be stored by default Desktop E G My Documents E Carl Zeiss 3 My eBooks Ta My Music ala My PalmData E My Pictures My Videos Settings 18 08 2006 ES 4 My Computer ES a My Network Places Folder My PalmData Make Mew Folder i e Selectthe desired folder or create a new folder e Click on OK Info You can also decide not to select or create a new folder until later when you are ready to save an image 415109 2614 101 e 09 09 10 3 Saving images manually When images are stored manually one image is stored every time you enter an appropriate com mand If you are working in File Mode the images are stored i
29. um 566 1 x 312 1 um 472 4 x 304 2 um 62 4 x 62 4 um 62 4 x 62 4 um 331 8 x 394 0 um 492 0 x 331 6 um 382 6 x 214 6 um 296 7 x 165 8 um 296 7 x 165 8 um 296 7 x 165 8 um 296 7 x 165 8 um Position 100868 3 39715 7 100146 0 39895 1 100899 6 39029 1 100841 0 40156 5 39999 7 40211 1 101272 4 39481 6 99334 0 38845 8 39638 5 38791 2 39396 4 40059 0 99982 1 39267 1 101313 4 40101 9 100801 8 38479 0 100801 8 3831 3 2 100505 1 38479 0 100505 1 38313 2 753 694 um TE 4 38109 4 101 6 Ox 820 2 um om a 39258 9 The Element List window provides a summary in tabular format of all elements and their properties It includes a series of functions for preparing and managing your elements Depending on the slideholder used you will find at least two tabs one for displaying totals and one or more for your slide s in the example shown above there is one tab each for the 3 slides in a SlideHolder 3x1 0 To open the Element List window View E Element List F5 e Click on the menu item Element List in the View menu To close the Element List window File Close e Click on Close in the File menu in the Element List window 415109 2614 101 e 09 09 8 1 Items shown on the Slide tab The Show Types field allows you to decide whether all elements or only the elements of spe cific types are to be listed lines freehands dots ruler
30. window 5 Stage 106376 3 46662 0 um The system components are shown in the left hand column and the current status in the right hand column Columns X Y and Z provide information on the current coordinates of each System Stage Cut Laser CaptureDevice Trapl Laser Trap2 Laser TrapXY1 Trapxr2 component 24 System and control box states stl dle stldle stldle 1028370 Double click in the field at the rightmost end of the Status Bar ljassses jo 18100 stidle stide Wo Istldle stidle loser To 2805 stidle x Not available 13165 415109 2614 101 e 09 09 Launching settings 3 Settings When working with PALM RoboSoftware you will make various settings All of your settings will be retained until they are actively changed again even if you exit the program and relaunch it You can also save your settings in a file for non volatile storage and load them again later All factory default settings can be restored at any time This chapter contains information on the following settings settings for launching PALM RoboSoftware Changing between the Basic and Advanced display options switching software over for different slidehold ers turning automatic focusing on and off for saved and reloaded elements Settings for the saving of settings saving and loading settings unlocking sof
31. 1 1 Delta Ani dp 4 4 Foot switch Foot switch C Auto change Auto change Cut Delta LPC Cut Delta LPC Cutting iteration Cutting iteration Cycles Cycles Auto Change deactivated Auto Change activated The current settings are displayed in the fields of the Energy and Focus blocks e Set the required values for energy and focus using the sliders or the arrow keys To combine and separate laser energy and focus for the cut and catapult functions e Click on the checkbox in the Laser Tools before the Auto Change entry in order to select or deselect a combination of these set tings Info H The setting always applies to both parameters e Ifacombination is enabled enter the value for the difference between the values for energy or focus in the Delta fields Info The value for delta can be positive or negative 126 To set laser energy and laser focus with menu commands or with keyboard shortcuts Laser Energy Power more Fage Up Energy Powerless Page Down Focus up Home Focus down End Note Using menu commands or keyboard shortcuts change the settings for the laser that is select ed in the Laser Tools Cutting Laser MicroBeam or Trapping Laser MicroTweezers You also change the settings when the Laser Tools are not displayed however you then have no control over the changes that have been carried out Therefore before you use these commands switch to
32. 10 Adjusting the reflector offset papa a a SS GO E ee a E O E 164 14 11 Activating and deactivating the timer LL 165 14 12 Colibri fluorescence illumination with LEDS LL 165 15 Control Microscope Zeiss Axio Observer 000000 lt lt oooooooo 167 151 LHe MIGhOSCOPe TOOG ias el ds hee ad o A 167 15 1 1 Seting required magnifica ciO uma es dew A A AA A A A EA E he 167 Sd SelM ne VOCUS nl a Aa iia Bd 167 15 L 3 Defining and setting fixed positions for the fOCUS_ ooo a ee ee e 168 15 4 Making Mumi atcon settnds acrilico ara 169 15 2 Making pre settings for the microscope ooooooccocconoa ra 170 15 3 TRS MIEFOSCOPe WINAO Webs E A aa e a AO 171 15 4 Status display ADO TOME etica ad iaia e ER A A ate 171 15 5 Hardware contigua tdonNS sib adria det a a a ee dl de a 172 15 5 1 Setting up a hardware configuration ea a a a ra 172 15 5 2 Editing the list of hardware configurations LL 174 16 Multi channel fluorescence extended focus and time lapse options 177 16 1 Multi channel fluorescence recordings a ssa aoaaa rr 177 16 2 EX Lenda TO CUE AE aaa e e A a 179 16 3 TIME Lapse ciema e a Sie tah WE dep ade cra ta aida n e daa ERRATA 181 16 4 PIOVICW oi asa et ee ac ee rae o a ei era 182 16 5 Viewing images in the picture viewer asas a 182 17 INCUBATION OPHON Essas aa SAA RA aa A 185 17 1 Making settings for the logging asa bias cea aa da e 185 17 2 Making settings for the I
33. 3 the gradation curve is moved in parallel In this way you will change the brightness of the image evenly over all areas i e the entire image will become brighter or darker Movement to the left the image will become brighter very bright parts of the image may become completely white Movement to the right the image will become darker very dark parts of the image may become completely black Squares at the end of the curve 1 5 the gradation curve retains its shape but becomes steeper or shallower In this way you can change the contrast and the bright ness 36 If you move the square at the bottom left particularly the darker areas of the image will be changed If you move the square at the upper right particularly the brighter areas of the image will be changed Other squares 2 4 the shape of the gra dation curve will be changed The brightest and darkest points in your image will retain their brightness however the brightness curve between black and white will no longer be linear change to the gamma value To have the software find the optimal param eters automatically e Click on the BestFit button To reset the modified settings Linear e Click on the Linear button To set the black point and white point ES e Select the required value in the Thousandths field The histogram will be adjusted such that the se lected number of thousandths in the image exam ple 5
34. 7 x 548 4 um Comment Text Objective Fluar 520 25 Function ClogeCut AutoL PC Well manual Cancel e Enter your name in the Name field or your comment in the Comment field 84 7 9 Changing colors and line thickness PALM RoboSoftware includes a Color Palette and a window in which you can select and preset the col ors you want available In the Color Palette select a color for new ele ments to be drawn Working with this palette is particularly advantageous if you want to draw multiple elements of the same type which are to be in various colors In the Draw Preferences window you can define which colors are available in the Color Palette In parallel you can define the color for different element types in the Draw Preferences win dow I e if you select a different drawing tool the color defined here is set automatically These settings are overwritten later by any subsequent changes to the Color Palette In the Draw Preferences window you can also enter the line thickness and dot sizes for your ele ments on the monitor The settings will apply until you make new set tings Elements already drawn will not be changed How to subsequently change the color of elements already drawn is described in chapter 7 9 3 The settings are retained after the program is closed down If you restore the factory defaults refer also to chapter 3 8 all the changes you made to color and line
35. Deka 2 f 3 u m Number of elements to process Start with Field 14 v Number of elements per well until Field 1F v Remaining elements Cancel e Enter the required values in the Area and Delta fields e Selectthe co ordinates of the first and last well into which the elements are to be catapulted on the Start with Field and until Field drop down lists Info The order of the wells is defined by the setting in the Subtype Configuration field in the RoboMover window line by line or column by column refer to chapter 12 2 2 Info AII wells are filled one after the other as per the mathematical series If there are no long er enough elements available for a well the laser operation is interrupted Remaining ele ments are not catapulted Note Elements are catapulted into the individual wells until the area you have defined is ex ceeded Then PALM RoboMover is moved to the next well I e the actual area catapulted into a well is in general somewhat larger than the value you have specified Note that the area catapulted into each well can vary if your elements are of varying size 415109 2614 101 e 09 09 Defining wells for the next laser operation Operating Mode morphology conserving This operating mode is for catapulting elements that you have drawn with the Grid Rectangle Tool refer to chapter 7 3 8 During this process a matrix of rows and columns of rectangles is crea
36. Help as well as a tutorial for several very important functions the PALMRobo Instructor You can also call up information about the program and the manufacturer 2 4 1 Online Help Online Help provides information about the pro gram layout and the meaning of the icon buttons the menu commands and the tools To open the Online Help Help PALMA obo Help Fl e Click on PALMRobo Help in the Help menu The Help window opens e Click on the point in the Help window about which you have questions or enter a key word To close the Help window e Click on the Close button in the top right corner of the Help window 415109 2614 101 e 09 09 Help and information about PALM RoboSoftware 2 4 2 The PALMRobo Instructor The PALMRobo Instructor explains in detail how to carry out the following functions Calibrating the system Defining the objective offset Saving elements in a file Retrieving elements from a file Besides this the PALMRobo Instructor contains a link to directly open the trouble shooting page of the online help So you have a direct way to this information if you have problems or questions To open the PALMRobo Instructor Help PALMA obo Instructor e Click on PALMRobo Instructor in the Help menu The PALMRobo Instructor window will open PALMRobo Instructor What do you wantto do 7 Calibrate the system Q Define the objective offset I 9 cave elements on f
37. If you are saving your scan with tiles and the number of tiles is very high saving can take a relatively long time 415109 2614 101 e 09 09 Saving and reloading the scanned image How to load a saved scan e Click on the Load button The Open scanned picture window opens e Select the image you want e Click on the Open button The image is loaded and displayed Info To coordinate the positions in the scan and actual positions the slide must be at the same position in the slideholder and inserted with the same alignment as when scanned Info If the stage has been re initialized between saving and loading the scan there may be a misalignment between the scan and the actual positions 61 Displaying elements in the scan 6 7 Displaying elements in the scan Info Text elements are used for marking and RE information purposes they do not describe a EEN section of your sample in the way for instance Y Marker elements that freehand elements do They therefore always appear the same size on the screen irrespective of the magnification setting If you have chosen a low level of magnification it is therefore possible that text elements may e Select the Elements checkbox to display all elements except text elements e Select the Marker elements checkbox to dis cover areas that are significant for you In this play text elements case you can turn off the text elements other The elements appear both in
38. M Color Nr Name Type Laser function Objective Well Area un Grp cut shot 11 Freehand AutoLPC 5x Fluar 5x 0 25 402946 Slide 2 E 12 Freehand AutoLPC 5x Fluar 5x 0 25 265509 Slide 2 E 13 Freehand AutoLPC 5x Fluar 5x 0 25 391517 Slide 2 14 Slide Group 5x Fluar 5x 0 25 15 Referenc Sx Fluar 5x 0 25 Slide 2 16 Referenc 5x Fluar 5x 0 25 Slide 2 17 Referenc 5x Fluar 5x 0 25 Slide 2 Step 5a transfering elements to the target sample automatic adaptation Now transfer the elements which are to be pro cessed with the laser from the source to the target sample The reference points on the source sample will then be aligned with those of the target sam ple Edit Match Serial Section Group e Click on the menu item Match Serial Section Group in the Edit menu Match Serial Section Groups Select Source Group Select Destination Group Name Mr Grp Comment Name Mr Grp Comment Slide1 4 Slide1 4 Slide 14 EE E E gt Delete source group after matching elements into destination group The Match Serial Section Groups window opens e Click on the group in the table at the left of the window which you want to transfer to the other sample in the example Slide1 e Click on the sample in the table at the right onto which the elements are to be transferred in the example Slide2 Info In the example above only one group of ele ments exists on ea
39. Preferences and Configuration window Incubation tab You can open this window using the menu command Adjustments gt PALMRobo e Click on the button id e Select the folder in which your log file is to be saved The logging can be either event controlled or time controlled Event controlled logging only changes to the incubation parameters are logged independent of the time 415109 2614 101 e 09 09 Time controlled logging the current values are added to the log file at regular intervals e Click on the radio button for the required log ging type If you have selected Time controlled logging e In the Intervall field enter the intervals at which the log entries are to be made 17 2 Making settings for the incubator Devices E Incubation Device F2 e On the Devices menu click on Incubation Device The Incubation window is opened In this window you will find three tabs for the settings for temper ature atmosphere and Y modules see figures on page 186 Make the settings for all parameters in the same way The current value of the related parameter is dis played in the Measured field e Set the required setpoint for the related param eter in the Setpoint field e Click on the On or Off button in the Heating area to switch the control of the related parameter on or off 185 n z Incubation Incubation Incubation Temperature Atmosphere Y Module
40. Scale Bar and the Copyright Note You can change the appearance of the Laser Markers These settings will be retained after you exit and relaunch the program You resize the program window in the same way as in all Windows programs To change the size of the icon buttons Adjustments PALMAR obo e In the Adjustment menu click on the menu item PALMRobo The Preferences and Configuration window opens Appearance Size of the icons O Small Medium Large e Select the Appearance tab e Select the required size in the Size of the icons section 17 Customizing the user interface To display or hide all icon bars and tools View Show All Bars Altes Hide All Bars Altes e Click on menu item Show All Bars resp Hide All Bars in the View menu Info There is only one menu item Depending on the current state the entry switches into the other state The menu item description tog gles between Hide All Bars and Show All Bars To display or hide a bar or tool Adjustments PALMA obo e In the Adjustment menu click on the menu item PALMRobo The Preferences and Configuration window opens Appearance Bars ToolBar Microscope tools Laser tools Graphic tools Speed tools Color palette Camera control Status bar e Select the Appearance tab e In the Bars section click in the checkbox for the bar or tool that you want t
41. Start Laser function entry in the Laser menu With the keyboard command e Press F11 on the keyboard 415109 2614 101 e 09 09 Starting the automatic laser functions After the laser function has been released the stage moves automatically along the defined cut lines or from LPC dot to LPC dot the laser is acti vated and deactivated as necessary For each ele ment the objective is set and the laser function ac tivated that you have defined for this element refer to Laser function and Lens columns in the Element List window Provided your system is equipped with PALM RoboMover PALM RoboMover is moved such that each element to be catapulted is catapulted into the well defined When all elements for processing have been pro cessed the laser is switched off Depending on your selection refer to chapter 11 4 4 the stage is moved back to the start point for the laser oper ation or it remains at the position the last laser op eration occurred Your elements are displayed on the monitor again this only takes place during the laser operation if you have made the appropriate settings refer also to chapter 11 4 4 This display serves as a check of the laser function Elements that have been processed with the laser are displayed on a green background in the Element List window 133 Indications of the operating state of the laser 11 8 Indications of the operating state of the laser The operating st
42. The position of the Laser Marker may vary with dif ferent objectives 194 Laser Microdissection and Laser Pressure Catapulting LMPC This technology combines microdissection in which the samples are cut out with zero contact with Laser Pressure Catapulting LPC Laser Pressure Catapulting LPC With a single laser pulse objects marked with an LPC dot are aimed and catapulted into a cap LineAutoLPC Using this function a line shaped area is pre drawn and catapulted into your cap This element is therefore not catapulted in one piece but with several laser pulses Load Position Stage Position of the stage where the stage insert or a slide can be changed The stage can be moved to the Load Position and back using a menu com mand PALM RoboMover or PALM CapMover II Position of PALM RoboMover or PALM CapMover II at which the collector can be replaced PALM RoboMover or PALM CapMover II can be moved to the Load Position using a command Load Position Focus Default focus settings of the microscope which can be set by a menu command motorized micro scope only lowest possible position of the objec tive Before the stage is moved to its load position or to the CapCheck the load position of the objec tive must be set to avoid damage to the objective or the stage and the slide respectively Multichannel fluorescence With multichannel fluorescence two or more re cordings of the sample are made in succ
43. The scan is saved with its coordinates This means that you can use the reloaded image in exactly the same way as with a new scan You can set a parameter to specify that your ele ments will appear on the scans If you have used a corresponding slideholder in PALM RoboStage II you can also make scans of several samples and switch between them in the view Slide Navigation ddl Selected slide 2 New Slide Set ROl top left Set ROI bottom right Draw C Elements C Marker elements 53 Starting PALM Navigator 6 1 Starting PALM Navigator View Navigator Window e Click on Navigator Window in the View menu or press the function key F4 The Navigator window with the Navigation tab opens This contains an image of your slideholder see Figure on previous page shows 3x1 0 slide holder which can hold three slides slide 2 is cur rently selected here Info Note that the type of slideholder must also be set in PALM RoboSoftware see chapter 3 3 Info If you are working in Database Mode the slides for which you have not yet entered any data see chapter 7 2 2 are shown shaded 54 6 2 Making the settings for a scan To prepare a scan e In the Display Tools in the PALM RoboSoftware main window select the Display tab and click on Linear Info If the display is set to non linear the individual tiles of the image in the Navigator may be displayed with varying brightness
44. Type Information and Control Ck m Texas Red DoubleClick Color to change Note In the Basic configuration with the manual microscope the function Fluorescence Filter is not active Adjustments Fluorescence e Inthe Adjustments menu click on the menu item Fluorescence The Fluorescence adjustments window will open e Select the Fluorescence Filter tab refer to the figure at the top of this page On this tab you can select a fluorescence excitation filter assign names for the filters and view various items of information on the filters Information about the installed filter wheel Information and Control section Filter Type field Information on the manufac turer of the filter wheel Ludl or microscope Filter Error field This is where error mes Sages are displayed in the event of problems with the filter Actual Position field This shows which filter is currently in the fluorescence beam path You can also select the desired excitation filter here refer to page 160 160 Filter Colors and Names section Information about the existing filters up to six if a Ludl filter wheel is installed up to eight if a microscope filter wheel is installed You can make or change any entry yourself in the Color column see below If you have installed a microscope filter wheel the names that were specified in the control program for the microscope appe
45. You can check the calibration by drawing an ele ment and activating the automatic laser Cut function Calibration is correct when the cut line exactly follows the element you have drawn for 41 Calibrating the offset instructions on working with these functions refer to chapter 7 Drawing and measuring or chapter 11 Cutting and Catapulting Note The calibration only applies to the camera se lected and the magnification selected before the calibration If you intend to work with var ious magnifications you must recalibrate for each change of magnification 4 5 Calibrating the offset Note In a TwinFlex system Double Beam system with two movable laser beams both offsets Trap 1 and Trap 2 must be calibrated Recalibration is necessary if the position of the La ser Marker and the active laser position do not be have together in a proportionally consistent manner when in motion If they do move together in a proportionally consistent manner but do not coincide only the Laser Marker must be readjust ed refer to chapter 4 3 Calibration is done in several steps PALM RoboSoftware will guide you through this process Note Before you carry out the calibration you must have set the Laser Markers as described in chapter 4 3 e Insert a sample and focus the image Info The Laser Beam Positioning can only be cali brated with the trapping laser switched on Accordingly you should use a sample th
46. activated If there is no indication here you will control the stage or if this is at the CapCheck the collection device using the joystick 16 Slide The third field indicates which slide is being Shown on the monitor this only applies when you are using a slide holder that accommodates more than one slide or a message is displayed whilst elements are being calculated by PALM RoboSoftware If you double click on the field the Navigator window will open refer to chapter 6 ExpO6lli0 2 5ek The fourth field shows the name of the current active setup file file extension set If you have not saved or loaded any settings which means that you are working with the factory defaults this field is empty E 9 Elements The fifth field shows either the number of the drawn elements or the number of the element presently positioned at the center of the image x of and the number of all existing ele ments of y If you double click on the field the Element List window will open refer to chapter 8 Idle The sixth field shows the current program mode Cursor Mode Stage Mode Scrolling stage Calibration Laser Point Continuous Position Laser ON Trap 1 Trap 2 If you are just in the process of drawing or editing an ele ment its current size is indicated If no action is currently in progress then Idle is dis played D Stage 106376 3 46662 0 um The field on the right shows the coor
47. all elements then select the desired elements again 7 6 3 Selecting and deselecting elements in the Element List window The selected elements in the Elements List win dow are highlighted in blue To select or deselect all elements Edit Select All DeSelect All e Click on the menu item Select All or DeSelect All in the Edit menu Element List window To select or deselect single elements e If you want to select a single element click on the corresponding entry in the table of the Element List window If you want to select multiple elements keep the Ctrl or Shift keys pressed and click on the corresponding entries successively e Ifyouwantto deselect the elements click in an empty line in the table Info With this method you can select groups of ele ments of the same type and color by clicking on the corresponding entry in the table on the Summary tab Depending on the choice made in the Show summary for field refer to chapter 8 2 either all elements or the ele ments on one slide are shown 78 7 7 Deleting elements and restoring deleted elements PALM RoboSoftware provides various possible ways to delete elements all existing elements at once the last element drawn one or more selected elements In addition you can delete all elements that you have drawn for a slide if you have used a slide holder that can hold several slides e g the SlideHolder 3x1 0 The e
48. all images and video sequences are saved in a folder The date and time are used to create a name for this folder which is created automatically in the specified folder refer to chapter 10 2 If you are working in Database Mode the images will be stored in the database Show Log File button every time the recorder function is activated PALM RoboSoftware creates a file showing the sequence of events for the function This file is saved in the same folder as the images Click on the button to open this file the button remains disabled until the sequence has been completed Set the sequence of events for your recordings in the Time Lapse Definition Data section If you tick the check box all time data inter vals and durations in minutes then all times will be expressed in minutes rather than the default of seconds The Video Time Lapse 1 and Time Lapse 2 check boxes allow you to choose whether you wish to save a video sequence or a series of images You can also tick more than one check box if for instance you wish to record a video anda series of images or indeed two series of images with different settings Enter an interval between images to be stored in the Interval field applies only to series of images Enter the length for a video sequence or how long images should be stored in the Duration field For series of images the number of images can thus be calculated from the time interval betwe
49. area within which it can be moved Motion Goto Center e Drag the cursor on item Goto Center in the Motion menu e Click on the item Trap 1 or Trap 2 The selected movable beam is centered 149 Moving the trapping laser beam to a particular point 13 6 Moving the trapping laser beam to a particular point You can use the Centering Tool to position the trapping laser beam at any point in the microscope image on the screen Arrow keps Joystick mode o de ae O e Inthe Arrow keys Joystick mode area at the right hand edge of the program window select the check box for the laser beam you want to position using the Centering Tool If you select the check boxes for both beams the Centering Tool will simultaneously position both beams at the same point Info If the stage is at the CapCheck refer to chapter 5 5 the check boxes are not available S Center Trapping laser beam 1 is selected GF Center Trapping laser beam 2 is selected 85 Center Trapping laser beams 1 and 2 are selected e Click on the Centering Tool in the graphics toolbar e Click on the point in the image at which you want the trapping laser beam to be positioned The Centering Tool will continue as the active tool until you click on any other tool on the graphics toolbar 150 13 7 Settings for the footswitch For the footswitch you can specify whether the trapping laser is only to remain activ
50. as on the source sample and combine these into a group 5a Transferring elements to the target sample PALM RoboSoftware moves the reference points on the source sample automatically to the locations of the corresponding reference points on the target sample and recalculates all the transferred elements so that they match up with respective structures on the slide Similarly for the manual adaptation 1 3 as with automatic adaptation 4b Preparing the target sample position the tar get sample under the microscope You do not need to define any reference points in this in stance 5b Transferring elements to the target sample To do this move the reference elements of the source group manually to the position of the corresponding structures on the target sample PALM RoboSoftware recalculates all the other elements to bring them into line with the corresponding structures on the slide 415109 2614 101 e 09 09 The Serial Sections function Step 1 preparing the samples automatic and manual adaptation e Before cutting create three markings in your sample block that are easily recognizable under the microscope e g three pinpricks The mark ings should form a triangle i e not a straight line the further away they are from each other the more accurate the adaptation will be Make sure that you place the markings exactly vertical to the cutting plane e Produce the wafers from the sample e Se
51. be able to see the entire well To see other areas in the well you can move the well in the x and y direction For this purpose use the same features as for the stage in the Stage Mode refer to chap ter 5 1 Movement types e Position the well using the scroll function using the keyboard arrow keys or the Centering Tool If you have connected a joystick you can also use it to position the well Info Set the speed for the positioning of the well as described in chapter 12 4 1 Setting the mo vement speed Stage e Click on the Stage icon button in the main window of PALM RoboSoftware Once you have viewed your sample Note To avoid damage to the objective or to the sample Move the stage back again to the ori ginal position after observing the collection vessel For this proceed solely as described below e If you do not have a motorized microscope Move the objective down as far as possible Eh Point of Origin e Click on the Point of Origin icon button 415109 2614 101 e 09 09 Working with PALM RoboMover or PALM CapMover II 12 2 5 Stopping PALM RoboMover or PALM CapMover II in emergencies Stop e Click on the Stop icon button or e switch off PALM ControlUnit In both cases the movement is interrupted imme diately 12 2 6 Placing the collector in the Home position In the Home position you can park the collector if you do not need it Ea Home e Click on t
52. beams is deactivated You can move the PALM RoboMover using the joystick when the control of both trapping laser beams is deactivated and the stage is at the CapCheck refer to chapter 5 5 The stage retains its position during this process Setting using the PALM RoboSoftware Arrow keps Joystick mode Arrow keps Arrow keps Joystick mode Joystick mode o E 1 O es on M e 2 2 2 O sa 2 O If the checkbox at the top is activated you will control the stage If the stage is at the CapCheck refer to chapter 5 5 all buttons are deactivated and you will control the PALM RoboMover The stage retains its position during this process If the checkbox in the middle is activated you will control trapping laser beam 1 If the checkbox at the bottom is activated you will control the trapping laser beam 2 Ifthe checkbox in the middle and the checkbox at the bottom are activated you will control both beams simultaneously and in parallel 27 Setting the selection of the available laser functions You can also see which unit is moved using the joystick or the arrow keys in the Status Bar al ui mal ui a b C d a Stage or PALM RoboMover b Trapping laser beam 1 c Trapping laser beam 2 d Both trapping laser beams simultaneously To activate the joystick Adjustments PALMA obo e In the Adjustment menu click on the menu item PALMRobo The Preferences and Configuration win
53. but only if you have not moved the stage in the meantime As soon the stage is moved the fluorescence images are lost 415109 2614 101 e 09 09 Extended focus 16 2 Extended focus Before you can generate a recording with extend ed focus you must make the following settings Adda corresponding hardware configuration Make settings for the experiment Select an experiment To prepare a recording with extended focus Setting up the hardware configuration Ex Create hardware settings e Openthe Extended focus menu in the toolbar e Click on Create hardware settings The Setting editor window opens where you can make settings for your hardware E Setting Editor Stored hardware settings Selected Setting ser Alles aus zvhs Component setting Closed Manual PC 0 D0W Reflector 1 100 Mirror Reflector 4255 Device b Microscoj pe oT Microscoj pe B Microscoy pe Lr vv Remove all gt e Make your settings see chapter 15 5 e Click on the Save button to save the configu ration you have created Making settings for an experiment ES Create modity stack experiments e Click on the Create modify z stack experiments option in the Extended focus menu A window opens where you can make settings for a new experiment or change settings for an existing experiment 179 Extended focus Ei Multidimensional Acquisition C x Ja Experiment Ed c EE fe T Stac
54. by clicking on OK To remove elements from a group Edit Remove from Group e Select the elements you want to remove from the group either on the monitor or in the Element List window e Click on the menu item Remove from Group in the Edit menu in the main window or in the Element List window To ungroup elements e Inthe Element List window select the group you want to ungroup do not select the ele ments in the group Edit a Delete selected elements Del e Click on the menu item Delete selected ele ments in the Edit menu The selected group is ungrouped the elements are retained e Click on the menu item Renumber in the Edit menu to update the numbering of the remaining elements 89 Measuring To select single elements within a group On the monitor R Ctrl key Pointer Tool e Press and hold the Ctrl key e Click on the element within the group that you want to select In the Element List window e Click on the element you want to select 7 11 Measuring PALM RoboSoftware provides functions for calcu lating the length of lines or the size of areas Note The measuring functions of PALM RoboSoftware are not calibrated at the factory they are an estimated value only Carl Zeiss MicroImaging GmbH does not guarantee the accuracy of the performed measurements The measuring functions have to be checked and if necessary calibrated by the user for example wit
55. can take a long time several images must be recorded for each scan section and the image with extended focus calculated from each 415109 2614 101 e 09 09 Scanning a sample 6 3 Scanning a sample You can start the scan from the Navigation tab or the Scan tab Starting the scan from the Navigator tab is useful if you want to scan specified areas on dif ferent slides without any further actions Click ing scans the specified areas of all slide positions in turn For example this method enables you to run time consuming scans with high magnification on several slides unsuper vised overnight On the Scan tab you can only ever start one scan for a previously specified slide position However here you have the option of inter rupting the scan and then continuing for example to correct the focus You can then rescan tiles that have not been scanned sharply In addition the specified area fills the entire display area on this tab i e it is larger than on the Navigation tab How to scan several specified areas ROIS with one click Scan all AOls e Make sure that you have made all settings as described in chapter 6 2 e Onthe Navigation tab click on the Scan all ROIs button The scan begins Instead of the Scan all ROIS button the Stop Scan button appears Click on this button if you want to cancel the scan The specified areas of all slide positions are scanned in turn The areas alread
56. can then use to analyze ur sample see AxioVision manual Open the Navigator window Select the Navigation tab and define the area ROI that is to be analyzed as described in chapter 6 2 Select the Scan tab Select the Elements checkbox Navigator 64 Navigation Scan AxioVision Analyze VisD at e If necessary select the overview scan Section 0 or the section scan Section 1 in the Section Navigation area depending on which you want to analyze e Select the AxioVision Analyze tab Scripts created in AxioVision are shown in the Analyze scripts drop down list in the Navigator e From the Analyze scripts drop down list choose the script you want to use for the anal ysis e Click on the Adjust parameters button to set the parameters for your sample A preview window opens in which you can check the results of your parameter settings e If necessary activate the Use Auto Focus function or the Suspend scan for focus func tion on the Scan tab in the Navigator window as described in chapter 6 2 e Click on the Analyze ROI Region Of Interest button The selected area of your sample is analyzed Analyze scripts Workgroup PALM Training SL w Adjust parameters Analyze ROI Information Slide Section 1 36 tiles Scanned with 10x objective To set the navigation mode use of stored tiles the focus correction and drawing of elements use the co
57. displayed They cannot be used with the superimposed image d A ko la lo Do 3 Al 1 Optimum display of lightest and darkest gray or color value excluding specified maximum and minimum values 2 Optimum display of lightest and darkest gray or color value 3 Linear display of all possible values 4 Set gamma value to 0 45 for realistic color reproduction e Click on the relevant button to select one of the specified settings Display images as slideshow You can display the individual images as a slide show To do this select whether you want to see the images with different focus settings extended focus for a particular time lapse cycle or images for a particular focus level over all time lapse cycles You can also select whether you want to see a particular fluorescence channel or the super imposed image e Click on the Time Lapse or z stack button to select which series of images you want to dis play as a slideshow time lapse images with a particular focus setting or images with different focus settings from a particular recording time ST mi 1 42 Ez x gt 347 e Use the Z arrow buttons to select the focus level or the T arrow buttons to select the recording time for the images to be displayed au e Select the fluorescence channel or activate the superimposed images 184 e Click on the Timer button to set the display time for each individual image This opens a window in wh
58. e Select the required laser function for the next element to be drawn refer to chapter 7 3 1 you can change this selection again later e Select the required color for the display of the element on the monitor and in the Element List refer to chapter 7 9 e Draw the element e Repeat the last three steps for all elements that you require e Make all the necessary settings for the laser refer to chapter 11 4 e Start the laser 68 7 2 Creating a new set of elements The procedure varies according to which mode you are working in Database Mode or File Mode Info Database Mode is only an option if your system is configured accordingly Please contact Carl Zeiss Microlmaging GmbH to upgrade your system if required Info When the program is launched you decide whether you want to work in File Mode or Database Mode refer to chapter 1 8 It is not possible to switch between these two modes whilst the program is running If you want to change mode you must first shut down PALM RoboSoftware and then relaunch it If neces sary you must first modify the initial settings as described in chapter 3 1 7 2 1 Working in File Mode You can start drawing elements as soon as you have launched the program and adjusted the sam ple in the microscope If you have already drawn elements during your current session and you want to process another Sample you must first after saving it if you wish remove the existing el
59. elements within the group will be considered Al Element List File Edit Motion Laser Collection Device 288 0 e2xaQ z 7 X Slide Slide2 Slide3 Summary Show Types all yl Color Nr Name Type Laser function Objective Well Area ur Grp cut 1 Freehand Ref 1 AutoLPC 5x Fluar 5x 0 25 416385 Slide 1 H 2 Freehand Ref 2 AutoLPC 5x Fluar 5x 0 25 388527 Slide 1 3 Freehand Ref 3 AutoLPC Bx Fluar 5x 0 25 308903 Slide 1 4 Slide 1 Group 5x Fluar 5x 0 25 5 Reference Ref 1 5x Fluar 5x 0 25 Slide 1 6 Reference Ref 2 5x Fluar 5x 0 25 Slide 1 7 Reference Ref 3 5x Fluar 5x 0 25 Slide 1 A 8 Dot LPC 5x Fluar 5x 0 25 Slide 1 9 Freehand AutoLPC 5x Fluar 5x 0 25 254142 Slide 1 EH 10 Circle AutoLPC 5x Fluar 5x 0 25 278380 Slide 1 Step 4a preparing the target sample automatic adaptation Likewise you will define three reference points or reference figures on the target samples Proceed in exactly the same way as described for the source sample e Insert the slide onto which you want to trans fer the elements into the slideholder If you use a multiple slideholder it is advisable to leave the source sample in place and insert the target sample in a vacant position e Position the stage successively on the three markings on the target sample and draw an element on each in the same way as you did for the source sample Note When creating the reference elements choos
60. farther apart e g elements drawn with the Freehand Tool the distance between the start and finish dots is shortened by a straight line to the set value of the RoboLPC joint parameter If you want to deselect this function enter the value 0 124 Note Should the value of the RoboLPC joint param eter be too small or at zero then it can occur that automatic catapulting does not take place in the RoboLPC function as the sample pos sibly will not remain fixed within the tissue when cut OL Elements as they were drawn and would be cut with the Cut function OGL The same elements with laser function JointCut selected for element 1 the distance from start to finish dot is increased for element 2 it is re duced Close element 3 will be opened hal The same elements with laser function RoboLPC selected for element 1 the distance from start to finish dot is increased for element 2 it is reduced Close element 3 will be opened In the center of the joint the catapult point is dis played Distance between AutoLPC shots and distance of the AutoLPC shots from the element border line only applicable in the case of AutoLPC The number and position of the individual laser pulses are then automatically calculated by PALM RoboSoftware 415109 2614 101 e 09 09 Specifying laser settings Position of the AutoLPC shots You determine whether the positions of the laser shots are to be ar
61. in certain circumstances e Choose the desired magnification Info The duration of the scan is dependent upon the magnification set on the microscope and the size of the area to be scanned Region Of Inter est Set to a high magnification e g 100x the scanning process would take several hours Therefore you should adjust the magnification set on the microscope and the size of the re gion of interest to one another first select a low magnification on the microscope e g 2 5x or 5x In this way you can produce an over view scan within a few minutes Note Make sure that you have set the same magni fication on the microscope as is in PALM RoboSoftware see chapter 4 2 with a motor ized microscope the magnification will be automatically set to that selected in the soft ware e Insert the slideholder e Focus the image e Click on the Set Workpos button in the Microscope Tools motorized microscope only see chapter 15 1 3 Slide Navigation qb Selected slide 2 e If necessary select the desired slideholder on the Navigation tab in the Navigator window Click on the slide or click on one of the Slide Navigation arrow but tons in the top right to switch from one slide to another 415109 2614 101 e 09 09 Making the settings for a scan New Slide e Click on the New Slide button to delete an image already displayed e Repeat this process for the other slideholder
62. it has al ready been processed using the laser In this case the menu command is disabled To delete any elements Edit a Delete selected elements Del e Selectthe element you want to delete in PALM RoboSoftware main window or in the Element List e Click on the menu item Delete selected ele ments in the Edit menu in the main window or in the Element List If you select multiple elements you can also de lete multiple elements simultaneously with this command To delete all elements on a slide Edit Delete all elements Ckrl Y e Inthe Element List window select the tab for the slide with the elements you want to delete e g with the SlideHolder 3x1 0 the Slide 1 Slide 2 or Slide 3 tab e Select All on the Show Types drop down list e Click on the menu item Delete all elements in the Edit menu All elements on the selected slide are deleted if you are working in the Database Mode only those elements that have not yet been processed with the laser Info Elements hidden using the Show Types drop down list are not deleted I e you can use the function to delete elements of a specific type 415109 2614 101 e 09 09 To restore deleted elements Edit Undelete Alt BackSp e Click on the menu item Undelete in the Edit menu in the main window of PALM RoboSoftware The last performed deletion command is undone Info If you have deleted several element
63. made in the control program for the microscope apart from shut ter and lamp and can only be changed there If only the entry lt none gt is available in a list this means that the corresponding part is not motorized in your system and therefore cannot be set via PALM RoboSoftware 415109 2614 101 e 09 09 15 3 The Microscope window The appearance of the Microscope window and the settings that can be made in this window are dependent on the configuration of your system To open the Microscope window Devices vl Microscope FE e On the Devices menu click on Microscope Microscope Common Refle Transm St LightPath Mio Sideport L TO R S Bla Oa 100 Ocular Dito TO a Oo Oo Oa Baseport Objective alalalalala Microscope Manager Enable Parfocal Correction Enable Light Manager Light Manager Mode Objective Contrast Manager Mode Contrast retaining Enable Dazzle Protection Illumination You will find information on the settings in this window and how to make the settings in the AxioVision online help 415109 2614 101 e 09 09 The Microscope window 15 4 Status display ApoTome If your microscope is equipped with an ApoTome and if the ApoTome is configured in the Micro scope Toolbox a status display will appear below Microscope Tools The displays are as follows ApoT ome The ApoTome is set up in such a way that the light beam ca
64. obo e Inthe Adjustment menu click on the menu item PALMRobo The Preferences and Configuration window opens Laser Lazer function Display Elements during Laser function Return to start position after Laser function e Select tab Laser e Inthe Laser function section click in the checkbox Display Elements during Laser func tion or the checkbox Return to start position after Laser function to enable or disable these functions 128 11 4 5 Special laser settings for the LineAutoLPC AutoLPC and Close amp Cut AutoLPC functions Note These laser functions only apply for glass mounted samples Note A lower energy and focus should be selected for glass mounted samples than for mem brane mounted samples Using the LineAutoLPC AutoLPC or Close amp Cut AutoLPC functions the contents of a line shaped LineAutoLPC or area shaped element remain ing functions are catapulted into the collection vessel using single laser pulses You must make four settings specifically for these laser functions for the LineAutoLPC only the first the distance of the laser pulses to each other LPC distance the distance of the laser pulses from the border line of the element the arrangement of the dot configuration diag onal or parallel the selection if a laser pulse should be set to the center of the element AutoLPC Center Shot Note The distance settings always appl
65. of the comment background and flag as described in chapter 7 9 Text elements are ignored by the laser functions To place a marking e Select the Text Tool in the Graphic Toolbar e Click on the point at which you want to place your marking Text element Enter text Text element A marking will appear as a flag at the selected site The Text element window opens e Enter if required a comment and close the window by clicking on the OK button E a Text element Your comment will appear at the foot of the flag 75 Displaying and hiding elements 7 4 Displaying and hiding elements All elements are displayed in the default configu ration of PALM RoboSoftware You can hide the el ements To display or hide elements e Click on the menu item Show Elements in the View menu The menu command works as a toggle Info If the display has been switched off then it is switched back on automatically when new ele ments are drawn 7 5 Centering elements on the monitor The image on the monitor only represents a sec tion of your sample If you have drawn multiple el ements usually not all of these are visible on your monitor at the same time If you want to view a particular element you can have PALM RoboSoftware display it for you automatically at the center of the image In main window certain elements can be centered automatically using icon buttons or menu com mands
66. on how to proceed Otherwise you will not obtain the desired result Ctrl key Freehand Tool fw e Select the Freehand Tool e Press and hold the Ctrl key e Move the cursor to the start or finish dot of the element that you want to continue the start and finish dots of an element are indicated by a Small square with thick black lines and the Same color as the element Note Please take care that a square with thick black lines is displayed A square with thin black lines indicates an anchor point of the element If you continue drawing from one of these points you change the shape of the consisting elements see To change the shape of elements lines rectangles ellipses on page 82 e Keeping the mouse button depressed draw the desired extension 415109 2614 101 e 09 09 To join two elements lines rectangles ellipses Note Select the laser function Cut before joining the elements refer to chapter 11 3 for instruc tions on how to proceed Otherwise you will not obtain the desired result Ctrl key Freehand Tool fw 4 e Select the Freehand Tool e Pressand hold the Ctrl key e Move the cursor to the start or finish dot of the first element the start and finish dots of an ele ment are indicated by a small square with thick black lines and the same color as the element e Keeping the mouse button depressed draw the connecting line to the start or end dot of the second element
67. or vice versa the trapping laser will be switched off if you have switched the laser on using PALM RoboSoftware On the other hand the laser remains on if you switched it on using the footswitch in the Hold position refer to chapter 13 7 and are continuing to keep the footswitch de pressed The laser likewise remains on if you switched it on with the footswitch in the Switch posi tion refer to chapter 13 7 To activate and deactivate the trapping laser with PALM RoboSoftware Laser Trap Laser onsoff Trap A O e Click on the Trap Laser on off entry in the Laser menu or click on the On or Off button on the right border of the program window The Off button appears after the trapping laser is switched on Note Particularly living objects may suffer injury due to the radiation of the trapping laser Therefore you should not activate the trap ping laser until you actually need to use it and keep it activated only as while you are working with it 415109 2614 101 e 09 09 Activating and deactivating the trapping laser Info You can also switch off the trapping laser by clicking on the Stop icon button on the Tool Bar However this results not only in the trapping laser being switched off all other processes currently running will also be stopped The Stop icon button should therefore only be used to switch off the laser in the event of an emergency 13 9 Indications of
68. pro cesses do not run synchronously this tab thus Shows the status of all processes You can display separate tiles as on the Scan tab e Select the Stored tiles radio button Buttons appear for navigating between the tiles see chapter 6 8 Info In Live mode the image is re recorded using the current camera settings and then analyzed while in Stored tiles mode the saved images from a previous scan are analyzed 66 415109 2614 101 e 09 09 7 Drawing and measuring The automatic laser functions allow you to cut along defined lines and catapult individual points or areas out of your sample For a summary of the automatic laser functions refer to chapter 11 1 You can also lift parts cut from your sample from the slide plane using PALM MicroTweezers and transport them to a different place refer to chap ter 13 Before you use these laser functions you must define these cut lines or areas they are called el ements PALM RoboSoftware offers you a range of tools in the graphics toolbar for drawing elements You can draw elements one after another for dif ferent laser functions for example elements that are to be cut and catapulted completely RoboLPC beside those that are only to be cut Cut or those that are only to be catapulted LPC You define which laser function is applied to the related element prior to drawing the element however you can also still change the laser func tion selected l
69. radio buttons can also be used to specify the focus that menu or key commands will set see page 148 147 Finding suitable power and focus settings for the trapping laser To set the power and focus for the trapping laser using menu commands or using keyboard shortcuts Laser EnergyPower more Page Up EnergyPower less Page Down Focus up Home Focus down End Note Using menu commands or keyboard shortcuts change the settings for the laser that is select ed in the Laser Tools cutting laser or trap ping laser You also change the settings when the Laser Tools are not displayed however you then have no control over the changes that have been carried out Therefore before you use these commands switch to the Laser Tools display and select the laser function for which you want to change the energy and focus as described earlier in this chapter Note If you have installed a DuoFlex TwinFlex sys tem then menu commands and shortcut keys are used to set the focus of the trapping laser beam selected using the Wheel radio buttons left Trap 1 right Trap 2 see page 147 Setting with menu commands e Click on the items Energy Power more or Energy Power less in the Laser menu to increase or reduce the trapping laser power e Inthe Laser menu click on the items Focus up or Focus down to set the trapping laser focus higher or lower Setting with keyboard shortcuts e Use the Page Up and Page
70. specifying the distribution of the elements to be catapulted to the wells refer to chapter 8 6 Info Even when the Element List window is open you can still access all the functions that are offered in PALM RoboSoftware main window This means that you do not have to close the Element List to continue working 93 Allocating a laser function to elements 8 4 Allocating a laser function to elements The laser function that is applied to the related element when the cutting laser is started is stated in the Laser function column Initially this is the laser function that was selected when the element was drawn refer to chapter 7 3 1 You can change this setting for each element ClozeCut AutoL FC ClozeCut AutoL FC Robol PC Center obol PC e Select the elements to which you want to allo cate a different laser function e Open the drop down list with the laser func tions and click on the required function Info You can also allocate a different laser function to an individual element in the Element prop erties window Select the element and open the Element properties window using the Edit gt Change menu command 94 8 5 Allocating an objective to elements The objective used to process the element with the laser when the cutting laser is started is given in the Objective column Initially this is the objec tive that was in use when the element was drawn You can change this s
71. the Freehand Tool in the Graphic Tool bar e Move the cursor to the dot where you want to begin your drawing e Hold the mouse button down while you draw the required shape and release it at the end dot Info In Cursor Mode you can only use the Freehand Tool inside the Scroll Rectangle When you reach the edge of this rectangle the line stops automatically The program switches to Scroll Mode the stage is moved As soon as you release the mouse button and the stage stops moving you can continue the line as explained in chapter 7 8 In Freeze Mode you can continue to draw with the Freehand Tool outside the Scroll Rectangle for a detailed explanation refer to chapter 7 3 3 415109 2614 101 e 09 09 Drawing new elements 7 3 6 The Line Tool Line Tool Quadratic Attribute Use the Line Tool to draw straight lines e Select the Line Tool in the Graphic Toolbar If you select Quadratic Attribute as well the drawing function is limited to horizontal and vertical lines e Click once on the dot where you want to start your line e Click once on the point at which you want to set a corner dot e Double click on the dot where you want to end your line Info In Cursor Mode you can only use the Line Tool inside the Scroll Rectangle When you reach the edge of this rectangle the line stops auto matically The program switches to Scroll Mode the stage is moved As soon as you release the mous
72. the Laser Tools dis play and select the laser function for which you want to change the energy and focus as de scribed earlier in this chapter Setting with menu commands e Click on items Energy Power more or Energy Power less in the Laser menu to increase or reduce the laser energy e Inthe Laser menu click on items Focus up or Focus down to set the laser focus higher or lower Setting with keyboard shortcuts e Use the Page Up and Page Down keys to raise or lower the laser energy e Press Home or End to increase or reduce the value for the laser focus The values that are currently set are displayed in the Laser Tools 415109 2614 101 e 09 09 11 4 2 Setting number of passes and three dimensional cutting Cutting iteration Cycles e Enter the required number of passes for PALM MicroBeam in the Cycles field in the Laser Tools If you select more than one pass a further field appears Make the setting for three dimensional cutting see Three dimensional cutting on page 123 here Cutting iteration Cycles Z Cut z focus delta ENT e Enter the value by which laser focus is to be raised for each cutting pass in the z focus delta field 415109 2614 101 e 09 09 Specifying laser settings 11 4 3 Activating and deactivating autofocus Info This function is only available if your micro scope has the autofocus facility If the autofocus is switch
73. the image parameters is that there may be a delay before the image appears on the screen because each time a change is made e g the stage is moved the original camera image must be recalculated So it is worth trying to obtain a optimum result from the camera in the first place The settings can be seen immediately in the live image but cannot be seen in the Navigator refer to chapter 6 until the image has been scanned You can change the following parameters Contrast When contrast is increased light parts of the image become lighter and dark parts become darker i e differences in bright ness are accentuated Reducing the contrast has the opposite effect Brightness the entire image becomes lighter or darker You should be aware that if the bright ness setting is altered too much the light parts of the image may become completely white or the dark parts completely black Gamma corrects the linear nature of the brightness pattern between black and white Black and white point in this way you can define the percentage for the brightest and darkest pixels that will be displayed as com pletely white or completely black The settings are in the Display Tools and in the Live Image window 415109 2614 101 e 09 09 Display Tools The Display Tools are shown in the top left of the program window in the standard configuration of the PALM RoboSoftware e Click on the Display tab Display Camera
74. the overview scan elements will continue to be displayed and the section scan Info You may for instance use text elements to mark the area on the sample that you wish to scan In this way the section of interest can be found more easily on the overview scan Navigator Navigation Scan AxioVision Analyze VisDat Section Navigation A gt Co E C Continue at actual position Dorne a Fluar 10 0 50 M27 Information gt Slide Section 1 36 tiles Scanned with 10x objective Thumbnail Navigation Mode Live Stored tiles Focuscorrection O None O Suspend scan for focus Use Auto Focus every 5 tiles Draw Elements 62 415109 2614 101 e 09 09 Displaying a particular tile on the monitor 6 8 Displaying a particular tile on the monitor Navigation Mode Live Stored tiles Tile Navigation KI lt gt DI In the Navigation Mode section of the Scan tab click on the Stored tiles radio button Click on an icon button in the Tile Navigation area First tile Previous tile Next tile Last tile or click on a tile in the overview image The corresponding tile is displayed in the PALM RoboSoftware main window instead of the live im age This tile has a green border in the Navigator Info The stage is not moved when you change from one tile to another Info Double clicking on a tile reverts to live mode and moves to the tile you hav
75. to edit an element refer to chapter 7 8 1 it is gen erally advantageous to select the element on the monitor If you want to select several elements at the same time this action is normally easier in the Element List window as here you have an over view of all elements drawn 7 6 1 Selecting elements using menu commands in the main window You can either select all elements at the same time or all elements that belong to the current slide if you have inserted a slideholder that can hold sev eral slides e g the SlideHolder 3x1 0 To select all elements Edit Select all all positions e Click on the menu item Select all all posi tions in the Edit menu main window To select all elements on the current slide Edit Select All current position only Ctrl e Click on the menu item Select all current posi tions only in the Edit menu main window 415109 2614 101 e 09 09 Selecting and deselecting elements 7 6 2 Selecting and deselecting elements on the monitor On the monitor selected elements are displayed with a surrounding white rectangle At all corners and in the center of each rectangle side a white Square is displayed Info The white selection rectangle partly covers the lines of the elements You cannot see the se lected element completely if you have select ed a rectangular element for example you see the selection rectangle only To select a single element
76. to be drawn for one wafer and are then automatically or if required manually transferred to subsequent wafers When you transfer the elements they are adjusted in such a way that the corresponding structures in the target sample cover them exactly You can also mount the samples at any angle on the slide Any differences in the positioning will be compen sated for in the course of the adaptation Make sure however that on assembly the samples are not stretched or contracted The Serial Sections function enables series of samples especially those whose structures only become visible following a specific treatment such as coloring to be processed significantly more quickly only a single sample needs to be treated accordingly You then only need to draw the ele ments for this one sample and then transfer them to the other samples Through the adaptation when transferring elements your elements will also be placed on the other samples on the desired spots Note that the adaptation can only function satis factorily if the relative position of the structures on the various samples is identical If the tissue block out of which you cut the samples contains a diag onal structure for example and the other struc tures are vertical the differences cannot be automatically compensated for You have the pos sibility to correct such variations by hand after the transfer procedure To keep any necessary correc tions small it is
77. to chapter 9 1 If you wish to save your elements in a folder other than the specified folder e Click on the button mp The Please enter select a file name window opens e Select the desired folder enter a file name and then click on the Save button To load an elements file File E Open Elements e Click on the menu item Open Elements in the File menu in the PALM RoboSoftware main window or in the Element List window The Load Elements from file window opens The exact appearance of this window depends on which slideholder you are using chapter 3 3 ex plains how you should notify the software of which insert you are using The following description ap plies to the SlideHolder 3x1 0 You can follow the same procedure for other slideholders Load elements from file Slide1 d PALMDatalElementsitestslide1 palm diPALMDatalElementstestslide2 palm slides d PALMDatalElementsitestslide3 palm all active If you are using a slideholder that can accommo date more than one slide this window can be used to load the elements for one or more slides at the same time 415109 2614 101 e 09 09 e If you have saved the elements for several or all slides with the same name and now want to load them Check the all active box enter the common file name in the adjacent field to the right if necessary uncheck the check box for slides for which you do not wish to
78. to use for the analysis e Select the Parameter tab A number of sliders for setting various parameters now appear on the left hand side of the window Info The parameters that you can set here depend on which set of rules you have chosen As a rule one does not know at first which param eter settings will deliver the optimum analysis result e Configure initial values for the parameters Analyze Ruleset Training SL KER 40 7 v Delete elements Analyze RDI Information Slide Section O O tiles Not scanned yet To set the navigation mode use of stored tiles and the focus correction use the controls on the scan tab 65 Analyzing images using the Definiens software Delete elements Analyze AOI 2 Elements found in 13 sec The parameter settings are tested with the current microscope image The result is displayed below the buttons on the right hand side of the window e Optimize the parameter values if necessary When you have found the optimum values e If necessary activate the Use Auto Focus or Suspend scan for focus function on the Scan tab described in chapter 6 2 e Click on the Analyze ROI button ROI Region Of Interest The selected area of your sample is analyzed 4 Elements found in 10 sec Evaluate 5 of 63 Info The Job Status tab shows information about the progress of the analysis The Image acqui sition Analysis and Element import
79. until Capture Device has detected the collector In this window you can see along with the current message all messages that the program has out put since it was last started e Click on the more button The list with the message history is displayed Instead of the more button a less button appears e Click on the less button to hide the mes sage history list You can open the PALMRobo Information window at any time To open the PALMRobo Information window For Help press Fi e Double click in the field at the leftmost end of the Status Bar PALMRobo Information 1 Show the message history History 10 30 37 Start fluorescence 10 30 35 Start Control Box 10 30 35 Start Grabber 10 30 35 Start Grabber 10 30 01 Start AY modules 10 30 00 Startup Microscope 10 29 47 Initializing Zeiss MTB2004 10 29 47 Start trace 10 29 47 Start Capture Device 10 29 47 Start Control Box 10 29 47 Read registry data 10 29 47 Start trace 10 29 46 PALM Robo Start The PALMRobo Information window is opened 415109 2614 101 e 09 09 Help and information about PALM RoboSoftware less More Click on the less button or the more button to show or hide the message history 23 Status display 2 5 Status display PALM RoboSoftware States window provides you with details of the current status of your system components To open the States
80. vis ible on the monitor as an image Loading and saving of settings movement speed of the stage laser settings pre selected folders for storing images elements etc Processing of the defined elements with the cutting laser Automatic cutting along defined lines followed by catapulting Controlling the devices for positioning collec tion vessels PALM RoboMover or PALM CapMover II Processing of a series of samples with the cut ting laser i e the areas to be cut and cata pulted are defined on one sample and can be transferred to other samples If you have purchased a system with a motor ized microscope you can also create settings for the microscope via PALM RoboSoftware The software can be upgraded in line with your specific requirements e g with automatic image recognition software or special functions e g for Time Lapse experiments multichannel flu orescence recordings and recordings with extend ed focus 10 1 3 Basic and Pro Mode The PALM RoboSoftware is available in two license versions Basic and Pro Mode The Basic license provides you with all the basic functions for your work with PALM MicroBeam and PALM MicroTweezers The Pro version includes a series of more advanced functions including Autofocus Recorder Field of View Analyse If you have the Pro version you can switch be tween the Basic and Advanced display modes refer to chapter 3 2 You can access all functions
81. was last exited Laser energy and laser focus You can make independent settings for the Cut and LPC functions However you can also link the energy and focus values for cutting and catapulting to each other Set a Delta value and determine in this way the difference between the two values As soon as you change the energy or focus for Cut or LPC the value for the other laser function is automati cally adjusted to reflect the Delta difference The Delta value has to be set separately for energy and focus The set values apply to all laser functions with which you cut or catapult Speed at which the microscope stage moves during an automatic laser function You can set various values for the cutting pro cess and for the speed at which catapulted dots are sequentially targeted LPC RoboLPC joint This parameter is important for the functions RoboLPC and JointCut only It is used to define the length of the joint that remains between the start and finish dots of an element when the element is cut out refer also to the Description of the RoboLPC and JointCut functions in chapter 11 1 If you select the RoboLPC or JointCut function all elements of type Figure whose start and finish dots are closer together than the value of this parame ter are opened to this value including closed elements such as rectangles or squares and cir cles or ovals For elements whose start and finish dots lie
82. window you will find the Position Speed tool using which you can set the movement speed PALM RoboMover moves from one well to the next at this speed PALM RoboMover and PALM CapMover II move to the Load Position or to the Home Position and back using this speed e Set the required speed 415109 2614 101 e 09 09 12 4 2 Positioning wells in x y direction Info All settings that you make apply to the collec tor type currently used You must make the settings for each collector type you use The settings are saved automatically If you fit a different collector type the settings saved for this collector type are applied e Seta low magnification e g 5x e Only PALM RoboMover Click on the well A1 on the Operation tab in the RoboMover win dow e Select the Adjust tab Adjust Fine Adjust xy Positions back left a es right Vv front Save Position as delta position for the collector Set to default In the Fine Adjust x y Positions section you will find buttons for the fine adjustment of the x y po sition for the active well In this way you can align the well exactly over your sample if necessary If your insert has more than one well only PALM RoboMover the setting applies to all wells In this way you can compensate for manufacturing tole rances e Click on the arrow buttons to align the well over your sample e Clickon the Save Position as delta position for the colle
83. 0 7 11 8 1 8 2 8 3 8 4 8 5 8 6 8 6 1 8 6 2 8 6 3 8 7 Gi 9 2 9 3 9 4 9 5 10 10 1 10 2 10 3 10 3 1 10 3 2 10 4 10 5 11 11 1 11 1 1 11 1 2 11 1 3 11 1 4 11 2 11 3 11 4 11 4 1 Contents Selecting elements using menu commands in the main window LL 77 Selecting and deselecting elements on the Monitor 77 Selecting and deselecting elements in the Element List window 78 Deleting elements and restoring deleted elements 78 Processing elements dada daa daa iaia 80 EURO elements penis ae ene nea 80 Copyingrelements kili e AS aree 83 Enabling or disabling the element numbering display o o 83 Updating the element numbering aasan aeaa a a a 84 Adding a name or a comment to elements 84 Changing colors and line thickness 0 a ee ee ee ee ee es 84 Presetting colors and line thickness aaa ee es 85 Selecting colors for elements to be drawn NeW saasaa aa a a 86 Change colors of elements that have already been drawn 87 Working With Clement GrOU DS ie vagos dao ed A We ath E we ee 88 MOASUFING aia di Sete Bi lane Jos stay Jag Wh a eek a e Ges GB e s UE ae da da ate es A 90 Working with the Element List Window 0000 lt lt lt o lt lt 91 Items Shown om the Slide tabs as Se ee A 91 Items shown on the summary tab lt o ooo e uu 92 Functions in the Element List window LL 93 Allocating a laser
84. 07740 Jena Germany BioSciences Munich Location Phone 49 89 90 9000 800 Telefax 49 89 90 9000 820 E Mail palm info zeiss de www zeiss de microdissection SAP No 415109 2614 101
85. 101 e 09 09 119 Saving images and videos using the Recorder function 120 415109 2614 101 e 09 09 11 Cutting and Catapulting 11 1 Laser functions With PALM MicroLaser System you can cut on the microscope and catapult parts of the sample De pending on the objective of your work you will need variable options of this function or combina tions thereof 11 1 1 Automatic laser functions PALM RoboSoftware gives you the following auto matic laser functions Cut e a POD WACISE Ode ass z T o Da oa With this ineo the laser cuts your sample along pre drawn lines Figure elements Lines rectangles and circles so that a clearly split line between required and non required material is generated In the cut area all biological material is destroyed by ablation Thus a pure sample preparation can be carried out without fear of contamination Close amp Cut With this rico the par ico cuts your sample along defined lines Unlike with the Cut function however the start and end dot of a line element are connected here by a straight cut line 415109 2614 101 e 09 09 Laser functions JointCut As with function Close amp Cut ine laser cuts your sample along defined lines Start and end dot of a line element are connected by a straight cut line It remains a joint between start and end dot With this function geometrical figures can be cut The cut area remains fixed within the tissue The entir
86. 13 2 Setting power and focus for the trapping laser There are several possible ways of setting the power and focus for the trapping laser with the Laser Tools on the right side of the program window in default configuration using menu commands in the Laser menu using keyboard shortcuts There is another option for setting the focus with the scroll wheel on the mouse If you have installed a DuoFlex TwinFlex system you can set a different power value for each beam You can also set the focus differently for each beam or you can couple the focus settings togeth er If you change the focus for one trapping laser beam the focus of the other beam will automati cally be adjusted to the same value Note The optimal settings for power and focus de pend on the objective in use on the micro scope The settings that you enter always apply to the magnification that you have set in the program As soon as you have selected another magnification the values that were last used or the factory defaults are automatically applied This also applies for coupled focus settings Therefore prior to setting power and focus first select the magnification that you want to work with 146 To couple and uncouple the focus settings for both trapping laser beams DuoFlex TwinFlex system only e Click on the Tweezers tab in the Laser Tools e Click on the Simultaneous Focus checkbox to enable or disable the combina
87. ALM RoboSoftware and restart it if you wish to change to a different mode or database If necessary you must change the launch set tings as described above If more than one user is working on the sys tem and using various different databases or modes you are advised to choose the start up setting Database Mode with selectable DB e Close this window by clicking on OK 26 3 2 Basic and Advanced display modes If you have the Pro version you can switch be tween the Basic and Advanced display modes You can access all functions of the PALM RoboSoftware with the Advanced setting The Basic setting provides a simplified user interface Adjustments PALMA obo e In the Adjustment menu click on the menu item PALMRobo The Preferences and Configuration window opens e Select the Appearance tab Appearance Mode Basic Advanced e Select the required display mode 415109 2614 101 e 09 09 Setting slideholder 3 3 Setting slideholder With PALM RoboStage II you can use various in serts up to three slides 35 mm or 50 mm culture dishes or capillaries Several windows in PALM RoboSoftware contain tabs which depict the insert or which provide a separate tab for each slide refer to chapter 6 PALM Navigator or chapter 8 Working with the Element List Window The software must be notified about which insert you are using To set the type of slideholder that is
88. As soon as you have reached the start or finish dot a small square with thick lines in the color of the element appears here Both elements are joined and are assigned a num ber as a new element Info If you have added a comment to each of the two elements you want to join the comment of the element with the higher sequential number is deleted by joining the comment on the ele ment with the lower sequential number is as signed to the new element If the elements you have joined were in two different colors the new element will be dis played in the color of the element that had the lower sequential number 81 Processing elements To change the shape of elements lines rect angles ellipses Note Select the laser function Cut before changing the shapes of the elements refer to chapter 11 3 for instructions on how to pro ceed Otherwise you will not obtain the de sired result Ctrl key Freehand Tool fw Y e Select the Freehand Tool e Press and hold the Ctrl key e Move the cursor to the area you want to edit The nearest anchor point of the element is indicat ed by a rectangle with black lines e Keeping the mouse button depressed draw the changed line As soon as you have reached the element again the nearest anchor point is displayed as a rectan gle with black lines and the color of the element e First release the mouse button then the Ctrl key 82 The new drawn line is joine
89. CICK LPC i ca sura ge da ELA a a i ew 123 Three dimensionalCUttild a tada DS ae IL we Se add A a DA 123 Non automatic laser fUNCLON ss RD di A A E a 123 NA A LV Re 123 Selection of automatic laser functions LL 124 Speci yng laser SeLtINOS neg o seca sas a a ra ae 124 Seting energy and FOCUS asi sea da e EAS a DAA A AS e n dr 125 415109 2614 101 e 09 09 5 Contents 11 4 2 11 4 3 11 4 4 11 4 5 11 4 6 11 5 11 6 11 7 11 8 11 9 11 10 11 11 12 12 1 12 2 12 2 1 12 2 2 12 2 3 12 2 4 12 2 5 12 2 6 12 3 12 4 12 4 1 12 4 2 12 4 3 12 4 4 13 13 1 13 2 139 13 4 155 13 6 13 7 13 8 13 9 13 10 13 11 13 11 1 13 11 2 13 11 3 13 11 4 13 12 14 14 1 14 2 14 3 14 4 14 5 14 6 Setting number of passes and three dimensional cutting LL 127 Activating and deactivating autofocus LL 127 Other general laser settingS sp nes Ega US pa AA A A eB 128 Special laser settings for the LineAutoLPC AutoLPC and Close amp Cut AutoLPC functions 128 Special laser settings for the RoboLPC function s sa soaa a e a 130 Finding s itable laser settings a orea ds e n a eae a a a 130 Selecting elements for the next laser action 132 Starting the automatic laser functions LL 133 Indications of the operating state of the laser nanao ea a es 134 SODI CONCIME E E SE CO e n 134 Working with the laser function Oneclick LPC 0 o ooo oooooose su 135 Cutting and catapulting without automatic laser fun
90. De MitoTracker Orange m ce Name MitoTracker Orange E Extended parameters Exposure Era Mode Measure Auto Fixed Camera Hardware Settings During acquisition User ReflectorPos3 E After acquisition User BrightField bl e Current Channel Actions Pe Channel pool 3 Duplicate e Make your settings refer to AxioVision online help 178 Selecting an experiment c Select multi channel experiment e Click on the Select multi channel experiment option from the Multi channel fluorescence menu A window with a list of the existing experiments opens Load Experiment Location Name Channels ZStack Timelapse Positions 8 User 2_channels 2 Channeli 2 User 3_channels 3 Channeli amp User z neu 1 Channeli 3 3 000000 8 User z_stack_9 ebe 1 Channeli 5 6 000000 e Select the experiment you want Info With multi channel fluorescence recordings you can also use the Extended focus function see chapter 16 2 To generate a multi channel fluorescence recording a Multi Channel e o e Click on the Multi Channel icon button The multi channel fluorescence recording is started A Multidimensional Acquisition Preview window opens showing the progress of the recording see chapter 16 4 As soon as the recording sequence is complete a fluorescence image appears on the monitor PALM RoboSoftware aut
91. Display 0 4095 al G 0 45 wE On the tab there is a histogram with a gradation curve and several buttons The settings are de scribed below Live Image window Advanced Settings e Click on the Advanced Settings button in the Display Tools Camera tab Live Image EE Camera selection Measurement toole Display AxioCam4R3 0 4095 54 2 bu E ge This opens the Live Image window 35 Set image parameters e Click on the Display tab On the tab there is a histogram with a gradation curve and several sliders and buttons To change how the histogram is displayed e Click on the Log button to switch between logarithmic and linear display of the histogram The Skip function is particularly useful if there are large very bright and or very dark areas in your microscope image In such cases the histo gram is primarily defined by these pixels In the remaining area of the histogram it is very difficult to detect differences Using Skip these extreme values in the histogram are hidden and the histo gram is re scaled e Click on the Skip button to hide the brightest and darkest pixels in the histogram To set brightness contrast and gamma using the histogram 1 2 3 4 5 e Click on one of the black squares on the histo gram and drag it up down to the left or to the right with the mouse button pressed Moving a square has the following effects Square in the middle
92. Down keys to raise or lower the trapping laser power e Press Home or End to increase or reduce the value for the trapping laser focus The values that are currently set are displayed in the Laser Tools 148 13 3 Finding suitable power and focus settings for the trapping laser The settings that you require for various samples will vary to some degree In order to determined the optimum power and focus settings you must experiment on similar samples with different set tings The factory defaults may serve as suitable starting points for your experiments If in doubt start at very low power and adjust upwards grad ually until you obtain the desired effects The focus can be set using a slide for example with an area you have colored with a black felt pen With sufficient power a hole appears in the paint film which should make the effect quite sim ple to assess The more precisely the focus is set the smaller the hole should be 415109 2614 101 e 09 09 Defining a reference position 13 4 Defining a reference position If you have moved the trapping laser beam to any position during your work you can instruct the system to return it to the Reference Position This function is useful if for example you have moved the beam into an area outside the image section displayed on the monitor and you are perhaps no longer certain of the exact location of the beam You can change the Reference Position at an
93. FARA 7 2 2 LaS 73 14 Da Dez TED 7 3 4 13 5 7 3 6 7 3 7 7 3 8 7 359 7 3 10 7 3 11 7 312 7 4 T9 7 6 RESetcallDratiO y do o a e a a A he eo aa o a E 43 Adjusting the Objective Offset LL 43 Resetting the Objective Offset 00 0 o oooooononcnaaa ee es 44 POSITIONING the Stage ici A AAA A AR ee 45 Movement YDES ic teri ds aaa 45 Positioning the stage with the mouse Stage Mode 00 ee ee ee es 45 Positioning the stage with the mouse Scroll 2 a ee ee ees 46 Positioning the stage with the scroll bars anaua oaoa 46 Positioning the stage with the keyboard aaaea a 46 Positioning the stage with the joystick aosa a 46 Positioning the stage with the Centering TOO 47 Moving the stage during an automatic cutting laser function LL 47 Further possibilities for positioning the stage ooo o 47 Stopping the stage in an emergency LL 48 SOtund SPECO aid lA EC RA AT AAA eae AS 48 The Reference Position of the Stage a 50 Capone area ada aaa nai 51 Laa POSITION e aa a a A A Rea de 52 Centeri a LNe stade sa AA A eth ea 52 PALM Navigator saia ra A ERA See 53 Starting PALM Navigator as RD E o A era A A a a 54 Making the settings fora scan 0 cee eee ee ee ee ee es 54 ScannindraSampl SS ake a ae as See ari Ge ais 57 Stopping and canceling or continuing a scan in progress aasa oaa a a 59 Scanning a section from the overview scan saasaa oaa a a 60 Saving and relo
94. How to set a focus correction during the scan As soon as you execute the scan command the sample will be scanned section by section For this the focus setting will be applied which was valid before the command was sent You can choose whether and how the focus setting should be corrected for each scanned section automati cally only if you have a motorized microscope with automatic focusing manually or not at all You can set the focus correction type on the Navigation and Scan tabs Focuscorection None Suspend scan for focus O Use Auto Focus e Select None if you do not want any focus cor rection during the scan Select Suspend scan for focus if you want to manually readjust the focus for each section scanned Select Use Auto Focus if you have a motorized microscope with automatic focusing that you want to use If you have selected Suspend scan for focus or Use Auto Focus you can specify after how many tiles each correction is to be carried out Focuscorection None Suspend scan for focus Use Auto Focus every tiles e Select the Every tile checkbox and enter a value for the number of tiles If the checkbox is not selected correction will be carried out before scanning each tile Info If you readjust the focus for each section to be scanned you will get a far sharper overall re sult However the duration of the scanning process will increase consider
95. Laser function Laser gt Start Laser function Capture Device Devices gt Capture Device Edit gt Select All DeSelect All Current position only Edit gt Copy Motion gt Speed faster Keyboard Shortcuts Ctrl K Ctrl N Ctrl S Ctrl V Ctrl Y Alt P Alt Enter Alt F Alt X Backspace Alt Back space Del End ESC Home Page up Page Down B Icon Button Menu Command Motion gt Goto CapCheck Return New Elements File gt New Elements Motion gt Speed slower Edit gt Paste Es Edit gt Delete all unproc essed elements Toggle switch between standard pointer and reference pointer Er Change Edit gt Change se Freeze Mode Motion gt Freeze Mode View gt Hide All Bars View gt Show All Bars Edit gt Delete last element Delete last element Edit gt Undelete x Delete selected elements Edit gt Delete selected elements Laser gt Focus down Stop Motion gt Stop Laser gt Focus up Laser gt Energy Power more Laser gt Energy Power less Page backwards through multi channel fluorescence images Page forwards through multi channel fluorescence images 415109 2614 101 e 09 09 Index A Alarm timer 31 ApoTome status display 171 Autofocus activate 127 automatic focusing 167 deactivate 127 for element with laser function 29 AxioVision 13 64 Brightness value microscope image 37 C Calibration movement of
96. PC or CenterRoboLPC and trigger the laser with varying settings for the LPC Energy and LPC Focus Disable the combination of Cut and LPC Energy as well as Cut and LPC Focus in order to be able to perform independent settings for these functions Note The optimal settings for energy and focus also depend on the objective in use on the micro scope The settings that you enter always apply to the magnification that you have set in the program As soon as you have selected another magnification the values that were last used or the factory defaults are automatically applied Therefore prior to setting laser energy and la ser focus first select the magnification that you want to work with To find the optimal laser settings for the cutting functions Info To ascertain your optimal laser settings you have to know how you can set the laser focus the laser energy and the speed of the stage for the different types of movement If necessary refer to chapter 11 4 Specifying laser settings and chapter 5 Positioning the stage or the User Manual for instructions on how to pro ceed e Insert a sample that has the same qualities as the sample that you want to examine select the required objective and adjust it into focus e Select the magnification set on the microscope in the Lens menu of PALM RoboSoftware 415109 2614 101 e 09 09 e Switch on the display for Laser Tools and Speed Tools if these have been hi
97. TdS r SIE2MIJOSOAOY W1Vd SUOI PN4y3 SUI bunelisdo September 2009 This document is only supplied to those persons who are trained and authorized by Carl Zeiss MicroImaging GmbH No part of this document may be reproduced or distributed in any form or by any means without the prior written consent of Carl Zeiss Microlmaging GmbH 11th edition September 2009 Regulatory Notice PALM MicroLaser Systems are intended for research use only and are not approved for medical applications in the United States and Canada LMPC is a trademark of Carl Zeiss MicroImaging GmbH PALM is a trademark of Carl Zeiss MicroImaging GmbH The following technologies have been patented by Carl Zeiss MicroImaging GmbH Laser catapult technology Laser Pressure Catapulting LPC Patents US 5 998 129 EP 879408 B1 and others Three dimensional laser beam positioning system Patents US 5 689 109 EP 679325 B1 and others Element List Patent US 6 930 764 Additional patents pending Carl Zeiss MicroImaging GmbH 07740 Jena Germany BioSciences Location Munich Phone 49 89 90 9000 800 Fax 49 89 90 9000 320 E mail palm info zeiss de www zeiss de microdissection Carl Zeiss Microlmaging GmbH 2009 All rights reserved 2 415109 2614 101 e 09 09 Contents Contents Chapter Page 1 TALFOGUICHON NS UE A A a ad 9 1 1 Functionality of PALM MicroLaser systems anaana aaa a a a 9 1 52 PALM RODOSORWa E a a RD AS AL A A Ged Riches BOW Go
98. Trap 2 e Click on Trap 1 or Trap 2 in the Laser Tools depending on which laser beam you want to move with the mouse Info The cursor is not displayed in TrapXY 1 TrapXY 2 Mode In TrapXY 1 TrapXY 2 Mode you can alter the speed of the Laser Beam Positioning and the power of the laser beam using keyboard commands and also change the focus with the scroll wheel on the mouse Note that you can only change the set tings for the trapping laser beam that is select ed in Laser Tools To switch between TrapXY 1 and TrapXY 2 Mode during a movement 1 e Traper 1 mode i Traper 2 mode e Open the context menu by right clicking the mouse e Click on the TrapXY mode to which you want to change To switch from TrapXY Mode back to Cursor Mode e Click once with the left mouse button 415109 2614 101 e 09 09 Moving objects along defined paths 13 11 3 Moving the trapping laser beam with the keyboard Arrow keps Joystick mode o de 7 e O i e Inthe Arrow keys Joystick mode area at the right edge of the program window click on the button for the laser beam you want to adjust using the arrow keys If you select the checkboxes for both beams you can control both beams simultaneously and in parallel Info If the stage is at the CapCheck refer to chapter 5 5 the buttons are not available e Pressoneof the four arrow keys The laser beam will continue to move until you release t
99. Using this mode one element is catapulted into the first well 1 X elements into the second well 1 2X elements into the third well etc The num ber X can be selected as required Distribution Calculator Operational Configuration Distribution calculation Operating Mode sequence 1 14 1 2 Number of selected well s Number of used well s sg 2 Number of elements to process Start with Field 14 v Number of elements per well v Remaining elements until Field e Enter a value for X in the X field e Selectthe co ordinates of the first and last well into which the elements are to be catapulted on the Start with Field and until Field drop down lists Info The order of the wells is defined by the setting in the Subtype Configuration field in the RoboMover window line by line or column by column refer to chapter 12 2 2 Info AII wells are filled one after the other as per the mathematical series If there are no long er enough elements available for a well the laser operation is interrupted Remaining ele ments are not catapulted 97 Defining wells for the next laser operation Operating Mode area per field With this mode you define the number of wells into which a defined area is catapulted Distribution Calculator Operational Configuration Distribution calculation Operating Mode area per field Number of selected well s Der Number of u
100. a ee UE 10 1 3 Basie anda Pro Mode re e ele e a Re RO a AA A ae 10 1 4 INtoOrmatonhCenter Za oa a aaa ate Go AA a ad 10 las ABOUT THIS User Manta sia RR LIRE A Ee hE eee 6 11 1 5 1 CONCENUS ss sadios A a A ADE E AA ee eS RA iena 11 1 5 2 CONVENLONS 4 mm aee a RL tabelle el aa 11 1 6 INstallationNizs lt sida nei ar ad Aa IRE 11 1 7 File Mode and Database Mode 0 ooo rr 11 1 8 Launching and exiting the program LL 12 1 9 Launching other programs from PALM ROboSoftware lt lt oo ee eee eee o 13 1 9 1 IRrormationCent hz a Sara Ace ee AS ew ees a Gov BG ws aa dE Se ae quase be raid 13 1 9 2 AXIOVISION and CelenGer ag usa nei ae ee RS we eG aie ee a a 13 2 Program layout and operation 00 000 eee eee 15 251 PALM RODOSOTt Ware layout sos hag ros AA A A ee 15 22 Customizing the user interface nasasa a a rr 17 2 3 Operation nada alal pai area E A es ee ee Some ag 20 2 351 MOUSO i uil ie al Aa E q a ae a ee a ee ee ee ee en gees ee 20 2 3 2 COMTEX Mens ds oud ae Sete la ea asar a AM eS a 468 bane bole E ees 20 2 3 3 SOFWate MOES raora e ae ae E A A a A A A 20 2 4 Help and information about PALM RoboSoftware saasaa asa aaa a a e 21 2 4 1 OnE RED es i aeaa di dl a ea he BE cae ee me RR Sh wee 21 2 4 2 ThePALMRODO Instructor sm so apo a ad a ARA OI na 21 2 4 3 Program license and manufacturer 2 ee 22 2 4 4 SYSULERMINTOMIMALION ya oS ae ata ok Boe RA bee GG Ae Se She ae e
101. abase Mode No settings need to be made in order to save im ages in Database Mode All images are stored in the database The file name is generated by PALM RoboSoftware manual followed by the next number in sequence The sequential number is in cremented by one every time you save an image To save your image Info Please note that the image is stored with the elements unless the option to display them has been switched off The elements are stored in the same way as they are displayed on the screen i e perhaps with their area content specified with or without the element number and if relevant with changed image parame ters refer to chapter 3 11 The Copyright Note and scale bar are only in cluded with the saved image if they are dis played on the screen However other features such as Laser Markers crosshairs etc are not saved File A Save Image e Click on the menu item Save image in the File menu 116 10 4 Saving images automatically during a laser function When saving images automatically immediately before and or after a cutting laser operation the current microscope image will be saved automati cally If you are working in File Mode refer to chapter 1 7 the images are saved in the specified folder refer to chapter 10 2 If you are working in Database Mode the images will be stored in the database Adjustments PALMA obo e In the Adjustment menu click on th
102. ably 56 How to specify the type of scan You can choose between the following scan types Live image Scan of the normal microscope image as is displayed on the monitor Multi channel Scan of a multichannel fluo rescence recording Extended Focus Scan of a recording with an extended depth of field for thick samples Info The Multi channel and Extended Focus op tions are only available if your system has an appropriate license Please contact Carl Zeiss MicroImaging GmbH if you wish to upgrade your system Note To use the Multi channel and Extended Focus scan types you must have made the re lated hardware settings as well as defined and selected an experiment see chapter 16 You can set the scan type on the Navigation and Scan tabs Scan all AOls ka Navigation tab Scan tab Extended Focus e Open the drop down list next to the Scan all ROIs button on the Navigation tab or next to the Scan button on the Scan tab and select the type of scan you want to perform Info For multi channel fluorescence recordings only the superimposed image made up from the separate channels is displayed in the Navigator However the information for the individual channels is stored in the saved tile images They can be used for script analyses in Stored Tile mode or viewed in the picture viewer see chapter 16 5 Note that especially scans of the Extended Focus type
103. ading the scanned image saaa a e 61 Displaying elements in the scan agua rai a al ei 62 Displaying a particular tile on the monitor LL 63 Positioning the microscope stage biliari die ai 63 Analyzing images using the AxioVision SO TWare o ooo a 64 Analyzing images using the Definiens software o oo ooo 65 Drawing and measuring sia rai a A A A A AA AA A a 67 General Procedi Ori ta Lad pa e A a AR e Been 68 Creating a new set of elements iL 68 VV Ol UG FIE MOC as e earn ae en 68 WOFKING In Database MOUE sissi ian AA i 69 Drawing Mew elements daa ilaria ah Boe a Sige AAA ei 71 Selectina a laser TUNCUON tada a a da RARA de 72 Derining xWel forcataPUltiida spa daa a da eke nh 72 Drawing in the various program modes ooooooooooe es 72 The Reference PONE TOO cis cdi Dai E Leni 73 re Freena a TOOL 5 sm e pi E Sp ida Se a Asa 73 As A A 73 TherRetctanale TOOL at AR iero ee AA 73 TNS Grid Rectangle TOOL teta a da da EA A E 74 WMeseircie To0l so Seco dels 4 Boa eae a id dea da i Aa 74 FAEDO TOON aerea a A Ren Pa 75 Testa TOO sei o AS Ss a e A A 75 ie exc TOO daa dad ea aaa 75 Displaying and hiding elements 0 a ee ee 76 Centering elements on the monitor aasa a a 76 Selecting and deselecting elementS o oo ee aaa 77 415109 2614 101 e 09 09 7 6 1 7 6 2 7 6 3 Lo 7 8 7 8 1 7 8 2 7 8 3 7 8 4 7 8 5 7 9 7 94 7 9 2 7 9 3 7 1
104. alibration e Click on New Calibration in the Adjust ments gt Calibration menu A submenu will open e Click on the menu item Trap 1 e Click on the actual position of the laser beam with the mouse The program will switch automatically to Trap 1 Mode e Use the mouse to move the laser beam to the crosshairs at the image center and click the left mouse button The cursor will reappear the program automati cally switches out of Trap 1 Mode e If you purchased a system with two movable laser beams calibrate the second laser beam in a similar manner to the first laser beam Note The calibration only applies to the camera se lected and for the magnification selected before the calibration If you intend to work with var ious magnifications you must recalibrate for each change of magnification 415109 2614 101 e 09 09 4 6 Reset calibration You can reset the calibration to the factory default setting at any time Adjustments Calibration k Factory Calibration e Click on Factory Calibration in the Adjustments gt Calibration menu 415109 2614 101 e 09 09 Reset calibration 4 7 Adjusting the Objective Offset If you have drawn elements with one magnifica tion and then select another magnification a dif ferent objective the microscope image on the monitor may appear to be shifted slightly al though the stage has not been moved Your ele ments are no longer in the correct posit
105. ams 415109 2614 101 e 09 09 4 4 Calibrating the movement of the stage Calibration is done in several steps PALM RoboSoftware will guide you through this process e Insert a sample and focus the image e Set the chosen magnification for the micro scope as described in chapter 4 2 e In Stage Mode choose a low or a medium speed setting for the stage refer to chapter 5 3 Note Do not use the Invert Stage Motion function when calibrating refer to chapter 5 1 1 Adjustments Calibration k New Calibration e Click on New Calibration in the Adjustments gt Calibration menu A submenu will open e Click on the menu item Stage e Look fora dot preferably as small but as sig nificant as possible in your image Note The farther the dot is from the image center both vertically and horizontally the more accurate your calibration will be e Note this dot and click on it with the cursor tip The cursor will disappear and the program will switch automatically to Stage Mode e Use the mouse to move your selected point to the crosshairs at the image center and click the left mouse button The cursor will reappear the program automati cally switches out of Stage Mode The following message will appear Calibration is finished Check position of Laser Marker This message will disappear automatically after a few seconds e Check the position of the Laser Marker as described in chapter 4 3
106. an button The scan in progress is interrupted How to cancel a scan The stopped scan is canceled if you start a new scan Clicking on the Scan button which appears again after stopping discards the previous scan ning result and the scanning progress starts over 415109 2614 101 e 09 09 topping and canceling or continuing a scan in progress C Continue at actual position Objective Fluar 10 0 50 M27 Information Slide Section O 110 tiles Scanned with 10x objective Thumbnail Navigation Mode Live Stored tiles Focuscorrection O None O Suspend scan for focus Use Auto Focus Y every 5 tiles Draw C Elements C Marker elements How to continue a stopped scan Continue at actual position e Select the Continue at actual position check box The Continue button appears instead of Scan You can then continue the scan from the point at which you interrupted it However you can also click on any of the tiles already scanned The scan will then be continued from the selected tile The existing images of this and the subsequent tiles al ready scanned will be discarded For example this can be useful if you notice that some tiles have not been scanned sharply e If necessary click on the first tile to be rescanned The corresponding section of your sample is dis played in the PALM RoboSoftware main window e Correct the focus setting e Click on th
107. and click on the OK button Info It is not essential to allocate a name to the group As you will no doubt later draw further elements and create further groups you will thus be easily able to retain an overview 102 amp Element List File Edit Motion Laser Collection Devi E gt fa E BERRA RoboLPC v Fiuar 5x 0 25 AR X Slidel Siide2 Slide3 Show Types Ta Color Nr Name Laser function Objective Well Area un Grp cut shot Co ER ER SE E E ES E Freehand AutoLPC 5x Fluar 5x 0 25 388527 Slide 1 E E Freehand AutoLPC 5x Fluar 5x 0 25 308903 Slide 1 4 Slide 1 Group 5x Fluar 5x 0 25 The next two steps are omitted if you have drawn elements of the Reference Point type In this case continue with Step 3 Drawing and Process ing Elements Edit Define Group Reference Figure e Select the three drawn elements and click on the menu item Define Group Reference Figure in the Edit menu Select a Group From List Mame Mr Grp Comment Slide l 4 This opens the Groups window e Choose the group to which the elements belong and click on the OK button to close the win dow PALM RoboSoftware creates three new elements of the Reference type 415109 2614 101 e 09 09 amp Element List File Edit Motion Laser Collection Device 1 PERU ARA e IE Slide1 Slide Slide3 Summary Show Types all gt Color Nr Name Type Laser function Objective Well Ar
108. ar in the Name column If you have installed a Ludl fil ter wheel this column is empty To define a name for the fluorescence excita tion filter used You can specify names for your installed fluores cence excitation filters e g color names If you subsequently select an excitation filter the names you specified will appear in the selection list e Inthe table double click in the row of the filter for which you want to change the color informa tion contained in the Color column e Enter the desired name for the filter e Repeat for all filters which you want to rename To select a fluorescence excitation filter e Open the drop down list Actual Position and select the desired position In the drop down list all possible filter settings are listed If you have entered names for the fil ters as described above the names will be listed otherwise they will be numbered from 1 to 6 The filter wheel will move automatically to the selected filter position 415109 2614 101 e 09 09 Editing reflector settings 14 8 Editing reflector settings Fluorescence adjustments Fluorescence Fluorescence Filter Reflector Fluorescence Shutter Reflector Colors and Names Mo Color Mame Lazer E posure 100 Mirror TES 54 00 DAPI TES 20 00 FITC TES 20 00 Rhodamine TES 20 00 Texas Red TES 0 00 none TES 0 00 Double Click Color or Laser columns to change Adjustments Fluorescence e Inthe Adjustm
109. at you no longer need for your work A slide that has been colored with a black felt pen is particularly suitable e Set the magnification selected in the micro scope in Microscope Tools as described in chapter 4 2 Note Note that you can only use the trapping laser in conjunction with oil objectives 60x 63x and 100x e If itis hidden enable the screen center display click on item Screen Center in the View menu e Forthe Laser Beam Positioning choose a low to medium speed setting TrapXY refer to chapter 13 11 11 e Choose low power and focus settings for laser beam Trap 1 as described in chapter 13 2 42 e Activate the trapping laser click on the On button on the right border of the monitor e Adjust the power and focus so that you can see an effect of the laser beam if you are using a slide colored with a black felt pen a hole in the colored coating will be visible at the appropriate settings e Switch to Trap 1 Mode click on the item Trap 1 Mode in the Motion menu The cursor will disappear every movement of the mouse will be reflected in a movement of the laser beam e Move the laser beam by dragging the mouse into a corner of the image in your image win dow Note The farther the dot is from the image center both vertically and horizontally the more accurate your calibration will be e Double click there to exit Trap 1 Mode again Adjustments Calibration k New C
110. ate of the laser is indicated on the right side of PALM RoboSoftware main window The cutting laser is deactivated C ut Activate el The cutting laser is activated C ut gt The cutting laser is activated and has been started CI ut Deactivate e Other indications The laser interlock has been tripped rey please check microscope support The laser is not ready for use this indica tion appears during the laser warm up phase or if the interlock is tripped 134 11 9 Stop continue In case of emergency you can stop all functions and movements in progress To stop all current functions The quickest way to stop all functions is to use the following commands Stop e Click on the Stop icon button in the Tool Bar in PALM RoboSoftware main window or in the Element List window see chapter 8 Or e Press Esc on the keyboard you can only use these commands if PALM RoboSoftware main window is the active window In addition you can also stop the movements us ing a menu command Motion d Stop ESC e Click on the menu item Stop in the Motion menu in PALM RoboSoftware main window or in the Element List window All current operations are stopped immediately If you want to continue an interrupted operation you must re launch the operation 415109 2614 101 e 09 09 11 10 Working with the laser function Oneqick LPC With this laser f
111. ated at defined time intervals This chapter briefly describes how to prepare and use the functions You will find more detailed infor mation on this topic in the AxioVision online help 415109 2614 101 e 09 09 16 1 Multi channel fluorescence recordings Before you can generate a multi channel fluores cence recording you must make the following preparations Adda corresponding hardware configuration Make settings for the experiment Select an experiment To prepare a multi channel fluorescence recording Setting up the hardware configuration c Create hardware settings e Open the Multi channel fluorescence menu in the toolbar e Click on Create hardware settings The Setting editor window opens where you can make settings for your hardware E Setting Editor are settings Duplicate Sh Execute Ir a e Make your settings see chapter 15 5 e Click on the Save button to save the configu ration you have created 177 Multi channel fluorescence recordings Making settings for an experiment E Create modify multi channel esperimenta e Click on the Create modify multi channel experiments option from the Multi channel fluorescence menu A window opens where you can make settings for a new experiment or change settings for an existing experiment Multidimensional Acquisition C Je Experiment Ed E ZII HA Channel Definition
112. ater As soon as you start the laser all elements or only the elements you have se lected are processed using the laser During this process the PALM RoboSoftware automatically ac tivates the laser function you have selected for the related element Previously drawn elements can be changed by their shape and size they can be numbered anno tated and deleted again You can calculate their area or length and you can select colors and line thicknesses in which your elements are to be dis played Note The measuring functions of PALM RoboSoftware and the scale bar are not cali brated at the factory they are an estimated value only Carl Zeiss Microlmaging GmbH does not guarantee the accuracy of the per formed measurements The measuring functions have to be checked and if necessary calibrated by the user for example with a ocular micrometer You can save your elements as a set or load a pre viously saved set of elements In addition you can save the properties of your elements in a file re load them in another program and process them further For instructions on how to do this refer to chapter 9 If your system includes database functionality and if you are working in Database Mode then your el ements will be saved automatically in a database and can be reloaded from there refer to chapter 1 8 and the InformationCenter program manual 415109 2614 101 e 09 09 In addition you can save the section disp
113. ay them has been switched off The elements are stored in the same way as they are displayed on the screen i e perhaps with their area content specified with or without the element number and if relevant with changed image parame ters refer to chapter 3 11 The Copyright Note and scale bar are only in cluded with the saved image if they are dis played on the screen However other features such as Laser Markers crosshairs etc are not saved File Di Save mage e Click on the menu item Save Image inthe File menu The Save Image As window opens the pre defined folder and the predefined file name with the additional sequential number 01 02 03 are preselected and offered e Selecta different folder if you do not want to save your elements in this folder e Enter a name for your image file if you do not want to use the preset name Note The name you enter is taken as a default set ting for all subsequent image saving opera tions and thus overwrites the original default setting e Choose the format in which you want to save your image Info You can choose between the formats bmp tiff and jpg The recommended formats are tiff of jpg since these formats allow addi tional information to be saved along with the image stage position microscope settings e Click on Save 115 Saving images automatically during a laser function 10 3 2 Saving images in Dat
114. be displayed as slightly offset In this case you will have to redefine the Objective Offset see chapter 4 7 415109 2614 101 e 09 09 To set the Reference Position of the stage Normally it is not necessary for you to reference the stage If however a disruption should occur e g you may have moved the stage unintention ally by hand you can reset the Reference Position of the stage Motion Initialize Unit e Click on the menu item Initialize Unit in the Motion menu e Click on the menu item Stage on the sub menu Note Note that the Reference Position for the stage has no relationship to the definition of a Reference Position for the movable trapping laser beam refer to chapter 13 4 415109 2614 101 e 09 09 CapCheck 5 5 CapCheck When you catapult samples into a collection device using the automatic laser functions you can then observe the contents of the collection device under the microscope For this purpose PALM RoboStage has a notch through which the microscope objec tive can be moved upwards The corresponding position of the stage is referred to as the CapCheck You can automatically target the CapCheck with a menu command Caution Set the objective to the lowest possible posi tion before you move the stage to or from the CapCheck If the objective is set too high the stage will collide with it This can cause serious damage to the objective and to your sample if you have
115. bed in the previous section To delete a set of settings e Click on the row in the table containing the set tings record that you want to delete e Click on Delete To change the order of the entries in the list 4 e Click on the row in the table that contains the record you want to move up or down e Click on the Up or Down button depending on the direction in which you want to move the entry The entry will be moved all entries will be renum bered 163 Adjusting the reflector offset 14 10 Adjusting the reflector offset If you have set or changed a reflector for fluores cence the microscope image on the monitor may appear to be shifted slightly although the stage has not been moved If you now draw elements and then change the reflector again your ele ments will not be positioned correctly The auto matic laser functions will then not process exactly the areas you wish You must compensate for this by setting the Reflector Offset With that you move all existing el ements Note Please note that on adjusting the Reflector Offset all existing elements are really moved not only the elements drawn with fluorescence illumination If you wish to draw elements un der normal as well as fluorescent lighting you should first check whether the Reflector Offset is set correctly It may be possible that you do not see the struc tures of the elements with normal illumination In this case you
116. being used Adjustments PALMA obo e Inthe Adjustment menu click on the menu item PALMRobo The Preferences and Configuration window opens The Configuration tab will be displayed Configuration Stage Tye Stage Holder SlideHolder 3x me e In the Stage section choose the slideholder you are using Info You can also set the slide using the menu command Motion gt Goto Loadposition refer to chapter 5 6 415109 2614 101 e 09 09 3 4 Settings for the joystick and the arrow keys If you have connected a joystick you can control the stage the PALM RoboMover or the trapping la ser beams using the joystick For this purpose the joystick must be activated refer to the end of this chapter You can also control the units mentioned using the arrow keys on the keyboard You can set which unit you control using the two buttons on the joystick or using checkboxes in the main window of the PALM RoboSoftware The set tings also apply to the arrow keys Setting on the joystick Using the bottom left button you can activate and deactivate the control of the trapping laser beam 1 Using the top right button you can activate and deactivate the control of the trapping laser beam 2 If the control of both trapping laser beams is activated you will move the two beams simul taneously and in parallel You can move the stage using the joystick when the control of both trapping laser
117. c Coordinates Micrometer Speed Logic Micrometer sec e Inthe Metric section click on the radio button Logic units percent or Micrometer sec units um sec 415109 2614 101 e 09 09 To change the speed of the Speed Tools tool e In Speed Tools select the movement type for which you want to change the speed speed Speed Urn Sec 2200 amo The Speed Tool for the selected movement type is shown in the example shown above Scroll The maximum selectable speed is dependent upon the chosen movement type e Enter the desired value To change the speed with menu commands or with keyboard shortcuts Motion Speed slower Chrl S Speed faster Ctrl F e Ifyou want to reduce the speed press key combination Ctrl S or click on the menu item Speed Slower in the Motion menu e Ifyouwantto increase the speed press key combination Ctrl F or click on the menu item Speed Faster in the Motion menu 49 Info Changes with keyboard or menu commands always refer to the type of movement that is currently selected in the Speed Tools tool Therefore activate the tool display before car rying out any speed changes with keyboard shortcuts or menu commands Otherwise you will not be able to see which movement type the changed speed setting will affect Info Using the keyboard commands you can change the speed in the following cases even during a m
118. can new collector type initiates a new scan of the collector Once the collector has been detected all the necessary data are read from the configuration file Click on Use same collector type if you want to continue to use the same collector type a new scan is not performed Use this selection if e g you have only replaced the insert Info In both cases all information on the wells for all elements is deleted Click on Cancel if you want to continue to use all the configuration data already known to the system including all information on the wells already used e Clickon the button with the required function 138 e Wait until PALM RoboSoftware has detected the collector type An illustration of the collector fitted appears in the RoboMover window The name of the collector used is displayed in the field at the top right The co ordinates of the wells are defined by rows A H and columns 1 12 Info On the use of microtiter plates the letters for labeling the rows A H are on the right si de the numbers for labeling the columns 1 12 run from right to left refer also to pro duct information on microtiter plates 3 RoboMover Subtype Configuration optional Column oriented Information Current field 000000000000 415109 2614 101 e 09 09 Working with PALM RoboMover or PALM CapMover II PALM CapMover II Change collector Now you can change
119. cells of interest 415109 2614 101 e 09 09 Working with the laser function Oneclick LPC 11 11 Cutting and catapulting without automatic laser functions Even without automatic laser functions cutting and catapulting are possible This is done by switching the laser on and off using the footswitch However to do so you must switch to Stage Mode and move the stage using the mouse refer to chapter 5 1 1 You can choose whether you wish to start the cut ting or catapulting function when you operate the footswitch To set the function of the footswitch Info You must perform this setting before switching to Stage Mode Laser III MicroBeam Tweezers Cut LPC Energy Energy 100 100 1 1 4 4 Focus Focus 100 100 1 1 4 oF 4 Foot switch e If you have a CombiSystem with an trapping laser click in the Laser Tools the MicroBeam tab e Click on the left radio button for Foot switch at Cut if you want to cut with the laser in the Stage Mode or click on the radio button on the right below LPC if you want to perform catapulting with the laser in Stage Mode 135 136 415109 2614 101 e 09 09 Opening the RoboMover or CapMover II window 12 PALM RoboMover and PALM CapMover II Using the laser functions you can catapult sections of your sample into a collection vessel Using PALM RoboMover and PALM CapMover II collection de vices you can automatically position c
120. ch element e Open the Well drop down list in the default configuration at the bottom left of the main window and select the required well the coor dinates of the well as displayed in the PALM RoboMover window are given You can also make this selection later or change it later refer to chapter 8 6 Info The coordinates given in the drop down list are dependent on the type of collector used Note that PALM RoboMover must be configured for well coordinates to appear in the drop down list refer to chapter 12 2 1 72 7 3 3 Drawing in the various program modes Info This section is concerned with the software modes in PALM RoboSoftware These are total ly unrelated to the Database and File operating modes As is described in chapter 2 3 3 PALM RoboSoftware works in a number of modes You can draw elements in Cursor Mode and in Freeze Mode Cursor Mode is the default mode for PALM RoboSoftware it is set automatically when the program is launched Chapter 14 5 includes in structions for switching into and out of Freeze Mode In both modes you can draw elements inside the Scroll Rectangle without limitations and with all graphic tools you can switch the display of the Scroll Rectangle on and off using menu command View gt Scroll Rectangle Outside the boundary of the Scroll Rectangle you can also draw with all graphic tools in Freeze Mode and with all graphic tools except the Freehand Tool in Cursor Mode
121. ch sample If you have cre ated several groups they will also appear in the tables Make sure you always choose the correct group e If you have inserted the target sample in PALM RoboStage Il in the same position that the source sample occupied before you should select the Delete source group after matching elements into destination group box As in these cases the elements are allocated to the same slide position they would appear double if you didn t check this box e Close this window by clicking on OK 415109 2614 101 e 09 09 amp Element List File Edit Motion Laser Collection Device 90259800 oRB x Slide Slide2 Slide3 Summary Show Types all y Color Nr Name Type Laser function Objective Well Area umf Grp E 11 Freehand Ref 1 AutoLPC 5x Fluar 5x 0 25 402946 Slide 2 IG 12 Freehand Ref 2 AutoLPC 5x Fluar 5x 0 25 265509 Slide 2 E 13 Freehand Ref 3 AutoLPC 5x Fluar 5x 0 25 391517 Slide 2 14 Slide2 Group 5x Fluar 5x 0 25 15 Reference Ref 1 5x Fluar 5x 0 25 Slide 2 16 Reference Ref 2 5x Fluar 5x 0 25 Slide 2 17 Reference Ref 3 5x Fluar 5x 0 25 Slide 2 E 19 t LPE 5x Fluar 5x 0 25 Slide 2 E 20 Freehand AutoLPC 5x Fluar 5x 0 25 297331 Slide 2 E 21 Circh AutoLPC 5x Fluar 5x 0 25 325701 Slide 2 A The elements are copied to the target sample At the same time an adaptation occurs The refer ence elements are moved to the respective posi tio
122. ch that of actual laser focus Only then the correct results can be achieved with the automatic laser func tions You should therefore check the position of the Laser Marker before you start work For the trapping laser also you can only achieve accurate results if the Trapping Laser Marker is exactly aligned with the laser focus Remember that the position of the Laser Mark ers may vary for use with different objectives To set a Laser Marker position correctly you must release the laser and determine its actual focus point Then you must set the associated Laser Marker to the focus point of the laser In order for you to be able to check the position of a Laser Marker and adjust it if necessary the Laser Marker must be displayed refer to chapter 2 2 40 To determine the focus point of the cutting laser e Using any of the methods described in chapter 5 move the stage so that an area of your sample which you do not require for your work appears in the middle of the monitor or insert a membrane coated slide Stage Mode e Switch to Stage Mode click on the icon Stage Mode in the Tool Bar e Release the laser with a single short application of pressure to the footswitch e Check the result You should see a small almost circular area where material has been removed This is most easily visible on the membrane Info In some circumstances you will not see any ef fect from laser activation or there w
123. croLaser Systems AxioVision can be accessed via Carl Zeiss MicroImaging GmbH Cellenger from Definiens can be accessed via Carl Zeiss MicroImaging GmbH 1 9 1 InformationCenter The PALM InformationCenter is a component of PALM MicroLaser Systems Its functionality is de scribed briefly in chapter 1 4 Further details can be found in the Manual of the InformationCenter To launch the InformationCenter program File Open Information Center FS e Click on menu item Open InformationCenter in the File menu of PALM RoboSoftware main window 1 9 2 AxioVision and Cellenger To allow you to enhance your system Carl Zeiss Microlmaging GmbH offers approved image pro cessing programs which for instance enable you to localize specific objects or structures within your microscope image with an automated search func tion these programs are AxioVision and Cel lenger from Definiens Both of these programs can be activated in Navigator refer to chapters 6 10 and 6 11 415109 2614 101 e 09 09 Launching other programs from PALM RoboSoftware 13 14 415109 2614 101 e 09 09 PALM RoboSoftware layout 2 Program layout and operation 2 1 PALM RoboSoftware layout 1 gs File Edit view Motion Laser Adjustments Devices Help 2 K lt 4 gt AEAR A RDA OI 0804 49900 eA e AxioCamMRm Rev 3 SN 2781 Exposure time y sec ner ner 1 1 i easure Microscope 4
124. ct appearance of the window and the setting options available depend on the selected script The following description relates to the Default script In this window you can mark the structures of relevance to you by setting a color threshold This action is performed either by simply clicking in the image using the color pipette appears automati cally or by setting threshold values in the three color channels After clicking on the OK button script execution is continued and a line is generat ed around the areas marked previously This enclosing line is displayed in the PALM RoboSoft ware main window and saved in the Element List Itis used as a cut line that the laser follows Some of the settings available in this window are described below You will find a detailed descrip tion of the function in the manual for the AxioVi sion program 415109 2614 101 e 09 09 AA Add color di Remove color To adda color to the selection e Click on the Add color button e Click on an area with the required color in the image To remove a color from the selection e Click on the Remove color button e Click on an area with the color to be removed in the image To undo all your settings e Click on the Reset button Undo ua al Redo To undo your last change e Click on the Undo button To redo a change you have undone e Click on the Redo button Show graphics plane To activate or deactivate display o
125. ctions 0 00 ooo ooo oo 135 PALM RoboMover and PALM CapMoverII 000000 nee eee eeu eee 137 Opening the RoboMover or CapMover II window o o ooo oo eee ee ee es 137 Working with PALM RoboMover or PALM CapMover II 138 Preparo a Colector eel cat ata del Sede eran ta el Gere a A we Ree 138 Defining the sequence of the wells for the next laser action only PALM RoboMover 139 Catapulting ClementS aw dara a de E oe eh RA AE A ERA ce aoe ae 140 Viewing the catapulted samples 0 eee ee 140 Stopping PALM RoboMover or PALM CapMover II in emergencieS oo oooooo o 141 Placing the collector in the Home position 0 00 cee ee te 141 DIFUSO OCI NN fissi caer Bech GO o ke ae 142 SONGS a e a kebab BO Sra bn ns ea Gees nh te te dos 142 Setting the movement speed anasa aoa a ees 142 Positioning wells in x y direction sussidi Aa Ge 143 Positioning wells in the z direction LL 143 Referencing PALM RoboMover or PALM CapMover II 0 00 eee ee 144 MicroTweezers Option musa a E A A A AAA E 145 Essential SeEtindas ar a da rain Ges cS 145 Setting power and focus for the trapping laser ooo 146 Finding suitable power and focus settings for the trapping laser 148 Defining a reference position sas gas A RARA AAA O 149 Moving trapping laser beam to the center o oo 149 Moving the trapping la
126. ctivate set of settings 158 adjust Reflector Offset 164 name of filter 160 name of reflectors 161 reset Reflector Offset 164 selecting a Fluorescence Excitation Filter 160 selecting a reflector 161 settings manual microscope 157 settings motorized microscope 157 shutter 159 415109 2614 101 e 09 09 timer 165 working with fluorescence 157 Fluorescence settings change sequence 163 create new set 162 delete 163 edit 163 FOCUS cutting laser 125 microscope 167 trapping laser 146 Footswitch cutting laser 135 trapping laser 150 Freeze Mode 158 Functionality 9 G Graphic Tool activate 1 circle 74 dot 75 freehand 73 grid rectangle 74 rectangle 73 Reference Point 73 ruler 90 stamp 75 text 75 types 71 Group of elements add elements 89 forming 89 remove elements 89 select single elements 90 ungroup 89 H Hardware configuration activate 172 add to the list 174 change name 173 change sequence 175 Help general 21 PALMRobo Instructor 21 Histogram 36 415109 2614 101 e 09 09 I Icon button change size 17 Import of elements 111 Incubation logging 185 InformationCenter functionality 10 start 13 Initial settings mode 25 Installation 11 J Joystick activate 27 PALM RoboMover 27 Stage 27 trapping laser 27 K Keyboard shortcuts summary 196 L Laser cutting laser continue 134 indication of the operating state 134 stop 134 Laser trapping laser
127. ctor button to save the setting To set the settings back to their factory default values e Click on the Set to default button 415109 2614 101 e 09 09 Settings 12 4 3 Positioning wells in the z direction The height of the collection vessel above the slide is set by the manufacturer If necessary you can change the setting Info All settings that you make apply to the collec tor type currently used You must make the settings for each collector type you use The settings are saved automatically If you fit a different collector type the settings sa ved for this collector type are applied To change the height settings e Position the collector over your sample e Select the Adjust tab in the RoboMover or CapMover II window Adjust Fine Adjust Asis Settings for current collector moving height APT working height In the Fine Adjust Z axis section you will find buttons for the fine adjustment of the Z position for your collection device You can define two height settings moving height the height for all movements in x y direction e g for the movement from one well to the next working height the height during a laser ope ration Note Note that for collection devices such as Sin gle tube collectors for dishes and slides the height for the movement in the x y direction must be set especially large to prevent collisi ons with the dish e Click on the Goto Button
128. d a message for the alarm 1 min Ma min 3 min Os min 5 10 min Onin One Alarm message optional pO Cancel e Select the required time and enter a message if you want to e Start the alarm timer by clicking on the OK button 415109 2614 101 e 09 09 Short term alarm 3 10 Setting camera parameters You can connect several AxioCam series cameras to your microscope To work with one of the cam eras connected you must select the required cam era and make a series of settings in PALM RoboSoftware The available settings depend on the camera selected The following examples show the procedure for the AxioCam MR3 You can make some important settings in the Display Tools You will find further settings in the Live Image window To make the basic camera settings in the Display Tools e In Display Tools click on the Camera tab e Select the required camera in the Camera drop down list Display Camera Display Camera AxioCamlCcl E Exposure time Gil Analog Gain db 4 White Balance Advanced Settings AxioCam ICc1 Display Camera Display Camera AxioCamMFic Rev 3 SN 504 e Auto Live Exposure time Seli Gain Factor _ Camera Gain 2x EH gt IRE White Balance R Advanced settings AxioCam MRc m Rev 3 31 Setting camera parameters Depending on the camera type the controls of the supported features are visible in the camera tool
129. d elements refer to chapter 10 4 On the other hand saved images can no longer be reloaded into PALM RoboSoftware window Howev er the supplied PALM RoboSoftware package also includes a program which you can use to observe and process your images the PALM InformationCenter Instructions for working with the InformationCenter are the subject of the pro gram s user manual 415109 2614 101 e 09 09 Insert a Copyright Note 10 1 Inserta Copyright Note You can specify a Copyright Note that will appear on the image on the monitor and on each stored image if you have opted to show it this option can be switched on and off using the menu command View gt Copyright Adjustments PALMA obo e In the Adjustment menu click on the menu item PALMRobo The Preferences and Configuration window opens e Click on the Images tab Images Copyright Info e Enter the desired text into the field Text and close the window by clicking on OK 113 Specify folder to store images in 10 2 Specify folder to store images in You can specify a folder for storing all images that are stored in File Mode either manually automat ically or using the recorder function If you are storing images manually then you can choose a different folder at the time you store them Note If you are working in Database Mode all your images will be stored in the database In this case the folder that has been specified for
130. d elements are shown on a blue back ground while elements that have already been processed by the laser are shown on a green back ground DE Number of Elements number of elements whose color matches Color column and type matches Type of Elements column Area um Area of the elements only ele ments of type Figure for elements of other types an area value has no meaning Remarks 415109 2614 101 e 09 09 The following summary information for all ele ments can be found below the table the total number of elements not including elements of the types Ruler Text Reference and Group the total area of all elements of type Figure To specify which slide the total values should be shown for only for multiple slideholder Show summary for e Click on the arrow on the right by the Show summary for field on the Summary tab A list containing all the slides will open example shows the SlideHolder 3x1 0 e Click on the name of the slide for which you would like total values to be displayed or click on over all if total values for all slides are to be displayed together 8 3 Functions in the Element List window The overview below shows which functions are available in the Element List window Many of these functions are also available in the main win dow for the PALM RoboSoftware Adding a new set of elements File Mode and Database Mode refer to c
131. d with the displayed an chor point see above The previous line between start and end dot of the correction is deleted Note If you first release the Ctrl key and then the mouse button the element between the start dot of your correction and the end dot of the el ement is removed see below To stretch elements lines rectangles ellipses R Pointer Tool e Select the element you want to stretch e Drag the Pointer on one of the squares at the corners or sides of the selection rectangle The cursor changes to a double headed arrow e Pull with depressed mouse button To rotate elements lines rectangles ellipses R Ctrl key Pointer Tool e Select the element you want to rotate e Drag the Pointer on one of the squares at the corners of the selection rectangle e Press and hold the Ctrl key The cursor changes to a circular arrow e Pull with depressed mouse button 415109 2614 101 e 09 09 To rotate and stretch elements lines rectangles ellipses R Ctrl key Shift key Pointer Tool e Select the element you want to stretch and rotate e Drag the Pointer on one of the squares at the corners of the selection rectangle e Press and hold the Ctrl and Shift keys The cursor changes to a circular arrow e Pull with depressed mouse button To restore a changed element Edit Restore e Select the element you want to restore e Click on the menu item Restore in the Edit men
132. dden e Setthe laser focus and laser energy at median values 50 50 or start with factory default set tings refer to chapter 3 8 Note If you reset to factory defaults not only laser energy and laser focus are reset but also all other settings that you have made refer also to chapter 3 The further procedure depends upon whether or not you want to use the automatic laser functions to find appropriate laser settings a Using the automatic laser functions e Set the Scroll function for the microscope stage speed so that you can easily follow the movement with the naked eye Move the microscope stage to test with the Scroll func tion refer also to chapter 5 1 2 Change the speed during movement in order to find an appropriate value Take a note of the value established and enter it as speed for the move ment type Cut e Draw an arbitrary Line image for example a spiral or a staggering line The longer the line is the longer you have the opportunity of con trolling and possibly changing the laser settings during the cutting operation e Setthe laser function Cut and select the num ber of operations to 1 refer to chapters 11 3 Selection of automatic laser functions and 11 4 2 Setting number of passes and three dimensional cutting e If you have drawn one or more elements open the Element List window press F5 for exam ple and select the element to be cut with a mouse click If you have on
133. dinates of the unit currently being moved or most recently moved Stage current coordinates relative to the point of origin of the drawing plane x y The point of origin of the drawing plane is permanently preset refer to chapter 5 4 Movement of the trapping laser moving trap coordinates of the laser focus relative to the point of origin for the movement 415109 2614 101 e 09 09 PALM RoboMover PALM CapMover II Coordinates of PALM RoboMover PALM CapMover II If you double click in the field the States win dow will open Amongst other things this win dow displays the coordinates of all units refer to chapter 2 5 415109 2614 101 e 09 09 Customizing the user interface 2 2 Customizing the user interface Info Please note that the following possible set tings described here may not be available depending upon the configuration of your PALM MicroLaser System PALM RoboSoftware offers a number of ways to customize the appearance of the program window to meet your requirements You can change the size of the program win dow The aspect ratio of the microscope image remains unaltered You can display or hide all icon bars tools and the Status Bar You can change the size of the icon buttons You can move all icon bars and tools to other positions even positions outside the program window You can display or hide the Crosshairs the Laser Markers the Scroll Rectangle the
134. dit an element group similar to editing single elements You can move stretch straighten and rotate or copy the entire group refer to chapter 7 8 You can edit or delete single elements within a group like ungrouped elements refer to chap ter 7 8 You can center a group of elements on the monitor by a menu command refer to chapter 7 5 You can add elements to a group You can delete elements from the group You can change the name defined for a group refer to chapter 7 8 5 Info If you want to merge elements into a group add elements to an existing group or remove elements from a group you can use the menu commands in the main window of PALM RoboSoftware or in the Element List window after selecting the elements Note that you have to use the corresponding menu command in the main window if you have selected the elements on the monitor If you have however selected the elements in the Element List window you need to use the corresponding menu command in this window 415109 2614 101 e 09 09 Working with element groups To merge multiple elements into a group Edit Create Group e Select the elements you want to merge into a group either on the monitor or in the Element List window e Click on the menu item Create Group in the Edit menu in the main window or in the Element List window E Element List File Edit Motion Laser Collection Device E y
135. djust General AxioCaml4P 3 Exposure Ca 2 100 dh al I I I F I pe AxioCamMR3 White Balance Cyan E ell e Red Magenta 5 el e 0 Green Yellows al fe Blue AxioCamMR3 Color Saturation Saturation gq 2 O Histogram Here you will find the settings for the following pa rameters Exposure time White Balance you can select a dot in your image for the white balance perform the white balance automatically set the color tempera ture to 3200K default value for halogen light or set the portion for each color channel sepa rately Color Saturation changes color saturation Reducing this setting results in a weakening of the colors leading ultimately to a black white image increasing it results in extremely inten sive colors You will find further information on the parameters in the text above and in the AxioVision User s Guide 33 Setting camera parameters e Click on the General tab Camera selection Measurement tools Display AxioCamMR3 Adjust General amp xioCamlR 3 Digital Gain as Index Gain factor 2 Index 0 5 jap gt Axio lamMR 3 Image Orientation AxioCamMR3 Black Reference Black Reference amp xioCamlR 3 Shading Correction Shading Correction Cam Enable Auto Automatic amp xioCamlR3 Sharpening Den AxioCamMR3 Shutter Enable Control Signal Low active 0 ms Acquisition Delay Here y
136. dow opens The Configuration tab will be displayed Configuration Joystick Activate Joystick Status e Click the Activate Joystick checkbox to acti vate the joystick 28 3 5 Setting the selection of the avail able laser functions In Cut Tools select the automatic laser function you want to apply to your sample refer to chapter 11 3 You can set which laser functions appear in Cut Tools Thus you can e g hide the laser functions you do not require Info All automatic laser functions are described in chapter 11 1 1 Adjustments PALMA obo e In the Adjustment menu click on the menu item PALMRobo The Preferences and Configuration window opens e Select the Appearance tab Appearance Laser function configuration Cut Joint Cut Close and Cut LPC Line Auto LPC Auto LPC Close and Cut and Auto LPC Robo LPC Center Robo LPC lt 8 x SJ SS xl E e In the Laser function configuration section select the laser functions you want to use 415109 2614 101 e 09 09 Turning on automatic focusing for elements 3 6 Turning on automatic focusing for elements In addition to the coordinates describing its posi tion on the drawing plane x and y coordinates each element also has a z coordinate This z coor dinate is represented by the focus value of the mo torized microscope When you save elements this z coordinate is also stor
137. e 22 2 4 5 PALMRODO IAfORMAUON awk twa Baw etek aw hee Wet be ae eee eee eho ed es 23 2 5 Status displaye Sot agi il RA EA AA a A 24 3 SEMOS sia e A ee ees ee aa 25 3 1 Caunening Settinas etarra eta de ARA a Ke r 25 3 2 Basic and Advanced display Modes 0 a e 26 3 3 setna Sid ende ias ek de oh a ay Gas ce Gy ai a Soh Pa A a ar a A PR ee a A A 27 3 4 Settings for the joystick and the arrow KEYS 27 3 5 Setting the selection of the available laser functions 0 0000 cee eee eee eee 28 3 6 Turning on automatic focusing for elementS 0 0c e 29 3 7 Saving ana loading Settings aura week a 4a Oe AA Swe BO RE a aN ee OR 29 3 8 Restoring SOLOS am rada e a a a a ee a Ai 30 3 9 e A sal 31 3 10 Setting Camera parameters o 31 3 11 Setimade parameters act al 2 A ke A ASE ere a ae ei 35 3 12 Displaying color and brightness values for the microscope image 0 oo a 37 4 Calibrating the Stemi a o RA A AE E ee A 39 4 1 Preliminary Femarks ira ee A AAA ds 39 4 2 setting the maghificatiO Me soria A A a e Gee ad 39 4 3 Setting the laser Marker ula greto PA ia we EAE aa Fa ee NE Rae 40 4 4 Calibrating the movement of the stage es 41 4 5 Calibrating Tne oriseti a ici la aa ARONA ete 42 415109 2614 101 e 09 09 3 Contents 4 6 4 7 4 8 Dad Seda sm Des 5 1 4 5S5 5 1 6 De 5 1 8 5 2 DIS 5 4 5 9 5 6 Deh 6 1 6 2 6 3 6 4 6 5 6 6 6 7 6 8 6 9 6 10 6 11 Lal 7 2
138. e Continue button Info You can also restart the scan from a selected tile once the scan has been completed 59 Scanning a section from the overview scan 6 5 Scanning a section from the To change between the overview scan and overview scan the scan of the section The Information section specifies whether you can currently see the section Section 1 or the overview scan Section 0 in the window You can select an area of an overview scan and then scan it This gives you an enlarged view of the selected area The overview scan itself is re tained Section Navigation e In the overview scan Section 0 on the Scan tab use the mouse to draw a rectangle lt around the area you want to scan Information Slide Section 1 36 tiles 1 W 5 ti H E ti e Click on the Scan button site n The selected section will be scanned Section 1 is gt displayed in the Information section The process and the available actions are the same as for an overview scan see page 57 and chapter 6 4 Information Slide Section 0 110 tiles Info e Click on the arrow button in the Section You can pull up a frame in the scan of a section Navigation area to the top right of the window in order to scan the area once more The to switch between the section and overview previously scanned section will then be lost scan 60 415109 2614 101 e 09 09 6 6 Saving and reloading the scanned Image You can save the
139. e area can be catapulted later by a single laser pulse With this function only the cells upon which you have drawn a Dot element are catapulted into the collecting cap LineAutoLPC A line shaped structure is catapulted into your col lection vessel using this function The line is there fore not catapulted in one piece but with several laser pulses The original structure of the material is not retained when using this function 121 Laser functions AutoLPC RoboLPC With this function the contents of a defined area of glass mounted samples are catapulted into your cap The area is therefore not catapulted in one piece but with several laser pulses The original structure of the material is not retained when us ing this function Both this function and the following one Close amp Cut AutoLPC can only be used for glass mounted samples i e these functions cannot be used if the sample is placed on a membrane LPC membrane With glass samples only a limited amount of cell material per laser pulse can be catapulted out with one laser shot Larger areas have to be catapulted with multiple laser pulses The exact position of the laser pulses is calculated by PALM RoboSoftware you have to define only the border line of the area and the distance LPC distance be tween the laser pulses as well as their orientation diagonal or parallel The required distance between the single laser pulses depends on
140. e edge of the slide If you draw elements here they will be deleted automatically by PALM RoboSoftware When installing your sample on the slide make sure that the areas to be processed do not come too close to the edge of the slide To activate one of the graphic tools e Click on the icon of the desired graphic tool in the Graphic Toolbar The tool selected remains active until you select another drawing tool or another function 71 Drawing new elements 7 3 1 Selectinga laser function Before you draw an element define the laser func tion that is to be applied to the element CloseCut Autol PC Robol PC CenterhoboLPE e Open the Cut Tools drop down list in the default configuration in the bottom left corner of the program window and select the required laser function If you do not select a laser function the laser function set prior to drawing will be assigned to the element Info You can define which laser functions appear in the drop down list as described in chapter 3 5 Info You can again change the laser function that is to be applied to the element after drawing refer to chapter 8 4 7 3 2 Defining well for catapulting With PALM RoboMover you can use inserts with several wells You can activate automatic distribu tion functions with which your elements will be au tomatically catapulted into the defined wells refer to chapter 8 6 2 Even before drawing you can define a well for ea
141. e if you have configured your experiment in such a way that images have been recorded at two times there will be a total of 28 images in the above example You can view all of these individually in the picture viewer To display a particular image select the fluorescence chan nel the focus level and the time lapse cycle 415109 2614 101 e 09 09 Viewing images in the picture viewer Multi channel fluorescence au 1 2 1 Switch between display in false colors On and original images Off 2 Select fluorescence channel for individual or superimposed image display Info For recordings with a monochrome camera only the false color images can be super imposed e Click on the relevant button to select the options described Extended focus Fz lt a 3 e Click on the arrow buttons to the right of the z stack symbol to select the desired focus level Time Lapse gaT ll dl 12 e Click on the arrow buttons to the right of the time lapse symbol to select the desired cycle Set the image size and section H aA A A B le 3 4 5 1 Move section displayed 2 Set image magnification to default value 3 Fit image to window 4 Zoom in 5 Zoom out e Click on the relevant button to select one of the specified settings 183 Viewing images in the picture viewer Setting image parameters Info The setting options described below are only active when individual fluorescence channels are
142. e illumination Devices x Microscope FE e On the Devices menu click on Microscope The Microscope window opens e Click on the Reflected tab Microscope Reflected Transm St Colibri Channels 1 400rm OL on Jo 19 2 455nm on for Jo 3 590nm D on of jx lt 4 E25rm OL on off 19 Colibri Light Source Colibri Illumination Mode Shutter external CH ME gt Reflectorttrl 100 Mirror ox li LE irc Rhodamine Texas Red 100 Mirror You can display a graphic of the light path for the LED illumination e Click on the Grafic button This opens the Colibri window You can make the Same settings in this window as in the Micro scope window Reflected tab The LEDs installed in your system and information on their wavelength are given in the Colibri Chan nels area 166 e Click on the All off All on button to switch all LEDs on or off e Click on an On or Off button to switch the related LED on or off e Adjust the light intensity of the related LED using the arrow buttons Colibri Colibri Control Colibri Channels OS 625 fm BCR6S 400 nm BC425 gt Alo E External O 455 nm Colibri Light Source Colibri Illumination Mode Shutter external of Closed gt In the Colibri Light Source area you can select whether you want to work with the LEDs or with an external l
143. e options As an option your PALM RoboSoftware can be licensed for the Multi channel fluorescence and Extended focus functions Please contact Carl Zeiss Microlmaging GmbH if you wish to upgrade your system With multi channel fluorescence two or more recordings of the sample are made in succession with different fluorescence excitation filters As a result a series of different images is available in which different structures in your sample are visi ble These images can be overlaid In this way structures in your sample can be made visible that would hardly be visible using a single fluorescence channel or perhaps would not be visible at all The Extended focus option is particularly suitable for thicker samples or samples that do not lie exactly on a plane Such samples cannot normally be displayed with full sharpness the focus must be changed for different areas of the sample Using the Extended focus function several images are taken of the sample with different focus settings The program analyzes these images and calculates from the sharp portions of each image a sharp overall image that is then dis played on the monitor You can also use both functions for scanning your Sample using the Navigator see chapter 6 2 You can also combine the two methods to create multi channel fluorescence images with extended focus You can also combine them with a time lapse function i e the recordings are automatical ly repe
144. e Beam System with one movable trap ping laser beam Trap 1 is the movable laser beam and Trap 2 is the fixed laser beam e Setthe required values for Power and Focus e g using the sliders If you wish to divide the power equally between the two beams 50 50 e click on the word Balance 415109 2614 101 e 09 09 Setting power and focus for the trapping laser If you wish to set the power of trapping laser beam 1 or trapping laser beam 2 to 100 e Click on item Tri or Tr2 The power of the other beam in each case will then be set to 0 If you wish to set different power values for each of the two beams e Setthe required relationship between the power values in the Balance section percent age of the total laser power To change the focus e Setthe required value in the related Focus section Info Regulate the energy and focus for the cutting laser using the settings in Cut identical to the related tools on the MicroBeam tab refer to chapter 11 4 1 To set the focus of the trapping laser using the mouse Laser JFS ss kicroBeam Tweezers e Wheel e Under Wheel in the Laser Tools click on the radio button for the trapping laser beam whose focus you wish to change using the mouse left for Trap 1 right for Trap 2 e Rotate the scroll wheel on the mouse until the desired value appears in the text box of the Focus Calibration Tool Info The Wheel
145. e Mode and Database Mode File Mode is always available Database Mode can only be used if an appropriate license has been purchased for PALM RoboSoftware In File Mode you can save microscope images and elements in folders on a data carrier e g your lo cal hard disk Elements are geometric figures that you draw You may for instance identify areas for processing with the laser In Database Mode PALM RoboSoftware is linked with a database If you save images then they are stored in the database Furthermore any ele ments that you draw are automatically stored in the database in exactly the same way as informa tion on samples experiments and microdissection operations The InformationCenter allows you to create databases and to display and edit their contents read the section in the manual about the InformationCenter for more details 11 Launching and exiting the program 1 8 Launching and exiting the To exit PALM RoboSoftware program File To launch PALM RoboSoftware e Click on Programs gt Palm gt PALMRobo in the Windows Start menu Exit PALMA obo Alt F4 e Click on Exit PALMRobo in the File menu If your system does not include a database license S Info then PALM RoboSoftware will be launched If necessary when the program is closed a If your system does include a database license prompt appears as to whether you want to then you can choose whether to work in database save your settings or your e
146. e Motion the stage will be moved in the opposite direction to the mouse movement In this case the motion of the image on the monitor is in the same direction as the movement of the mouse To invert stage movement Motion Invert stage motion e Click on the menu item Invert stage motion in the Motion menu to switch the function on or off Info You must invert the movement of the stage be fore switching to Stage Mode You cannot ac cess this command when you are in Stage Mode Note Do not use the function Invert stage motion if you want to calibrate the system To switch to Stage Mode Motion n Stage mode Fr e Click on the menu item Stage mode in the Motion menu Info The cursor is not displayed in Stage Mode In Stage Mode you can only change the speed and energy or power of the stage and the focus of the laser by means of keyboard commands note that you can only change the settings for the laser beam that is selected in Laser Tools You cannot access any other functions To switch from Stage Mode back to Cursor Mode e Click once with the left mouse button 45 Movement types 5 1 2 Positioning the stage with the mouse Scroll In Cursor Mode you can use the scroll function to move the stage e If you want to move the stage horizontally ver tically or diagonally move the cursor to an edge of the image or a corner of the image out side the Scroll Rectangle but still withi
147. e an element is targeted during a laser function and each time the lens is changed the image is fo cused automatically 168 15 1 3 Defining and setting fixed positions for the focus You can target three defined focus positions two of it are freely definable the third position is pre scribed Work Position freely definable The objective is set so that the focus is in the same plane as the slide In this position you can view your sample on the monitor or in the ocular of the microscope Info The exact setting of the focus is variable for various objectives Because of this you have to define the setting of the work position for all objectives However if you are working with slides or sam ples of different thicknesses you may have to readjust the focus Check Position freely definable The objective is set such that the focus is on the same plane as the cap into which your samples are cata pulted using the LPC functions refer to chapter 12 In this position you can check the results of the catapulting Load Position prescribed The objective is moved into the lowest possible position In this position you can for example move the stage to the CapCheck and back or change the slide without any fear of hitting the objective with the slide Caution Using a non motorized microscope Ifyou want to return the stage from the CapCheck you must set the Load Position manually so that the objec
148. e button and the stage stops moving you can continue the line In Freeze Mode you can continue to draw with the Freehand Tool outside the Scroll Rectangle for a detailed explanation refer to chapter 7 3 3 7 3 7 The Rectangle Tool O Rectangle Tool Quadratic Attribute O Centric Attribute Use the Rectangle Tool to draw rectangles The dot at which you begin your drawing becomes a corner of the rectangle e Select the Rectangle Tool in the Graphic Tool bar If you select Quadratic Attribute as well the drawing function is limited to squares If you select Centric Attribute as well the dot at which you begin your drawing becomes the center point of the rectangle or the square e Move the cursor to the dot where you want to begin your drawing e With the mouse button pressed down drag the rectangle or square out until it is the desired size and then release the mouse button 73 Drawing new elements 7 3 8 The Grid Rectangle Tool 7 3 9 The Circle Tool EM Grid Rectangle Tool O Circle Tool Configure Mm Quadratic Attribute You can draw a rectangle using the Grid Rectangle Tool this rectangle will be automatically divided into a matrix of smaller rectangles you have de fined Each individual rectangle is considered an independent element by the PALM RoboSoftware and has its own number in the Element List refer to chapter 8 1 You can now catapult the ele ments into PALM RoboMover wells such that t
149. e clicked on 415109 2614 101 e 09 09 6 9 Positioning the microscope stage Using the Navigator you can move the stage to specific points of your sample Slide Navigation AD Selected slide 2 e Onthe Navigation tab click or use the arrow buttons to select the slide you want to view e Scan your sample as described in chapter 6 3 if it has not yet been scanned Navigation Mode Live Stored tiles e On the Scan tab click on the Live radio button under Navigation Mode e Click on the point to be displayed in the PALM RoboSoftware main window either on the Navigator tab or on the Scan tab here you can select between the overview scan Section 0 and the section scan Section 1 if required see chapter 6 5 The stage will be positioned at the desired point 63 Analyzing images using the AxioVision software 6 10 Analyzing images using the AxioVision software The AxioVision software enables images of sam ples to be analyzed using scripts that have been defined in advance and also allows elements to be generated This helps with routine operations to au Th an yo tomate the selection of the areas of interest Info This analysis feature is only available if your system has the AxioVision modules for image analysis Please contact Carl Zeiss MicroImaging GmbH if you wish to upgrade your system e AxioVision program allows you to generate d save scripts that you
150. e color e Click on the Apply button the settings are applied the window remains open or click on the OK button the settings are applied the window is closed 85 Changing colors and line thickness To change the settings for the line thickness and dot thickness You can enter values for the following parameters in the three fields in the Select values for sec tion Line thickness thickness of Figure elements Dot thickness diameter of Dot elements Ruler thickness thickness of measuring lines e Enter the desired values in the corresponding fields and confirm your entries by clicking OK Info The set thickness of the lines or dots is not related to the width of the laser cut line or the diameter of the laser beam These are depen dent on the laser parameters setting laser en ergy laser focus and movement speed of the stage refer also to chapter 11 4 86 7 9 2 Selecting colors for elements to be drawn new You can either select colors for new elements to be drawn in the Draw Preferences window Element colors and thickness tab refer to chapter 7 9 1 or as described in the following from the Color Palette You can define which colors appear in the Color Palette To define the colors displayed in the Color Palette Colors e Click on Colors in the Graphic Toolbar The Draw Preferences window will open e Select the Color Names tab Draw Preferences Elemen
151. e dots the laser beam touches with the automatic laser function LPC in order to catapult the area AutoLPC Dot color of the dots the laser beam touches in the automatic function AutoLPC RoboLPC Line if an open element is envi sioned for the RoboLPC laser function but the distance between the two finish dots is greater than the value specified in Distance of RoboLPC joint refer also to chapter 11 4 the distance is automatically shortened to the preset value This auto matically determined line is displayed in the color selected here Lines that close open elements are also dis played in this color if the functions Close amp Cut or Close amp Cut AutoLPC are selected RoboLPC Dot color of the dot the laser beam touches with the automatic laser func tion RoboLPC in order to catapult the de tached area Ruler Line color of the lines for measuring refer also to chapter 7 11 Text Field background color of text ele ments the text itself is always black Number Field background color of the field with the element number and where appli cable the comment number and comment are always black e Select the desired color from the color palette in the right window pane Info Select the colors so that they contrast strongly with your sample This way your elements will be more readily recognizable e Proceed the same way for all element proper ties for which you want to change th
152. e exactly the same sequence as for the source sample Otherwise the references will not be correctly collated e Ifyou are using a multiple slideholder then you should click on the tab associated with your tar get sample in the Element List window Slide 2 in the example image shown below e Group the three elements and allocate the group a name e g Slide 2 103 The Serial Sections function amp Element List File Edit Motion Laser Collection Device Il pep 288 Ur ER K Slide Slide2 Slide3 Summary Show Types all Color j Nr Name Type Laser function Objective Well Area un Grp cut shot 11 Freehand AutoLPC 5x Fluar 5x 0 25 402946 Slide 2 A 12 Freehand AutoLPC 5x Fluar 5x 0 25 265509 Slide 2 a 13 Freehand AutoLPC 5x Fluar 5x 0 25 391517 Slide 2 14 Slide2 Group 5x Fluar 5x 0 25 e Select the three drawn elements and click on the menu item Define Group Reference Figure in the Edit menu not applicable if you have drawn elements of the Reference Point type This opens the Groups window e Choose the group to which the selected ele ments belong in the example Slide 2 and click on the OK button to close the window PALM RoboSoftware creates three new elements of the Reference type for the target sample Al Element List File Edit Motion Laser Collection Device A rd Yet 2288 0Uc ER K Slidet Slide Slide3 Summary Show Types all
153. e laser beams TwinFlex If you have purchased a system with laser beam positioning moving trap you can move a trapped object with the laser beam parallel to the plane of the slide The systems with two laser beams en able you to trap two objects at the same time and move one or both parallel to the plane of the slide PALM RoboStage II Using PALM RoboStage II it is possible to process up to three slides at a time a feature that opens up completely new possibilities for working with serial sections and that is not all It is also possi ble to use 35 mm or 50 mm culture dishes for pro cessing living cells 415109 2614 101 e 09 09 Functionality of PALM MicroLaser systems Devices for positioning caps or vessels Carl Zeiss MicroImaging GmbH offers various pos sibilities for positioning different caps PALM RoboMover PALM RoboMover is our special solution for working at high throughput rate Time saving automatic sample capture and additional sort ing functions of the captured samples enable above all deployment in the routine labora tory PALM RoboMover makes automatic posi tioning of caps over your sample possible There are various inserts collectors available each of which contain one or more vessels All of PALM RoboMover functions can be controlled by PALM RoboSoftware PALM CapMover II PALM CapMover II makes automatic positioning of caps over your sample possible There are various inserts collec
154. e menu item PALMRobo The Preferences and Configuration window opens e Click on the Laser tab Laser Auto Image Before Cut After Cut Before LPL After LPC Save Elements in Image Image Format JPEG Files jpg The Laser function section contains four Auto Image checkboxes e Click on the Auto Image button to enable or disable the related function e Select the file format you want If the function is enabled images for each element processed with the laser will be saved every time the laser function is triggered Names have been assigned for the images as follows x BeforeCut x AfterCut x BeforeLPC x AfterLPC x stands for an element number that is assigned automatically by PALM RoboSoftware This ele ment number is incremented starting from 1 for each save operation If the images are to be saved together with the su perimposed elements e Click on the checkbox in front of the Save Elements in Image to enable or disable this function 415109 2614 101 e 09 09 Saving images and videos using the Recorder function 10 5 Saving images and videos using the Recorder function Info The Recorder function is only available if you have purchased an appropriate license Please contact Carl Zeiss MicroImaging GmbH if you wish to upgrade your system You can use the Recorder function to record video sequences Save series of images If you a
155. e while the switch is being depressed or whether it is to func tion as an On Off switch Info You can only use the footswitch when you are in Trap Mode or Stage Mode refer to chapter 13 11 2 Moving the trapping laser beam with the mouse TrapXY 1 TrapXY 2 Mode and chapter 5 1 1 Positioning the stage with the mouse Stage Mode To set the function of the footswitch T_ e pu Laser Trapping foot switch Configuration Hold Switch e Inthe Adjustments menu click on the menu item PALMRobo The Preferences and Configuration window opens e Select tab Laser If the footswitch is to operate as a pressure switch e Click on the Hold radio button The trapping laser remains switched on as long as you keep the footswitch pressed If the footswitch is to operate as an on off switch e Click on the Switch radio button The trapping laser is switched on or off by pressing once 415109 2614 101 e 09 09 13 8 Activating and deactivating the trapping laser You can specify whether the trapping laser is acti vated and deactivated by the computer or by the footswitch You can only use the footswitch when you are in Trap Mode or Stage Mode refer to chap ter 13 11 2 Moving the trapping laser beam with the mouse TrapXY 1 TrapXY 2 Mode and chap ter 5 1 1 Positioning the stage with the mouse Stage Mode Info If you switch from Stage Mode to Trap Mode
156. ea un Grp cut a 1 Freehand Ref 1 AutoLPC Bx Fluar 5x 0 25 416385 Slide 1 ll 2 Freehand Ref 2 AutoLPC 5x Fluar 5x 0 25 388527 Slide 1 3 Freehand Ref 3 AutoLPC Bx Fluar 5x 0 25 308903 Slide 1 4 Slidel Group 5x Fluar 5x 0 25 5 Reference Ref 1 5x Fluar 5x 0 25 Slide 1 6 Reference Ref 2 5x Fluar 5x 0 25 Slide 1 7 Reference Ref 3 5x Fluar 5x 0 25 Slide 1 lt Info n With the command Undefine Group Reference Figure you can undo the definition of an element as a reference element The original element along with the Reference type element created by PALM RoboSoftware are preserved just the connection between the two elements will be deleted Within each of the three drawn elements the re spective reference element created by PALM RoboSoftware will be shown SI UNE IPO UE i 4 beJ y di ke As to PF y wee A aa a i Ri y n P i jil Ei boy E You have now created your reference for transfer ring the elements to further samples 415109 2614 101 e 09 09 The Serial Sections function Step 3 drawing and processing elements automatic and manual adaptation e Draw the elements which are to be processed on all samples e Add these elements to the reference group Select the elements and click on the menu item Add to Group in the Edit menu Note If you transfer elements to another sample as described below only those
157. ed a Single Beam System and or a system without Laser Beam Positioning Please contact Carl Zeiss MicroImaging GmbH to upgrade your system 415109 2614 101 e 09 09 Essential settings 13 1 Essential settings Before you can work with your PALM MicroTweezers System you must make a number of preliminary settings Optimum results can only be achieved if the initial settings are correctly car ried out You must align the Trapping Laser Marker in the DuoFlex TwinFlex system both Trapping Laser Markers with the actual trapping laser position refer to chapter 4 3 You must set the power and focus for the trap ping laser refer to chapter 13 2 If you have a system with Laser Beam Positioning you must also calibrate the movement refer to chapter 4 5 set the speed with which the Laser Beam Posi tioning is performed refer to chapter 13 11 1 You only need to calibrate the Laser Beam Posi tioning and adjust the Trapping Laser Marker set ting if the results from using the laser are incorrect For systems with positioning you can define a ref erence position for each trapping laser beam refer to chapter 13 4 In certain circumstances you may need to make the settings for power and focus for the trapping laser and the speed with which the movement is made separately for each sample and sample type The following section describes how to proceed 145 Setting power and focus for the trapping laser
158. ed on then each time an element is targeted during a laser function and each time the lens is changed the image is fo cused automatically Autofocus can be turned on and off in the Micro scope Tools or on the Laser tab in the Preferences and Configuration window In the Preferences and Configuration window autofocus can be turned on and off as follows Note To ensure that the Autofocus function actual ly produces a sharp image you must first have defined the actual objective for the work posi tion refer to chapter 15 1 3 and the parfocal ity must be correctly adjusted on the microscope e In the Adjustment menu click on the menu item PALMRobo The Preferences and Configuration window opens e Select tab Laser Lazer Auto Focus AF active e In the Laser function section click on the Auto Focus AF active box to turn the function on or off To turn autofocus on or off in Microscope Tools proceed as follows JAF active e The function can be turned on or off by clicking on the AF active box in the Microscope Tools 127 Specifying laser settings 11 4 4 Other general laser settings You can define whether the elements are to be dis played on the monitor while the laser function is executed and whether the stage is to be moved back to the start position when the laser function is complete To enter these settings proceed as follows Adjustments PALMA
159. ed with each element refer to chapter 9 for details of how to store ele ments As you approach elements the focus is automati cally set to the appropriate value However focusing is also temperature dependent This means that if the temperature is different when an element is approached than it was when the element was drawn there is a possibility that the focus setting will be incorrect Correct focusing is only possible if the work area has a stable tem perature and if your system has been moderated for a sufficient length of time see microscope manual for further information The fluctuation in room temperature must not exceed 1 degree and your system should be moderated for a minimum of four hours In order to avoid focusing problems you can turn the automatic mode on and off the system will then automatically use a stored value To turn automatic stored value focusing on and off Adjustments PALMA obo e In the Adjustment menu click on the menu item PALMRobo The Preferences and Configuration window opens e Click on the Elements tab Elements Positioning Elements Go to the element 2 focus e Tick the check box Go to the element z focus if focusing is to be carried out automatically 415109 2614 101 e 09 09 3 7 Saving and loading settings You can save your settings in a file and reload them for future work This also means that a back up copy is created of al
160. efine Columns window opens 112 415109 2614 101 e 09 09 10 Save Images You can save the portion of the stage that is cur rently displayed on the monitor You can save in three ways Manual saving every time you enter the corre sponding command the current image will be saved refer to chapter 10 3 Automatic saving every time you release a laser function an image will be saved for each element that has been processed with the laser before and or after processing refer to chapter 10 4 Recorder function optional this can be used to automatically store series of images or video sequences refer to chapter 10 5 You can specify the time interval between images in a series and the duration of a recording You can specify a Copyright Note If you have opt ed to display the Copyright Note then it will be in serted in every image that you store refer to chapter 10 1 If you save an image with the trapping laser switched on the image is always saved with the Trapping Laser Markers If you are working in File Mode refer to chapter 1 7 you can specify a folder or create a new folder for images to be stored in You can also specify a file name refer to chapter 10 3 If you are working in Database Mode your images will be stored in the database The file name is generated automatically For automatic storage of images you can specify whether your images should be stored with or without the superimpose
161. elected Operating Mode are not chosen correctly not all elements selected to be catapulted will actually be catapulted Using the Distribution Calculator it is easy to find the right parameters The Distribution Calculator au tomatically calculates the distribution of the ele ments to be catapulted over the number of wells defined The results of the calculations are dis played in the Distribution calculation area in the Distribution calculator window Info If you have selected the morphology conserving operating mode only the number of elements to be catapulted appears in the Distribution calculation area Distribution calculation Number of selected well z Number of elements to process Number of elements per well Number of used well g Remaining elements IC The information in the fields have the following significance Number of selected well s Number of selected wells defined by the selec tion in the Start with Field and until Field fields Number of used well s Number of wells into which elements are actu ally catapulted after the triggering of the laser function Number of elements to process Total number of elements that are to be pro cessed this is the number of elements that are selected in the Element List window or the number of unprocessed elements Number of elements per well Number of elements that are catapulted into each well For the n el
162. elected in the Speed Tools tool Note The settings that you enter always apply to the magnification that you have set in the program As soon as you have set another magnification the values that were last used or the factory defaults are automatically ap plied Therefore prior to setting the speed first select the magnification that you want to work with 415109 2614 101 e 09 09 Before you initiate a movement of the trapping laser beam all three setting options are available for both movement types The speed can be varied using keyboard commands during movement To select the unit for speed setting Adjustments PALMA obo e Inthe Adjustments menu click on the menu item PALMRobo The Preferences and Configuration window with the Configuration tab opens Configuration Metric Coordinates Micrometer Speed Logic Micrometer sec e Inthe Metric section click on the radio button Logic units percent or Micrometer sec units um sec 415109 2614 101 e 09 09 The moving trap To change the speed of the Speed Tools tool e In Speed Tools select the movement type for which you want to change the speed Speed Traps Arrow The Speed Tool for the selected movement type is shown in the example shown TrapXY The max imum selectable speed is dependent upon the cho sen movement type speed Speed Traps wt Sheps sec 6500 lt F
163. ement set from the monitor To prepare a new sample for processing in File Mode File New Elements Delete all Ctrl N e If you want to save previously drawn elements for later work save them as described in chap ter 9 2 e Click on New Elements Delete all in the File menu in either the main window or the Element List window If elements exist they will be deleted 415109 2614 101 e 09 09 7 2 2 Working in Database Mode In Database Mode after the program has been launched you can initially draw only a single ele ment using the Freehand Tool refer to chapter 7 3 5 in order to test the laser settings for instance To distinguish it from other elements this element is displayed on the monitor with a dashed line Before you can draw elements you must enter var ious details of your sample and your project With out this data your elements cannot be saved in the database When this data is entered distinctions are made between three hierarchical levels which you might use in the following way see illustration beneath Source Data represents the tissue sample that you wish to process Specimen Data represents a wafer of the tis sue sample that is placed on the slide for pro cessing Experiment Data represents the specific ex amination that you intend to perform on the wafer If you would like to create a new set of objects for the same slide you should enter a new experi men
164. ements per field Operating Mode this figure is the number of elements defined by you in the n field The calculated number is displayed for the spread elements evenly Operating Mode The number depends on the number of wells defined and the number of elements to be cat apulted 99 The Serial Sections function The number of elements catapulted into each well can be or is different for the Sequence 1 1 X 1 2X area per field and sequence area operating modes For this reason this field does not appear for this operating mode Remaining elements Number of elements that will not be catapulted with the settings selected The reason for this remainder is dependent on the selected Oper ating Mode n elements per field Operating Mode the number of wells defined is insufficient for all elements spread elements evenly Operating Mode remainder from the division of the number of elements to be catapulted and the number of wells defined sequence 1 1 X 1 2X Operating Mode either the number of wells defined is insuffi cient for all the elements or the remainder does not fulfill the condition for the mathemat ical series 100 8 7 The Serial Sections function With the function Serial Sections you can trans fer elements that you have drawn for a sample to other samples This method saves time when pro cessing consecutive wafers in a tissue sample The elements only need
165. en individual images and the overall duration Via the check box endless Mode only for series of images you can determine The Endless Mode check box only for series of images can be used to specify that images should be captured at a defined time interval until such point as the sequence is broken off manually The Use auto focus check box can be used to activate a mode in which the focus is read justed for each new image Above all this can be important if the stage is moved whilst the image is being captured and images of different parts of your sample are being stored You should be aware that it takes around 30 seconds to adjust the focus This means that if this function is chosen then it is not an option to have an interval of less than 30 seconds 118 If you check the Switch off transmitted light close transmitted light shutter between picture recording box then in the time between differ ent images being saved the microscope light is switched off and the shutter on the microscope is closed to prevent any light from reaching the sample Please note the it takes around 10 seconds to switch the light off and on again This means that if this function is chosen then it is not an option to have an interval of less than 10 seconds If you check the Close reflected light shutter between picture recording box which is only enabled if a fluorescence unit is installed then the fluorescence shutter is c
166. ent You can enable and disable the element number ing display View les Show Numbers e Click on the menu item Show Numbers in the View menu 83 Changing colors and line thickness 7 8 4 Updating the element numbering If you delete elements the remaining elements keep their respective numbers If you want to re number your elements sequentially you can per form a renumbering operation Edit E Benumber all e Click on the menu item Renumber all in the Edit menu in the main window or in the Ele ment List window 7 8 5 Adding a name or a comment to elements You can add a name and or a comment to all ele ments not only text elements The name of an el ement is displayed at its start dot for the number as well as in the Element List window in the Name column A comment is displayed in the Element List window in the Comment column You can edit a name or a comment at any time Pro ceed as you would for writing a name or comment To add a name or comment to an element Edit ET Change Alt Enter e Select the element to which you want to add a name or a comment in the main window or in the Element List window e Click on the menu item Change in the Edit menu in the main window or in the Element List window The Element Properties window will open Element properties Name Color MI green green Hr 13 cut shot Grp Area 149396 Type Freehand H ww 566
167. ents menu click on the menu item Fluorescence The Fluorescence adjustments window will open e Select the Reflector tab refer to the figure above On this tab you can select a filter make various settings and view information on the reflector How to calibrate the reflector compensate for the reflector offset is defined in chapter 14 10 all other settings are described in this chapter Information about the reflector Information and Control section Reflector Type field information about the manufacturer Reflector Error field this is where error mes Sages are displayed in the event of problems with the reflector Actual Position field this shows which reflec tor is currently in the fluorescence beam path You can also select the desired reflector here see page 161 Reflector Colors and Names section Information on the existing reflectors You can make or change any entry yourself in the Color column 415109 2614 101 e 09 09 Information and Control Reflector Type Microscope Reflector Reflector Error EG Actual Position Start Reflector Calibration To define names for the reflectors You can specify names for your installed reflectors e g color names When you select a reflector lat er the names you have defined appear in the drop down list otherwise the sequential numbers from 1 to 6 are used e In the table double click in the row of the reflect
168. equired Info The selection available depends on which ob jectives are fitted to your microscope and con figured in the MTB 2004 15 1 2 Setting the focus You can either adjust the focus almost constantly in small steps of about 1um each or you can de liberately target defined positions Load Position Working Position Check Position refer to chapter 15 1 3 If your microscope is equipped with autofocus you can perform the focusing auto matically To set the focus e Drag the slider in the Focus area to the required value or click on the left or right arrow button to adjust the focus up or down The current position is indicated in the field to the left of the arrow buttons You can also make the setting using the mouse e Position the mouse pointer on the slider With the right mouse button pressed coarsely adjust the focus using the mouse wheel you can make a fine adjustment using the mouse wheel with the left mouse button pressed To focus automatically Info This function is only available if your micro scope has an autofocus facility e Click on the Autofocus button 167 The Microscope Tools To activate deactivate the autofocus Info This function is only available if your micro scope has an autofocus facility e Move to the work position JAF active e The function can be turned on or off by clicking the AF active box If the autofocus is switched on then each tim
169. er Marker using the Line arrow but tons Info The line thickness setting only has an effect if you have selected line as the style e Select the required color for the Laser Marker from the Color list 19 Operation 2 3 Operation All program functions can be accessed using the menu bar various icon bars or using special tools There are additional keyboard shortcuts for sever al functions These are summarized in Appendix B Keyboard Shortcuts 2 3 1 Mouse You will almost always be working with the prima ry mouse button that is the left mouse button in the default configuration If it is necessary to use the secondary mouse button the right button in the default configuration this will be indicated ex plicitly 2 3 2 Context menu Several functions can also be accessed via the con text menu To open the context menu e Right click on any dot in the image 20 2 3 3 Software modes Info This section refers to the software modes of PALM RoboSoftware These are not related to the operating modes Database Mode or File Mode or to the license versions Basic Mode or Pro Mode and are completely indepen dent of these features The PALM RoboSoftware operates in various modes Cursor Mode Stage Mode Freeze Mode Scrolling stage Calibration Laser Point Continu ous Position Laser ON Trap 1 Trap 2 The mode that is currently active is indicated in the Status Bar You can and in fact must s
170. erial number 1214000109 Control Box Vers 1 Connection Type RS232 via CanServer Connection COM1 38400 8E1 NONE Stage Stage llb Capture Device Cap Mover ll UV Laser CryLaS Port 4 IR Laser Quantum IR Beam double Vers CS IV Vers CS IV Microscope Filter Shutter v Export Details Fluorescense Support contact palm info zeiss de Info You can click on the eMail entry and in this way open your e mail program and send an e mail provided you are connected to the net work Info You can create a file with information on your system by clicking on the Export Details but ton You can e g send this file to service at Carl Zeiss MicroImaging if you have questions about your system configuration 22 2 4 4 System information To display a range of information about the system Help PALMAR obo Systeminto e Click on PALMRobo Systeminfo in the Help menu e A window containing information about your system will open PALMRobo Systeminfo K Frame grabber AsiolamkdAc Rew 3 SH 4 Resolution 1344 SEO Color RGB 24 bit Frame rate 8 0 sec Screen rate 8 0 sec Screen size 1358 970 Stage type Stage Ilb Memory usage 39184 Bytes 415109 2614 101 e 09 09 2 4 5 PALMRobo Information The PALM RoboSoftware displays messages in cer tain situations These appear in the PALMRobo Information window PALMRobo Information A Please wait
171. ession with different fluorescence excitation filters As a result a series of different images is available in which different structures in your sample are visi ble These images can be overlaid In this way structures in your sample can be made visible that would hardly be visible using a single fluorescence channel or perhaps would not be visible at all 415109 2614 101 e 09 09 Onecick LPC With this laser function a designated element will be located in the screen center and lifted up by a single laser pulse by one click Thus single cells or sperms are isolated fast and easily simply by clicking on the cell of interest Reference Position trapping laser The reference position for the trapping laser can be defined as required The laser beam can be moved back to the reference position under pro gram control Reference Stage PALM RoboSoftware uses the Reference Position of the stage as the point of origin for calculating the coordinates of your elements With each start of the controller the stage is moved to the end positions in x and y direction This position is defined as a reference point auto matically The Reference Position is retained for as long as the controller unit remains switched on and even if you shut down and restart PALM RoboSoftware RoboLPC With RoboLPC an element is detached and cata pulted into a cap in a single operation only possi ble with membrane mounted samples The drawn
172. etting for each element Plan Neofluar 020 4 Lor Fluar 50 25 Fluar 10x 0 50 LO Plan Meofluar 200 4 Korr LO Plan Meofluar 400 6 Korr LO Plan Meofluar 6350 75 Ko Plan Neofluar 100 30 Oil F e Select the elements to which you want to allo cate a different objective e Openthe drop down list with the objectives and click on the required function Info You can also set a different objective for an in dividual element in the Element properties window Select the element and open the Element properties window using the Edit gt Change menu command 415109 2614 101 e 09 09 Defining wells for the next laser operation 8 6 Defining wells for the next laser operation Info The functions described in the following are only available if your system is equipped with PALM RoboMover Please contact Carl Zeiss MicroImaging GmbH if you wish to upgrade your system For each element you can manually define the well into which it is to be catapulted or you can have the PALM RoboSoftware automatically calculate and implement the distribution of the wells For this purpose first select the elements for which you want to define the distribution in the wells Then select an operating mode and define various parameters The exact procedure and the function of the different operating modes are described in the following Chapter 8 6 3 describes how the PALM RoboSoftware calculates the automatic dis tribution of the element
173. evel as that of your sample With a higher energy setting you cannot just cut within the laser focus but also above or below If the laser focus happens to be in the glass smaller or larger glass segments are catapulted out The catapult function requires a different energy and focus setting to that of the cut function For catapulting the laser beam is extended to an ap propriate value de focused You can select sepa rate values forthe cut and for the catapult function or combine the values for the cut and catapult function When you then set the values for en ergy and focus for a function these values are automatically changed for the other function by an amount that you can set Note that the cutting effect of the laser is not only dependent on the energy and focus settings but also on the speed at which the stage is moved The slower you move the stage with a fixed energy set ting and defined focus the greater is the effect of the laser since it works with a fixed pulse frequen cy This means that there are three parameters to set that reciprocally influence each other Work up to the optimal settings successively Essentially there are two options for ascertaining the optimal laser settings for the cutting function without using the automatic laser functions Enter Stage Mode move the stage with the mouse release the laser with the footswitch refer also to chapter 11 10 and change energy and focus
174. f the selected areas e Activate or deactivate the Show graphics plane checkbox 415109 2614 101 e 09 09 Field of View Analysis 191 Field of View Analysis 192 415109 2614 101 e 09 09 Appendix A Glossary AutoLPC Automatic catapulting of larger areas of samples mounted on glass With glass samples only a lim ited amount of cell material per laser pulse can be catapulted out with one laser shot Larger areas have to be catapulted with multiple laser pulses The distance between the individual laser pulses on the cut is dependent on the properties of the tissue and is pre selected in the Draw Preferenc es window LPC Distances tab or in the Prefer ences and Configuration window Laser tab Moving trap movement of the trapping laser Using the Laser Beam Positioning function on the trapping laser you can move trapped objects in both the x and y axes ie parallel to the plane of the slide Center The center point of the microscope image dis played on the monitor It may be marked with crosshairs The crosshairs can be displayed or hid den with the Screen Center command in the View menu Center RoboLPC Cuts an element of the freehand line rectangle or circle type and catapults the entire area to the cen ter of the element with a laser pulse CapCheck A position of the stage If you move to the CapCheck the circular opening of the stage is po sitioned within the beam path of the micr
175. ferences and Configuration window Saving Elements Folder C Documents and Settings AdministratorkPy Save Elementtiles automatically e Click on the checkbox Save Element files automatically option in the Saving Elements section to enable or disable this function 107 Saving and loading elements in File Mode With the function enabled the elements are saved in a file when the program is closed If you have already saved your current elements and made changes to them since the last save e g drawn new elements or deleted existing elements the element file is automatically saved using the name already defined If you have not yet saved your current elements a new file is created e Close the Preferences and Configuration win dow by clicking on OK 108 9 2 Saving and loading elements in File Mode Info In the Element List window you can specify that only elements of selected types are to be displayed in the window ie elements of other types are not included in the table in the Element List refer to chapter 8 1 This has no effect on the Save operation all existing el ements will be saved under all circumstances To save your elements in a file In PALM RoboSoftware main window or in the Element List window e Click on the menu item Save Elements in the File menu The Save elements to file window opens The exact appearance of this window depends on which slideholder yo
176. for testing 1 5 2 Conventions To help you find your way around the text it has been organized into distinctive sections Indented paragraphs with a dash provide a swift overview of various functions or proper ties of PALM RoboSoftware e Indented and bulleted paragraphs provide instructions for executing certain functions Menu names menu commands and the names of icon buttons and windows are set in inverted com mas In addition you will always be referred to the menu or bar where the command or button is lo cated Special markers in the left margin indicate text that you must not fail to read Caution This indicates a potentially dangerous situa tion You must observe the precautions marked like this to avoid injury or damage 415109 2614 101 e 09 09 About this user manual Note This indicates important information Read this information to maintain your system s high level of performance and safety and to prevent malfunction or damage to your sam ple Info This indicates tips on correct use and offers useful notes for getting the best use of PALM RoboSoftware 1 6 Installation PALM RoboSoftware is already installed on the computer upon delivery or initial operation of your system If you experience problems or if you want to work with another computer please contact Carl Zeiss MicroImaging GmbH 1 7 File Mode and Database Mode PALM RoboSoftware recognizes two operating modes Fil
177. function to elements LL 94 Allocating an objective to elements 0 0 cee ee es 94 Defining wells for the next laser operation LL 95 Denning Wels Manuals rindo par pei usi Grae esa A asi dead 95 Setting automatic distribution of the elements to WellS 0 ooooo oo 96 Automatic calculation of the distribution of the elements o oo ooooooooo 99 The serial Sections function ia esa ei SiS da E Ga eR GS 100 Saving and printing elementS 00 coooooooooo oo uu 107 Settings for saving elements in File Mode lt lt ee es 107 Saving and loading elements in File Mode 0 o o ooo ooo ooo 108 Saving and loading elements in Database Mode om 110 Exporting and importing elements suina A ee a li 111 PANUNG CST CUS marea ds a AA eee dedi SOS 112 SAVE IMAGES us sines DE AA a ee ae 113 Insert Copyright Note sai dae ia ios 113 Specify folder to store iMageSin ooooooconcon aaa 114 Saving images Manually diia da EA A AAA a 115 Saving images in FIS MOS ir dd ed ia a 115 Saving images in Database Mode 0 ee ee rr 116 Saving images automatically during a laser function 0 ooo ooo oo 116 Saving images and videos using the Recorder function o o ooo 117 Cutting and CatapultiNd sa sas ssa a aaa RI 121 Laser UNCU S a io A AAA E DE ae Len AS 121 Automatic laser UNCU ONS dig we dA a a eee q SC E 4 121 Laser tunction ONE
178. functions Configuration Advanced the simultaneous application of fluorescence and laser functions is possible For more information about the application and the basic settings refer to the User Manual Along with the standard fluorescence illumination fluorescence illumination using light emitting diodes LED is also available For more detailed information refer to chapter 14 12 14 1 General introductory remarks The procedure to use for working with flu orescence illumination depends on the way your system is configured The differences concern the manipulation of the microscope motorized or manual version and the fluorescence shutter or filter wheel respectively If your system is equipped with a motorized micro scope you can control all the elements that you need to adjust specifically in order to use the fluo rescence illumination via PALM RoboSoftware When using a manual microscope you have to make the settings on the microscope manually the functions for the filter wheel and the shutter are controlled by PALM RoboSoftware Ie you can select the desired excitation filter or close and open the shutter via a program command 415109 2614 101 e 09 09 14 2 Settings for working with flu orescence manual microscope To use the fluorescence illumination you must make the following settings switch on the fluorescence illumination refer to User Manual select the desired fluorescence excitati
179. ge is pre vented After editing the freezed image you can exit the Freeze Mode to perform a laser function or to observe other areas with fluorescence Note Before switching on a laser function a position with a beam splitter suitably coated for this wavelength must be swiveled into the reflector revolver of the microscope For systems with the fluorescence configuration Basic this is the first position only This position is adopted automatically on motorized microscrope sys tems 415109 2614 101 e 09 09 Opening and closing the shutter 14 6 Opening and closing the shutter Fluorescence adjustments Fluorescence Fluorescence Filter Reflector Fluorescence Shutter Information and Control Shutter Type fi ud ee Shutter Ena Actual state CLOSED O o You can open and close the shutter via a menu command or an icon button However you can use the timer function chapter 14 11 to preselect a time after which the shutter is to be closed again automatically after you have opened it Note In the Basic configuration with the manual microscope the function Shutter is not ac tive In this case the shutter has to be oper ated manually at the microscope stative To open and close the shutter on the filter wheel via icon buttons D Open Shutter shutter is currently closed O Close Shutter shutter is currently open Info There is only one button for opening and clos ing the shutter Its appea
180. gonal AutoLPC Center shot 1001 Distance of RoboLPC joint 415109 2614 101 e 09 09 The rest of the procedure is the same in both win dows e Select the magnification for which you want to make the settings from the for Objective list e Enterthe required value for the dot distance in um in the Distance of AutoLPC shots field e Enter the required um value for the distance of the points from the border of the elements in the field Distance of AutoLPC shots from line e Activate the AutoLPC shots diagonal check box to change the arrangement of the dots A tick in this box indicates that the dots will be ar ranged diagonally If there is no tick in the box the dots will be arranged vertically or horizontally e Click in the box AutoLPC Center shot if a laser pulse should be set to the center of the element 129 Finding suitable laser settings 11 4 6 Special laser settings for the RoboLPC function Note These laser functions only make sense with membrane mounted samples If you release the RoboLPC function an area el ement will be cut out but a small RoboLPC joint will be established between the start and end dots of the cut line A laser pulse on this RoboLPC joint will catapult the area contents into the cap This function requires a special setting establish the length of the RoboLPC joint remaining after cutting To establish the length of the remaining joint for RoboLPC You can
181. guration Distribution calculation Operating Mode spread elements evenly J iv Number of selected well s Number of used well s Number of elements to process Start with Field Number of elements per well until Field Remaining elements Cancel e Selectthe co ordinates of the first and last well into which the elements are to be catapulted on the Start with Field and until Field drop down lists Info The order of the wells is defined by the setting in the Subtype Configuration field in the RoboMover window line by line or column by column refer to chapter 12 2 2 Info Select the number of wells such that the num ber of elements to be catapulted is an integer multiple of the number of wells Put another way the number of elements to be catapulted must be divisible by the number of wells in which they are to be catapulted without a re mainder If there is remainder from this divi sion the elements cannot be evenly distributed over the wells In this case the number of elements catapulted is limited to the number that ensures the same number are catapulted into each well The remaining elements are not catapulted refer to chapter 3 6 3 Example if you have defined 10 wells and 54 elements are to be catapulted 5 elements will be catapulted into each well The remaining 4 elements are not catapulted 415109 2614 101 e 09 09 Operating Mode sequence 1 1 X 1 2X
182. gure The element types Line Freehand Rectangle and Circle are known collectively as element type Figure JointCut As with function Close amp Cut the laser cuts the sam ple along defined lines Start and end dot of an open path figure are connected by a straight line It remains a joint between start and end dot With this function figures can be cut The cut area re mains fixed within the tissue The entire area can be catapulted later by a single laser pulse on this joint Laser Marker There is a Laser Marker for each laser beam Its tip should display the point where the laser beam meets the sample when released For a fixed laser beam such as the cutting laser and the trapping la ser beam without Laser Beam Positioning this is roughly the middle of the monitor for a beam that is movable this point may be located elsewhere PALM RoboSoftware calculates the cutting func tions of the cutting laser from the position of the Laser Marker The position of the Laser Marker should therefore always match that of actual laser focus Only then the correct results can be achieved with the automatic laser functions Like wise using the trapping laser you can only capture objects without difficulties and if your system is appropriately configured move precisely using the adjustment if the position of your Laser Marker is correctly set You should therefore check the posi tion of the Laser Marker before you start work
183. h a ocular micrometer Correct determination of lengths and areas requires that your system is correctly calibrat ed If in doubt refer to chapter 4 4 To measure the length of lines Ruler IL Quadratic Attribute e Click on the Ruler icon button in the Graphic Toolbar If you select Quadratic Attribute as well the drawing function is limited to horizontal and vertical lines e Click on the start dot of the line whose length you want to determine and keep the mouse button depressed e With the mouse button depressed move the cursor to the end dot of the line to be measured and release the mouse button 90 To show and hide area sizes Element areas e Click on the Areas icon button in the Graphic Toolbar Area sizes of all drawn Figure type elements will be shown in um At the same time the areas measured will be displayed with a transparent col or marking e To hide the area display click again on the Areas icon button in the Graphic Toolbar Note If elements are not drawn closed the start and end points of the elements are connected by a straight line for calculating the area Info Only the area of elements of type Figure line freehand rectangle circle is indicated The displayed size of Dot type elements has no relation to the diameter of the laser beam or the size of the catapulted area the display of an area size for these elements therefore has no practical use
184. hapter 7 2 Saving elements and loading saved elements File Mode refer to chapter 9 2 Exporting and importing the elements refer to chapter 9 4 Printing of elements to a file or on a printer refer to chapter 9 5 Selecting and deselecting elements refer to chapter 7 6 3 Changing properties of elements refer to chap ter 7 8 5 Adding a name or a comment to ele ments and 7 9 3 Change colors of elements that have already been drawn Deleting individual elements or all elements if you are working in Database Mode it is only possible to delete elements that have not yet been processed using the laser refer to chapter 7 7 Updating element numbering refer to chapter 7 3 4 415109 2614 101 e 09 09 Functions in the Element List window Copying elements refer to chapter 7 8 2 Forming and editing groups of elements refer to chapter 7 10 Transferring elements from one sample to an other sample refer to chapter 8 7 Centering an element on the monitor refer to chapter 7 5 Selecting the laser function for an element refer to chapter 8 4 Selecting the objective with which the element is to be processed using the laser refer to chapter 8 5 Selecting elements that are to be processed us ing the laser refer to chapter 11 6 Starting the laser refer to chapter 11 7 Stopping a running laser function refer to chapter 11 9 Defining operating mode for PALM RoboMover and
185. hat you want to rename in the Stored hardware settings field e Click on the Rename button and enter a new name To activate a hardware configuration icf Apply e Select the required configuration in the Stored hardware settings field e Click on the Apply button 173 Hardware configurations 15 5 2 Editing the list of hardware configurations The Hardware setting adjustments window will Assign HW Settings e Click on the Assign HW settings button in the Microscope Tools on the HW Settings tab gt Hardware setting adjustments Hardware Settings Configue the Hardware Setting Buttons Mo Button description Hardware setting name 1 Binocular 3 EDok 2 new Select an Entro and DoubleClick to Change C Documents and Settings 4 dministrator my Documents Carl Zeis 2 JEDI C Documents and Settings Administrator Documents Carl Zeis C Documents and Settings dministrator bmp Documents Carl Zeis open To add an additional configuration or replace a configuration with another configuration Insert If you want to add a further hardware configura tion e Inthe table double click in the row with the entry new or click once in the row with the new entry and then click on the Change button or Click on the Insert button Change Button Assignment Hardware Setting Button Assignment dr rr Di Daten Eigene Dateien Tom Carl ZeissiData Setting S
186. have to draw a reference element on a point of your sample that is to be seen with both types of illumination To adjust the Reflector Offset e Draw the required elements with fluorescence illumination Draw at least one element on a point that is to be seen with normal illumina tion also Suitable for this is any type of ele ment for example Freehand or Dot refer to chapter 7 3 5 The Freehand Tool or chapter 7 3 10 The Dot Tool This element serves as a reference for the Reflector Offset e Switch off the reflector and the fluorescence illumination e Move the stage so that the reference element and the corresponding point on your sample is displayed on the monitor R Pointer Tool e Select the Pointer Tool in the Graphic Toolbar Adjustments Calibration k New Aeflector Offset e Click on the menu item New Reflector Offset in the Adjustments gt Calibration menu or click on the Calibrate button in the Fluores cence adjustments window Reflector tab refer to chapter 14 8 164 All existing elements are selected automatically and enclosed in a selection rectangle white line e Click on any dot within the selection rectangle and hold the mouse button down Info If you have drawn elements that are currently positioned outside of the monitor section some or all of the borderlines of the selection rectan gle will also be outside the monitor section In this case the border lines are no
187. he O Centric Attribute Use the Circle Tool to draw circles or ellipses The dot at which you begin your drawing becomes a corner of a perceived rectangle enclosing the circle or the ellipse morphology is retained i e their arrangement in e Select the Circle Tool in the Graphic Toolbar the wells is exactly the same as the arrangement If you select Quadratic Attribute as well the in the sample drawing function is limited to circles e Click on the arrow beside the tool If you select Centric Attribute as well the dot e Click on the Configure menu entry at which you begin your drawing becomes the center point of the ellipse or the circle e Move the cursor to the dot where you want to begin your drawing e With the mouse button pressed down drag the oval or circle out until it is the desired size and then release the mouse button The Morphologic grid configuration window opens Morphologic grid configuration Number of columns Number of rows Orientation E OK e Enter the number of rows and columns into which the rectangle to be drawn is to be divided e Select the required numbering sequence for the elements produced row by row or column by column in the Orientation list e Close the window e Draw the rectangle 74 415109 2614 101 e 09 09 Drawing new elements 7 3 10 The Dot Tool Dot Tool Use the Dot Tool to mark for example individual cells that are to be catapulted w
188. he Home icon button 141 Diffusor option 12 3 Diffusor option Some collectors e g the CapturePlate Collector 96 have an integrated diffusor to improve the image quality You will find more information on this topic in the product information supplied with the collectors If the collector fitted has a diffusor this is shown in the illustration in the RoboMover or CapMover II window In addition the Tool Bar contains a correspon ding icon button Diffusor e To use the diffusor Click on the diffusor in the image or Click on the Diffusor icon button You can now pre select your samples 10 1i Pa MS 000 ee Si E 0 o EO 0 00000000 F0 000000000 60 0000002000 HO 000000000 Diffusor Diffusor SingleTube SOOCM Info You can also use the diffusor if you do not want to catapult In this case it is sufficient to fit the collector on its own i e without insert into PALM RoboMover or PALM CapMover II 142 12 4 Settings 12 4 1 Setting the movement speed Info As a rule it is not necessary to change the movement speed However if you are wor king with large amounts of liquid it is recom mended to reduce the speed The maximum possible speed is pre set by the manufacturer e Select the Adjust tab in the RoboMover or CapMover II window Adjust Speed Control Position Speed um sec ser Piceni 100000 In the Speed Control
189. he key If you press two arrow keys simultaneously eg 15 you can also move the laser beam diagonally 13 11 4 Moving the trapping laser beam with the joystick Note that the joystick must be activated to be able to use it refer to chapter 3 4 You can activate the control of the laser beams us ing the joystick in the same way as using the arrow keys refer to chapter 13 11 3 or using the but tons on the joystick Using the bottom left button you can activate and deactivate the control of the trapping laser beam 1 Using the top right button you can activate and deactivate the control of the trapping laser beam 2 If the control of both trapping laser beams is activated you will move the two beams simul taneously and in parallel The speed of the movement is dependent on the deflection of the joystick The speed you have set for the TrapXYArrow function refer to chapter 5 3 is also the maximum speed for move ments using the joystick 415109 2614 101 e 09 09 13 12 Moving objects along defined paths Using PALM MicroTweezers you can transport trapped objects along a defined path You can use this function e g to collect several objects at one point The path is defined by an element As soon as you trigger the function the trapping laser beam is moved to the start of the element and moved along the element to the end of the element You can deposit the object there by switching off the la
190. he posi tion and shape of elements automatically You can also reload the most recently saved ele ments using the list of most recently used element files on the File menu if you haven t saved any elements this list will be empty File 1 testslide palm on Position 2 testalide palm on Position 1 3 S32 4yE lements palm 4 193440E lements palm 109 Saving and loading elements in Database Mode e Inthe File menu click on the name of the ele ment file listed there which you wish to reload Info If you have used a slideholder that can accommodate more than one slide then the position occupied by the slide when the ele ments were saved is shown to the right of the file name However you can also load the el ements file for a different position The loaded elements will appear on your monitor and the name of the elements file will be displayed in the title bar of the program window Any ele ments that were already on the monitor will be de leted Note Take particular care to insert your sample care fully and correctly If the slide is canted or twisted the loaded elements will not match to the corresponding structures of your sample 110 9 3 Saving and loading elements in Database Mode In Database Mode you do not need to save your elements manually every drawn element is saved immediately and automatically in the database To load elements from the database File S Enter Select Data
191. her you draw elements with the freehand tool or set reference elements with the reference point tool If you have drawn elements you will create a ref erence element in the center of the elements using the Define Group Reference Figure command This point is omitted if you work with the reference point tool refer to the following description It is advisable to use the freehand tool to draw el ements The subsequently necessary allocation of the other samples to the respective markings will then as a rule be more exact This procedure is described in the following e Position the stage so that one of the three markings in your sample is displayed on the monitor e g with the help of the PALM Navigator e Select the desired drawing tool the Freehand Tool is recommended and draw an element to the shape of your marking e Position the stage on the second marking and draw an element here also to the shape of this marking e Proceed likewise with the third marking e Open the Element List window Your three elements are now listed there e Select the three elements Edit Create Group e Inthe Edit menu click on the menu item Create Group The three elements are joined together to form a group The group appears as a fourth element in the Element List window Allocate a name to the group e g Slide 1 e Double click on the element group The Element Properties window will open e Type a name
192. hich you later want to reload into PALM RoboSoftware refer to chapter 9 2 In Database Mode you do not need to save the elements explicitly they are saved automati cally in the database and can be reloaded from there refer to chapter 9 3 Info In the Element List window you can specify that only elements of selected types are to be displayed in the window ie elements of other types are not included in the table in the Element List refer to chapter 8 This has no effect on the Export operation all existing elements will be exported under all circum stances Info If you are using a slideholder that can accom modate more than one slide all the existing elements are saved in one file irrespective of which sample you have drawn them for To export elements File E Export Elements e Click on Export Elements on the File menu in PALM RoboSoftware main window or in the Element List window The Export Elements window will open e Selecta folder to store your elements as neces sary e If you have saved your elements previously the name of the file in which they are saved is offered by default If you want to overwrite this file do not change the name in the input field Or enter a new name for your element file e Click on the Save button You can now open the file with a text editor and read it print it or edit and save it again 415109 2614 101 e 09 09 Exporting and imp
193. hutter zu zvhs Button Text Hit setting 174 Info An additional configuration is added at the end of the list above the new row An additional configuration is inserted above a marked configuration using the Insert but ton You can change the order of the configurations as explained below If you want to replace a configuration with a differ ent configuration Double click on the row with the related config uration or click on the row with the related configuration once and then click on the Change button 415109 2614 101 e 09 09 Hardware configurations To delete a configuration from the list e Clickon the row with the configuration you want to delete in the table in the Hardware setting adjustments window refer to page 174 e Click on Delete Info Only the entry in the list is deleted not the configuration itself To change the order of the configurations in the list El e Click on the row with the configuration you want to move up or down in the table in the Hardware setting adjustments window refer to page 174 e Click on the Up or Down button depending on the direction in which you want to move the configuration The configuration is moved all configurations are re numbered sequentially 415109 2614 101 e 09 09 175 176 415109 2614 101 e 09 09 Multi channel fluorescence recordings 16 Multi channel fluorescence extended focus and time laps
194. ich you can enter the time in milliseconds e Click on one of the buttons shown below to start the slideshow iJ DI Db 1 2 3 1 Display from last image to first 2 Display from first image to last 3 Display from first image to last and then from last image to first The slideshow automatically starts over as soon as all images have been shown e Click on the Stop button to end the slideshow 415109 2614 101 e 09 09 17 Incubation option You can add an incubator to your system The in cubator is controlled using the program AxioVision The PALM RoboSoftware has an inter face to this program This chapter describes how you can make the set tings for controlling the incubator You will find a description of the parameters to be set in the AxioVision online help The settings for the incubator parameters are logged in a file 17 1 Making settings for the logging A Configuration Start Logging View Log file e Open the menu for the Incubation icon but ton e Click on the Configuration menu entry The Incubation parameter logging configuration window opens Incubation parameter logging configuration Configuration Elements Images Laser Appearance Microscope Setup Incubation Time Lapse Logging File location nents and Settings dminiztratorMy Documents My PalmD ata cad O Event controlled logging Time controlled logging Info The logging settings are also available in the
195. icroscope lamp to 3200 K 32006 e Click on the 3200K button The microscope lamp is automatically adjusted to a color temperature of 3200 K This is the default value for a halogen lamp and will produce a micro scope image with a coloration that is closest to the image in the microscope s ocular To open and close the shutter transmitted light shutter optional B Open Shutter shutter is currently closed O Close Shutter shutter is currently open e Click on the Shutter button to open or close the shutter 169 Making pre settings for the microscope 15 2 Making pre settings for the microscope You can specify a range of settings that will be made automatically on the microscope when PALM RoboSoftware is started and quit Reflector Condenser Filter Shutter Lamp Baseport and Sideport Adjustments PALMA obo e In the Adjustment menu click on the menu item PALMRobo The Preferences and Configuration window opens e Select the Microscope Setup tab Preferences and Configuration Configuration Elements Images Laser Appearance Microscope Setup Incubation Time Lapse Define the Microscope Settings for program Startup Shutdown dontcare e dont care e dontcare e e Shutter Reflected don t care se don t care Reflector don t care Condenser don t care Filter don t care Shutter Transmitted don t care
196. idual images in the picture viewer see chapter 16 5 415109 2614 101 e 09 09 To save your images and display them on the monitor amp Save z shack as 2vi image e Click on the Save z stack as zvi image option in the Extended focus menu if you want to save the images save the image in jpg bmp or tif format as described in chapter 10 The images are saved in a file as a multi dimensional image Note The image with extended focus is not a live image of your sample PALM RoboSoftware therefore automatically switches to Freeze Mode as soon as you start an experiment You can exit Freeze Mode at any time to see the live image from the camera on the moni tor see chapter 14 5 Using the commands described here you can return to the image with extended focus but only if you have not moved the stage in the meantime As soon as the stage is moved the image with extended focus is lost Extended Focus Image e Click on Extended Focus Image to display the calculated image on the monitor again 415109 2614 101 e 09 09 Time Lapse 16 3 Time Lapse You can combine multi channel fluorescence recordings and images with extended focus with a time lapse function e the recordings are repeated at defined time intervals e Click on the Create modify multi channel experiments option in the Multi channel fluorescence menu The Multidimensional Acquisition window opens e Select the T tab gt Mu
197. ight source white light Select the external light source if you need an excitation wavelength that is not available from the LEDs e Click on the LEDs button or the External button depending on which exposure type you require 415109 2614 101 e 09 09 The Microscope Tools 15 Control Microscope Zeiss Axio Observer If you have purchased a system with a motorized microscope you can also control settings via PALM RoboSoftware You can specify that certain set tings on the microscope are made automatically when the PALM RoboSoftware is started and quit To make settings while you are working you can use the Microscope Tools in the main window as well as the Microscope window You can open it by menu command or via an icon button The Microscope Tools enable you to make the most frequently used settings the Microscope window offers additional setting options If you are using an ApoTome on your microscope its current status is displayed on the monitor 15 1 The Microscope Tools Microscope Common Reflected Light Hw Settings Objective Focus Focus Light AF active 1000C um 100 an lie You can make the most frequently used settings directly in the main window on the Common tab in the Microscope Tools 415109 2614 101 e 09 09 15 1 1 Setting required magnification Microscope Common Reflected Light Hw Settings Objective olalol la e Click on the button for the objective r
198. ile Y Load elements from file Read troubleshooting e Click on the function about which you want more detailed information The PALMRobo Help window opens the required help topic is displayed The PALMRobo Instructor window stays open If you arrange both windows side by side on your monitor you can select several or all topics in the Instructor one after the other and have them dis played in the Help window To continue working with PALM RoboSoftware you must close the PALMRobo Instructor window again You can leave the PALMRobo Help window open 21 Help and information about PALM RoboSoftware 2 4 3 Program license and manufacturer To view information about the program your license and the manufacturer Help 9 About PALMRobo e Click on About PALMRobo in the Help menu A window with information on your system opens hardware software components licensed and in stalled license information of the dongle About PALMRobo Carl Zeiss Microlmaging PALM CombiSystem zess Release 4 3 SP License Pro Mode including Auto Focus Recorder FOV Analyze Database LMPC Module Extended Focus Multi channel Fluorescense VisDat Force Measurement Copyright Carl Zeiss Microlmaging GmbH 2009 Version and Configuration Information Software l PALMAR obo 4312 MTB 1 6 0 5 Local DLL 1 6 0 5 AxioVision 4 8 0 0 Control Box 1 169 140C T Hardware S
199. ill be a large hole in your sample In this case check that there is nothing obstructing the laser beam in the microscope s laser light path or conduct preliminary tests to determine the op timum settings for laser energy and focus For instructions on how to do this refer to chapter 11 5 To determine the focus point of the trapping laser e Inserta slide that has been colored with a black felt pen e Switch the trapping laser on briefly toggle the On Off button in the right border of the pro gram window to switch the laser on and off Check the result You should see a small almost circular area where color has been removed Info In some circumstances you will not see any ef fect from laser activation or there will be a large hole in your sample In this case check that there is nothing obstructing the laser beam in the microscope s laser light path or try again with different settings for laser power and focus refer to chapter 13 2 415109 2614 101 e 09 09 Calibrating the movement of the stage To set the position of a Laser Marker Laser Position Laser Marker P ego Cut Laser 1 i ae Trap 1 Lazer i Trap 2 Laser e Inthe Laser menu click on the menu item Position Laser Marker e Click on the name of the Laser Marker whose position you want to change e Click in the center of the laser focus for that laser beam Note Be sure to assign the Laser Markers to the cor rect laser be
200. in the Motion menu The stage is moved to the Load Position The PALMRobo Select Holder window will open PALMRobo Select Holder The loadposition is reached Now please insert the slideholder and select the correct type To Stage Holder SlideHolder 3x e y Return to the working area e Change the slideholder and or the slide and choose the corresponding entry from the Holder field Note To avoid damage to the objective or to the sample Move the stage back again to the original position after changing the slide and the slideholder For this proceed solely as described below 52 To return the stage to its original position Return to the working area e If you do not have a motorized microscope Move the objective down as far as possible e Click on Return to the working area in the PALMRobo Select Holder window 5 7 Centering the stage You can move the stage to the center point of the area in which it can be moved using a menu com mand Motion Goto Center e Drag the cursor on item Goto Center in the Motion menu e Click on the menu item Stage The stage is centered 415109 2614 101 e 09 09 6 PALM Navigator The PALM Navigator will provide you with an over view of your sample or a section of your choice First of all scan your sample A picture of the sam ple is then displayed in the PALM Navigator win dow By clicking at any point in scanned image you can
201. indow in the Arrow keys Joystick mode area Info If the stage is at the CapCheck refer to chapter 5 5 the button will not be available In this case you will not move the stage using the arrow keys but your capture device e Pressoneofthe four arrow keys The stage will continue to move until you release the key If you press two arrow keys simultaneously eg 15 you can also move the stage diagonally 5 1 5 Positioning the stage with the Joystick You can move the stage in any direction using the joystick Note that the joystick must be activated to be able to use it refer to chapter 3 4 Arrow keys Joystick mode o de o a o wt e Click on the button at the top right edge of the program window in the Arrow keys Joystick mode area Info If the stage is at the CapCheck refer to chapter 5 5 the button will not be available In this case you will not move the stage using the joystick but your capture device The speed of the movement is dependent on the deflection of the joystick The speed you have set for the Scroll function refer to chapter 5 3 is also the maximum speed for movements using the joystick 415109 2614 101 e 09 09 5 1 6 Positioning the stage with the Centering Tool The Centering Tool can be used to shift any point on the microscope image into the center Arrow keps Joystick mode o O e o ill e Click on the top button at the right edge of the program w
202. indow in the Arrow keys Joystick mode area Info If the stage is at the CapCheck refer to chapter 5 5 the button will not be available D Center e Click on the Centering Tool in the graphics tool bar e Click on the point in the image that is to be moved to the center The Centering Tool will continue as the active tool until you click on any other tool on the graphics toolbar Note Do not confuse the Centering Tool with the Onedick LPC tool The One LPC tool also moves the point you click on to the center However it also simultaneously triggers a laser pulse refer to chapter 11 10 415109 2614 101 e 09 09 Movement types 5 1 7 Moving the stage during an auto matic cutting laser function As soon as you start the cutting laser the stage is automatically moved along the defined cut lines or from point to point You do not have to select any particular type of movement in this case 5 1 8 Further possibilities for positioning the stage Aside from the mentioned possibilities you can move or precisely position the stage using the fol lowing methods Centering elements The stage is so positioned that a previously selected element will be dis played in the center of the monitor refer to chapter 7 5 To move to the Load Position refer to chapter 5 6 When the stage is in the Load Position you can easily change the slide and the slideholder To move to the CapCheck refer to chap
203. ing on one of the buttons refer to chapter 14 4 Adjustments Fluorescence e Inthe Setup menu click on the menu item Fluorescence This opens the Fluorescence window The Flu orescence tab is displayed refer to the figure at the top of this page 162 Se ee ae Laser Light es default ON Tes OFF y OFF OFF To create a new set of settings Insert e Inthe table double click in the row with the entry new or click once in the row with the new entry and then click on the Change button or click on the Insert button Info A new entry is always added at the end of the list over the new row If you have already created entries you can select one of the exist ing entries with a click of the mouse If you then create a new record using the Insert button a new record will be inserted above the marked record You can change the order of the records as will be explained later This opens the Fluorescence Entry window When you create a new settings record every field is populated by default with don t care This set ting means that no change will be made to the cur rent setting for the respective part when you activate the settings record as described in chapter 14 4 e Enter any name for your settings record in the Name field 415109 2614 101 e 09 09 Editing a fluorescence settings record e Open the drop down lists Filter Shutter Reflec
204. ings button in the Display Tools Camera tab Live Image Camera selection Measurement tools Display AsioCamMAS Framegrabber mono RGB horizontal vertical flip Sideport 100 A e Exposure mz 1 60000 415109 2614 101 e 09 09 Setting camera parameters This opens the Live Image window You will find the settings for the camera on the Camera selec tion and in this example AxioCamMR3 tabs e Select the camera required in the Framegrabber drop down list on the Camera selection tab For some cameras e g for the AxioCam MR3 you can select whether you want to display a black and white image mono or a color image RGB e Click the mono or RGB radio button e Select the beam path required from the Side port list e In the Exposure section set the required exposure time Info You can also set the exposure time on the AxioCamMR3 tab and in the Display Tools You can also set the exposure time automat ically there To make further camera settings Info The following example is based on the AxioCam MR3 The settings for other cameras may vary from those described Advanced Settings e Click on the Advanced Settings button in the Display Tools Camera tab This opens the Live Image window e Click on the AxioCamMR3 tab 415109 2614 101 e 09 09 e Click on the Adjust tab Camera selection Measurement tools Display AxiolamMA3 A
205. ion The automatic laser functions will then not process ex actly the areas you wish You must compensate for this difference by setting the Objective Offset Note These differences between the objectives are adjusted for the objective supplied by a factory default setting for the Objective Offset You only need to adjust the Objective Offset if the elements are moved when altering the magnification To adjust the Objective Offset e Select the Cut laser function refer to chapter 7 3 1 e Drawan arbitrary cut line at the magnification currently set e g using the Freehand Tool e Trigger the cutting laser refer to chapter 11 7 e Checkthe cut line It should be exactly aligned with the cut line drawn If not adjust the Laser Marker as described in chapter 4 3 and restart from the beginning e Choose the desired magnification R Pointer Tool e Select the Pointer Tool in the Graphic Toolbar Adjustments Calibration k New Objective Offzet e Click on the menu item New Reflector Offset in the Adjustments gt Calibration menu All available elements are automatically selected and surrounded by a selection rectangle the se lection rectangle is indicated by a white line Info If you have drawn elements that are currently positioned outside of the monitor section some or all of the borderlines of the selection rectan gle will also be outside the monitor section In this case the border lines are not vi
206. ith the AutoLPC automatic laser function Info The Dot Tool only appears on the Tool Bar if you have enabled the AutoLPC laser functions in the configuration refer to chapter 3 5 e Select the Dot Tool in the Graphic Toolbar e Click roughly in the middle of the cell you later want to catapult The selected location is displayed as a dot 7 3 11 The Stamp Tool 2 Stamp Tool Select new template Using the Stamp Tool you can copy existing ele ments and place the copy in the required position with a mouse click e Click on the arrow beside the tool e Click on the Select new template menu entry e Click on the element you want to copy The mouse pointer changes to a hand e Click on the position in your microscope image where you want to place the copy The copy is positioned such that the center of the element is at the position clicked on If you use the Center RoboLPC laser function for the element refer to chapter 11 1 the laser catapult shot is placed at the position clicked on You can continue placing copies of the selected el ement until you select another element or another drawing tool 415109 2614 101 e 09 09 7 3 12 The Text Tool e Text Flag Use the Text Tool to add markings with or without textual comments to your image The marked sites are displayed as flags the comments in a rectan gle Comments are always displayed on one line You can select or subsequently change the color
207. k Settings Slices o To generate a recording with extended focus Z stack Click on the z stack icon button Slice distance 3 000 pm The recording is started The Multidimensional Acquisition Preview window opens showing the Mode e Center Start Stop progress of the recording see chapter 16 4 As soon as the recording sequence is complete E the sharp image calculated from the individual 6962 376 um images appears on the monitor PALM um RoboSoftware automatically switches to Freeze Z Stack height 6 000 pm Mode _ all Channels per Slice Additional options appear in the Extended focus Le menu which you can use to call up the following Z Stack navigation functions lili Step Go Position A 6965 376 pm Index a Settings before after 2 Stack before 2 stack after Z stack e Make your settings refer to AxioVision online help Selecting an experiment Ed Select 2 stack experiment for Extended focus e Click on the Select z stack experiment for Extended focus option in the Extended focus menu A window opens with a list of the existing experi ments see related illustration in chapter 16 1 e Select the experiment you want 180 Save the recording as a multi dimensional image Display the calculated sharp image in the PALM RoboSoftware main window if you have exited Freeze Mode in the meantime Display indiv
208. l requirements Please contact Carl Zeiss MicroImaging GmbH if you wish to upgrade your system Info If the camera image does not appear when the program is launched check that all connections to the microscope and also the settings on the microscope have been made correctly A YO Depending on the activated laser in the area of the camera image you will see various markings and information chapter 2 2 describes how you can activate and deactivate or change the items shown Darkred crosshairs these mark the exact cen ter of the image A solid blue triangle the Cutting Laser Marker a red Y shape marking as well as if you pur chased the DuoFlex TwinFlex system a green square the Trapping Laser Marker The Laser Markers indicate the target for the laser You will see a dotted line rectangle on the bor der of the image this is the Scroll Rectangle At the bottom on the left a scale bar and Copyright Note if specified are displayed refer to chapter 10 1 The Status Bar at the bottom of the program win dow provides you with information on a number of current settings Some of the fields feature a win dow that opens when the field is double clicked For Help press Fi If you double click on the field the PALMRobo Information window will open refer to chapter 2 4 5 sl si Thereis an icon in the second and or third field if control of the trapping laser beams with the joystick is
209. l the settings you have made To save settings you can specify a folder When you save your settings later this folder will be selected automatically However you do not have to accept this specification You can also save your settings later in another folder You can also create a new folder when saving specify whether the settings should be saved automatically when the program is closed To specify a folder for saving the settings Adjustments PALMA obo e In the Adjustment menu click on the menu item PALMRobo The Preferences and Configuration window opens The Configuration tab will be displayed Configuration Saving Setting Files Folder C Documents and Settings AdministratorM y cod The name of the currently set folder appears in the Folder field of the Saving Setting Files section e Click on the button ed The Select new setting folder window opens A directory tree structure is displayed in the window and the currently set folder appears underneath in the Folder field e Select the desired folder and click on OK to close the window 29 Restoring settings To save your settings automatically when exiting the program Info PALM RoboSoftware essentially saves the set tings which are valid on exiting the program These settings are automatically loaded at the next start and remain valid until you change them or load a previously saved set tings fi
210. layed on the monitor with the elements on it in a file for documentation purposes Elements stored in this way cannot be reloaded into PALM RoboSoftware For instructions on how to do this refer to chapter 10 3 Saving images manually and chap ter 10 4 Saving images automatically during a la ser function In order to draw elements you must display the section of your sample on the monitor that you want to process Refer to chapters 5 or 6 9 for in structions on how to move the stage in order to display the required section of your sample on the monitor However you can also draw elements that extend beyond the section displayed on the monitor Chapter 7 3 3 includes instructions on the factors you must consider for this When processing elements you will often be work ing with the Element List window This window provides a summary of your elements and their properties and allows you to make changes to them and to perform various program commands refer to chapter 8 67 General procedure 7 1 General procedure e Insert your sample in the slideholder e Add a new set of elements chapter 7 2 e If you want to catapult prepare the PALM RoboMover or PALM CapMover refer to chapter 12 Select the well into which you want to catapult the element refer to chapter 7 3 2 you can also make this selection again later or change it refer to chapter 8 6 e Select the required magnification refer to chapter 15 1
211. ld In this way you can define how many elements are catapulted into a well As soon as the number of el ements set has been catapulted into the first well defined PALM RoboMover moves automatically to the next well Distribution Calculator Operational Configuration Distribution calculation Operating Mode Number of selected well s 0 Number of used well s n Start with Field Number of elements per well 0 Number of elements to process 1 1 E until Field Remaining elements Cancel e Inthe n field enter the number of elements that are to be catapulted into each well e Select the co ordinates of the first and last well into which the elements are to be catapulted on the Start with Field and until Field drop down lists Info The order of the wells is defined by the setting in the Subtype Configuration field in the RoboMover window line by line or column by column refer to chapter 12 2 2 Info As soon as the last well has been reached and filled the laser operation is interrupted even if further elements were selected for catapult ing refer to chapter 8 6 3 415109 2614 101 e 09 09 Defining wells for the next laser operation Operating Mode spread elements evenly With this mode you define the number of wells into which the elements are catapulted The elements are divided evenly over these wells Distribution Calculator Operational Confi
212. le The function described here saves your set tings in a file that can be reloaded any time even if you have changed the settings in the meantime Configuration Saving Setting Files Folder Ci Documents and Settings 4 dministrator My Save Setting file on program exit e To toggle this function off or on tick the Save Setting file on program exit check box in the Save Setting Files section of the Configura tion tab in the Preferences and Configuration window Note The Save Setting file on program exit de fault setting is only effective if you have al ready saved your settings when you were working or you have loaded a settings file If you subsequently change the settings they will be saved automatically to the current set tings file when you exit the program The name of the current settings file appears in the Status Bar The current file will also be overwritten 30 To save your settings Adjustments Save Settings e Click on Save Settings in the Adjust ments menu The Save Settings as window opens The folder that you have specified for your settings files opens automatically e Selecta different folder to store your settings files as necessary e Enter a name for your file and click on Save The window will close The new file name will ap pear in the Status Bar To load a settings file Adjustments Load Settings e Click on Load Settings
213. lect a sample for the source sample and treat it if necessary with a coloring substance or similar Step 2 creating reference elements on the source sample automatic and manual adaptation e Insert the source sample into the slideholder of the stage Info The following image examples relate to the SlideHolder 3x1 0 The source sample was in serted in position 1 the target sample in po sition 2 Slide 1 and Slide 2 tab in the Element List window Info Before further processing it is advisable to create an overview scan of the sample with the help of the PALM Navigator refer to chapter 6 This makes the targeting of indi vidual areas of your sample far easier Reference Tool Reference Point l Freehand Tool PA Now you will create the reference elements There are two options available Insert an element of the type Reference Point into the center of each marking Draw three elements which correspond exactly to your markings For this it is advisable to use the freehand tool With which you can repro duce the shape of the markings exactly PALM RoboSoftware then calculates the center of each element in succession and at each of these points automatically inserts an element of the type Reference Point Info It is advisable not to mix both methods Either set or draw three reference elements 101 The Serial Sections function The procedure is essentially the same whet
214. lement s Info The selected color only applies to the type of el ements that you selected prior to choosing the color If for example you have selected a color for Line type elements and then change to the Dot graphic tool new elements of this type will be displayed in the color that is select ed in the Draw Preferences window Element colors and thickness tab for LPC Dot 415109 2614 101 e 09 09 7 9 3 Change colors of elements that have already been drawn The colors of elements that have already been drawn can be changed Using menu commands in the main window using commands in the Element List Using an icon button and the Color Palette To change the color using a menu command Info For text elements the color of the flag can be changed as described below How to change the color of the text background is described further below Edit ET Change Alt Enter e Select the element whose color you want to change in the main window or Element List window e Click on the menu item Change in the Edit menu in the main window or in the Element List window The Element Properties window will open Element properties ET Pr dark green Grp lightblue Type blue blue darkblue Comment lightyellow yellow Test DO darkyellow A lightcyan cyan darkcyan lightmagenta Function magenta darkmagenta Fm Objective well e Select the required color on the Color li
215. lements for later or File Mode refer to chapter 1 7 Depending on use only in File Mode in Database Mode your the initial settings refer to chapter 3 1 PALM elements are saved automatically refer to RoboSoftware will be launched chapter 3 7 Saving and loading settings or either in File Mode chapter 9 2 Saving and loading elements in or in Database Mode with the database pre File Mode selected under Settings or there will be a dialog for you to specify which mode you wish to start in Operating Mode Selector Please select database Database DB Mode e Inthis case you should select the database you wish to work with and click the DB Mode but ton or click on the File Mode button if you wish to work in File Mode Note When PALM RoboSoftware is launched you can choose between Database and File Mode Once the program is running this is no longer possible Once the program is running it is also not possible to change the database You should exit PALM RoboSoftware and restart it if you wish to change to a different mode or database If necessary you must first modify the initial settings as described in chapter 3 1 12 415109 2614 101 e 09 09 1 9 Launching other programs from PALM RoboSoftware PALM RoboSoftware has interfaces with other pro grams The programs concerned are programs for organizing and editing images and detecting struc ture InformationCenter a component of the PALM Mi
216. lements on the other slides are retained Info In the Database Mode refer to chapter 1 7 it is not possible to delete elements already pro cessed with the laser To delete all elements Edit a Delete all elements Ctrl e Click on the menu item Delete all elements in the Edit menu of the PALM RoboSoftware main window All elements are deleted if you are working in Database Mode only the elements that have not yet been processed with the laser Info If you are working in File Mode refer to chapter 1 7 you can also delete all elements by clicking on New Elements Delete All on the File menu In this case any elements that have already been processed by the laser will also be deleted If you are working in Database Mode and cre ate a new record group refer to chapter 7 2 2 or select a new record group see chapter 9 3 your elements will likewise disappear from the screen They are however saved in the data base and can be retrieved at any time 415109 2614 101 e 09 09 Deleting elements and restoring deleted elements To delete the last drawn element Edit Delete last element BackSp e Click on the menu item Delete last element in the Edit menu of PALM RoboSoftware main window Info You can repeat this function many times Each time the latest of the remaining elements will be deleted If you are working in Database Mode you can not delete the last element drawn if
217. load elements and then click on the OK button Your elements will be loaded for all the selected slides e If you want to load the element files for one or more slides that you have saved with different names Select the related checkboxes in the example Slide1 Slide2 and or Slide3 in each case enter a file name if necessary select the required file using the button and click on the OK button Info If you have drawn and saved elements for a slide and now wish to continue working on the same slide you need not necessarily insert it at the same position it occupied when the ele ments were saved If for instance you had the slide inserted at position 1 you could now also insert it at posi tion 2 You must bear in mind however that the elements you had saved for position 1 must now be loaded for position 2 Info This method can also be used to transfer ele ments from one sample to another in that you draw and save elements for one slide and then load them for a different slide for two consec utive tissue samples for instance Remember though that the position and probably also the shape of the elements will not be exactly right for the second sample and you will have adjust the position and shape by hand It is therefore recommended when transferring elements from one slide to another that this is done using the Serial Sections function refer to chapter 8 7 This function adjusts t
218. locate laser function to element 94 allocate objective to element 94 allocate well to element 95 open 91 overview of functions 93 Slide tab 91 Summary tab 92 Element types dot 71 grid rectangle 71 line 71 Reference Point 71 ruler 71 text 71 Elements add comment 84 add elements to a group 89 center 76 change color 87 change comment 84 change correct shape 82 copy 75 83 create new set 68 definition 193 deselect monitor 78 display 76 dot thickness 86 draw 71 edit 80 export 111 group 89 hide 76 import 111 load 109 merge 81 naming 84 number 84 print 112 process 80 remove from a group 89 reposition 80 restore 79 83 restore changes 33 restore deleted 79 resume 81 rotate 82 save 107 select 77 select within a group 90 set color 84 set color for new elements 87 set line thickness 84 settings for saving File Mode 107 show anchor points 80 stretch 32 stretch and rotate 83 switch on and off numbering 83 types 71 ungroup 89 z Focus 29 Export of elements 111 Exposure time automatic adjustment 32 Extended focus generate recording 180 prepare recording 179 save image 181 show image 181 F Factory defaults 30 Field of View Analysis 189 integrate script 190 run analysis 190 select script 190 File Mode change to Database Mode 12 InformationCenter 10 load elements 108 PALM RoboSoftware 11 save elements 108 save images 115 Fluorescence a
219. losed during the interval between images being saved In this way you can protect delicate samples against fading Info You can either check the check box for micro scope shutter control or the one for fluores cence shutter control In the Trigger Point Definition area you define which event will be used to start the recorder func tion Manually the sequence is started manually by clicking on the button Start Recorder manu ally see page 118 Foot switch Cut LPC the sequence is started as soon as you activate the laser using the foot switch chapter 11 10 explains how you can set the footswitch for Cut or LPC LPC laser function the sequence is started as soon as you activate an automatic LPC laser function Scheduled you can set a time for the function to be started automatically In accordance with the start event that has set up one of the following symbols is displayed in PALM RoboSoftware main window Th Start Recorder manually Foot switch Cut LPC or LPC Laser function ne Scheduled 415109 2614 101 e 09 09 Saving images and videos using the Recorder function To start the recorder function manually nny Start Recorder manually e Click on the Start Recorder manually icon button To cancel the recorder function During video recording and the storage sequence a STOP icon button appears on the Tool Bar ie Stop e Click on the STOP icon button 415109 2614
220. low changes with a movement operation in progress You can set the speeds of all types of movements with the Speed Tools tool Speed changes with keyboard or menu commands always refer to the type of movement that is currently selected in the Speed Tools tool Note The settings that you enter always apply to the magnification that you have set in the program As soon as you have set another magnification the values that were last used or the factory defaults are automatically ap plied Therefore prior to setting the speed first select the magnification that you want to work with 415109 2614 101 e 09 09 Setting speed Before you initiate a movement of the stage all three setting options are available for both move ment types While the stage is moving you can only change the speed of the Cut movement type using the Speed Tools tool In the case of the other movement types the speed can be changed with keyboard commands Info If you have selected too fast a speed for Cut Mode it may happen that your sample is not cut through completely In this case reduce the cut speed To select the unit for speed setting For the speed setting you can select between two different units percent and um sec Adjustments PALMA obo e Inthe Adjustments menu click on the menu item PALMRobo The Preferences and Configuration window with the Configuration tab opens Configuration Metri
221. ltidimensional Acquisition C EI Je Experiment E Be 2 COT Time Lapse Interval Settings Maximal Speed Interval 10 000 e Duration Settings Orcs 2 Duration Settings before after Timepoint before time point after time point e Make your settings refer to AxioVision online help 181 Preview 16 4 Preview As soon as you start a multi channel fluorescence recording or a recording with extended focus the Multidimensional Acquisition Preview window opens Multidimensional Acquisition Preview HE amo Acquisition C 2 T 1 2 6 Pos 1 A AR Els A wx a 16 5 Viewing images in the picture viewer F Picture Viewer The progress of the recording is shown in the top left In the bottom left there are various buttons that can be used to interrupt or cancel the recording The last image recorded appears on the right The buttons below the image can be used to influence how it is displayed for the function of the buttons see chapter 16 5 2s de a WR 2A LR UD e gt Sica BT ud ar 172 32 q li 414 771 133111 6 42883 1 um Gray For recordings with extended focus the PALM RoboSoftware main window only shows the overall image calculated from the individual images Extended Focus Image 182 In the picture viewer you can view all of the indi vidual images recorded in experiments with ex tended focus as well as images from multi
222. ly drawn one ele ment then that one is selected automatically e Start the automatic laser function press F11 or release the laser using one of the other meth ods explained in chapter 11 7 Starting the automatic laser functions The stage is moved below the laser the laser is re leased e If necessary increase the energy until the laser achieves a cutting effect You will get a cut line in the sample This line will initially be unclear and wide or the sample will only be cut incompletely as a result of an inadequate setting of the laser focus and energy You can change the settings during the cutting process as long as the cut line remains incomplete If the cutting process has been completed before 131 Selecting elements for the next laser action you have found your optimal settings you will have to draw a new element and restart the laser If possible try with multiple cutting operations e Change the laser focus and try to aim for a thin ner cut line e If you have achieved that then reduce the energy also with the intention of getting a thin ner cut line e Nowtry to continue optimizing your results by repeatedly changing both focus and energy e Once you have attained the best possible result you should either save or note the appropriate settings refer to chapter 3 7 b Without using the automatic laser func tions e As described in chapter 11 10 adjust the foot switch in such a way tha
223. make the settings in the Preferences and Configuration window or in the Draw Preferenc es window How to open this window is described in chapter 11 4 5 LPC Distances um for Objective E Distance of AutoLPC shots Distance of 4utoLPC shots from line AutoLPC shots diagonal AutoLPC Center shot Distance of RoboLPC joint e Select the magnification for which you want to make the settings from the for Objective list e Enter the desired value in um for the width of the remaining RoboLPC joint in the Distance of RoboLPC joint field Note Should the value of the RoboLPC joint param eter be too small or at zero then it can occur that automatic catapulting does not take place in the RoboLPC function as the sample pos sibly will not remain fixed within the tissue when cut 130 11 5 Finding suitable laser settings Perfect cut and catapult results can only be achieved with optimal parameter settings The op timal settings are not however the same for all samples You must select the best suited settings for each type of sample by experimentation To achieve cut lines that are as fine as possible you should enter the lowest possible energy set ting with which you can still achieve the required cutting effect sometimes multiple cutting along the same line with lower energy achieves better results than a single cut with higher energy In addition to that the laser focus should be at the same l
224. microscope It operates using an IR laser trapping laser and is available as a single beam or double beam each with or without movement moving trap on the DuoFlex TwinFlex system the laser beam is split into two beams using a beam splitter TwinFlex Double Beam with two movable trap ping laser beams With this system you can trap and move two objects independently of one another DuoFlex Double Beam with one fixed and one movable trapping laser beam With this system you can also trap and move two objects independently of each another with the limitation that one trapping laser beam is fixed and therefore one object can only be trapped and moved by moving the stage in the x and y axes MonoFlex Single Beam with Laser Beam Posi tioning With this system you can trap and move one object by moving the trapping laser beam or by moving the stage Mono Single Beam with one fixed trapping laser beam With this system you can trap an object and move it in the x and y axes by moving the stage In all four configurations you can lift trapped ob jects in the z axis away from the plane of the slide Thus in combination with PALM MicroBeam you can for example trap objects transport them to the cutting laser and process them with it The following chapters will explain how to operate your PALM MicroTweezers together with PALM RoboSoftware A few of the functions described are not available if you have purchas
225. microscope image The mouse cursor changes to the Group Reference Pointer recognizable by the attribute R With the Group Reference Pointer you can select reference elements and then move them e Select the reference element with a mouse click and then drag it to the respective marking in your sample Note Make sure that you do in fact move the ref erence element to the respective marking If you move it to another marking the match will be faulty e Proceedin the same way with the other two ref erence elements By moving the reference elements the shape and position of the remaining elements will be auto matically matched e Click on the menu item Match Serial Section Point manual in the Edit menu The pointer reverts to the standard pointer Note Check the position of the matched elements if your tissue block contains diagonal struc tures Match the position if required the shape also of such elements by hand if necessary refer to chapter 7 8 1 105 106 415109 2614 101 e 09 09 9 Saving and printing elements PALM RoboSoftware has a number of options for saving exporting or printing the elements you have drawn or their properties to load them into PALM RoboSoftware later on again This allows to reuse a sample in the stage and superimposing the associated elements on the monitor to observe and edit the element properties in another program to enable the element prope
226. n a folder and when in Database Mode they are stored in the linked database refer to chapter 1 7 10 3 1 Saving images in File Mode You can specify a folder and a name for the image being saved refer to chapter 10 2 and text be low When you save images later the specified image name will automatically be supplemented with a sequential number 01 02 If you want to save pictures subsequently these assignments are selected automatically However you can decide whether you want to accept them You can also save your images later in a different folder You can also create a new folder when sav ing and assign different names to your images Note If you select another folder or another image name than the one proposed for saving these settings are adopted as the default settings and used for all subsequent saving operations To enter a file name for saving images manually Adjustments PALMA obo e In the Adjustment menu click on the menu item PALMRobo The Preferences and Configuration window opens e Click on the Images tab Images Saving Images Folder ags AdministratorsM y Documents My FalmD atas e Entera name for images to be saved in the Filename field of the Saving Images sec tion 415109 2614 101 e 09 09 Saving images manually To save your image Info Please note that the image is stored with the elements unless the option to displ
227. n draw shapes along which you can later cut with the Cut laser function or the area content of which you can catapult later with the AutoLPC or RoboLPC auto matic laser functions Using these tools you can also identify line shaped structures that you can then catapult later using the automatic LineAutoLPC laser function e g dendrites Elements drawn using these tools are collec tively designated elements of type Figure Dot Tool Use the Dot Tool to mark individual cells for subsequent catapulting with the LPC laser function Grid Rectangle Tool Use this tool to draw a rectangle that is automatically divided into the number of smaller rectangles you define When the laser function is triggered the individual rectangles are cut and catapulted if necessary Using this feature in combination with the PALM RoboMover operating mode morphology con serving you can catapult sections from your tissue wafer into wells during this process the arrangement of the sections is retained refer to chapter 8 6 section Operating Mode mor phology conserving Stamp Tool Using this tool you can copy exist ing elements and position the copies in the re quired position with a mouse click Ruler Use the Ruler to measure zonal lengths The zone is shown as a line Text Tool Text With the Text Tool you can identify individual areas of your sample so that for example they can be easily located again a
228. n on the target sample Any differences in the position and shape of the elements between the two samples will be balanced out Note Check the position of the matched elements if your tissue block contains diagonal struc tures Match the position if required the shape also of such elements by hand if necessary refer to chapter 7 8 1 Now you can process your elements on the target sample with the laser Info Elements which have changed shape are identified by an asterisk following the ele ment type in the table in the Element List window Step 4b preparing the target sample manual adaptation e Insert the slide onto which you want to trans fer the elements into the slideholder Step 5b transferring elements to the target sample manual adaptation Edit Match Reference Point manual e Select the group which you want to match in the example Slide 1 e Click on the menu item Match Reference Point manual in the Edit menu Info This menu command is only available in PALM RoboSoftware s main window 415109 2614 101 e 09 09 The Serial Sections function e Inthe main window of PALM RoboSoftware move the mouse cursor to the menu item Goto Reference gt Group in the Motion menu and click the item Ref 1 Motion Goto Reference k Group Ref 1 1 Ref e Trap 1 2 Tione Ref 3 The reference element 1 will be displayed on the monitor e Drag the mouse cursor onto the
229. n pass through it unrestricted With this setting you can work with the laser without risk of damage to the ApoTome ApoT ome The ApoTome is set up in such a way that the grid is positioned in the beam path Info The laser beam would destroy the grid If the grid is swiveled in the laser is automatically switched off and no laser function can be start ed ApoT ome a The ApoTome controller is switched off In this case PALM RoboSoftware has no information on the status of the ApoTome Note Check the status of the ApoTome before switching the laser on The grid must not be positioned in the beam path as it would be de stroyed by the laser 171 Hardware configurations 15 5 Hardware configurations You can compile and save different hardware con figurations In a hardware configuration you define settings for the camera and microscope compo nents e g shutter reflector filter etc You can activate a saved hardware configuration with a click of the mouse I e if you want to change to a different system configuration you do not need to separately make the settings for each individual component e Selectthe HW Settings tab in the Microscope Tools Microscope Common Reflected Light H Settings Settings Assign HW Settings ICc1 Brightfield MAm DAPI To activate a hardware configuration e In the Settings area click on the button with the name of the required hardware co
230. n performed see Figure on next page to a 3x1 0 slideholder Slidel Slide2 or Slide3 Position of the slide to be scanned in the slideholder Section O Overview scan Number of tiles in this scan Scan status e Click on the Scan button The selected area is scanned section by section a section is referred to as a tile You can follow the progress in the window Depending on your setting for focus correction either the focus is corrected automatically or the scan stops so that you can adjust the focus manu ally The remaining duration of the scan is specified in the Information section Info The specified time remaining is a rough estimate The program cannot calculate in advance exactly how long any focus correction will take You can cancel or interrupt the scan e g to make corrections to the focus setting This gives you the option of rescanning tiles that have not been scanned sharply see chapter 6 4 58 415109 2614 101 e 09 09 Navigator Navigation Scan AxioVision Analyze VisDat 6 4 Stopping and canceling or continuing a scan in progress You can interrupt a scan in progress at any time After doing this you can completely cancel or continue the scanning process for example after making corrections to the focus setting How to stop a scan in progress While a scan is in progress the Stop Scan button appears instead of Scan Stop Scan e Click on the Stop Sc
231. n the image itself Info If the Scroll Rectangle is not displayed you can activate it by clicking on Scroll Rectangle in the View menu Scrolling with the mouse also works when the Scroll Rectangle is not displayed e Press and hold the mouse button down The stage is moved in the direction of the arrow You can also change the direction of movement during the movement itself by moving the cursor from an image edge towards an adjacent corner or vice versa e Release the mouse button when you have found the required area of your sample and positioned it in the correct place The Scroll function is only active as long as the cursor is outside the Scroll Rectangle Movement of the stage is therefore also stopped when you drag the cursor into the Scroll Rectangle with the mouse button depressed 5 1 3 Positioning the stage with the scroll bars Scroll bars are located on the bottom and right edges of the microscope image With these you can move the stage e Click once on one of the arrow icon buttons until the stage has reached the desired posi tion or drag the position cursor on the scroll bar to the desired position If you want to move the stage in steps of one im age width or height e Click beside the position cursor on the scroll bar 46 5 1 4 Positioning the stage with the keyboard Arrow keps Joystick mode o de o e O ll e Click on the button at the top right edge of the program w
232. ncubator em sda des MU A ac need DS de ne e 185 17 3 Starting and SEOPPINA ION sas er ce AAA SATA E DE AO 187 17 4 DUS AVY WAGE VOGT aaa nee eth ATE a as ES RE A A E O ee Bt 187 18 Field of View Analysis sacas A ea A AAA a ARA 189 Appendix A Glossary diras a ee ds Rn Le RR A 193 Appendix B Keyboard Shortcuts 00000 lt lt lt oooooo oo 196 Indek ia RARE AEREAS ee 197 415109 2614 101 e 09 09 7 415109 2614 101 e 09 09 1 Introduction 1 1 Functionality of PALM MicroLaser systems PALM MicroLaser Systems are modular in design and extremely flexible PALM MicroBeam PALM MicroBeam offers the greatest degree of pre cision in the field of non contact laser microdissec tion and manipulation It can be used in virtually all areas of molecular research Your PALM MicroBeam is designed to enable you to cut sections from your sample and detach them from the surrounding material under the micro scope and then to catapult them into a collecting cap PALM MicroTweezers With PALM MicroTweezers you can trap objects under the microscope and lift them away from the plane of the slide It can be employed as stand alone technology or in combination with PALM MicroBeam as PALM CombiSystem PALM MicroTweezers is available in four configura tions with a fixed laser beam Mono with a movable laser beam MonoFlex moving trap with one fixed and one movable laser beam DuoFlex with two movabl
233. nd annotated These comments are then displayed at the specified position on the monitor Reference Point Tool Use this tool to define ref erence points For example you need them for the Serial Section function refer to chapter 8 7 Using the Field of View Analyse function you can also generate elements automatically refer to chapter 18 Ruler Text and Reference Point type ele ments have no effect on the laser functions Select the required laser function for elements of other types before you draw the element 415109 2614 101 e 09 09 Drawing new elements The elements are displayed on the monitor as col ored lines dots or areas You can preset the colors and line thickness or the dot sizes for display and you can change them later refer to chapter 7 9 The program automatically assigns a number to each drawn element If you have not changed the default setting this number is displayed refer to chapter 7 8 4 Info In some cases the elements you have drawn will be changed automatically by the PALM RoboSoftware e g open elements may be closed or closed elements opened This action is dependent on the laser function selected and especially on the parameters that you can set for the individual laser functions refer to chapter 11 1 Laser functions and chapter 11 4 Specifying laser settings Note It is sometimes not possible for the laser to process elements located very close to th
234. nfigura tion 172 15 5 1 Setting up a hardware configuration Setting e Click on the Setting icon button The Settings Editor window is opened refer to figure on page 173 In the Available Components field you will find a list of all available components Show all e Click on the Show all button to display all sub entries The Hide all button appears in place of this but ton Hide all e Click on the Hide all button to hide the suben tries In the Stored hardware settings field you will find a list of all existing hardware configurations The elements and related settings for a new con figuration or a configuration selected in the Stored hardware settings field are displayed in the Selected Setting field To add a new configuration or change an existing configuration If you want to change a configuration e Select the required configuration in the Stored hardware settings field Mew If you want to add a new hardware configuration e Click on the New button Duplicate If you want to copy an existing hardware configu ration e Selectitinthe Stored hardware settings field and then click on the Duplicate button You can change the copy and save it with a new name 415109 2614 101 e 09 09 Hardware configurations E Settings Editor Stored hardware settings Location Setting 3 a User alles aus zvhs User Binocular zvhs a User Channel4 2vh
235. ng must have been cali brated the Laser Markers in the default setting the green triangle or the red and green squares in the middle of the monitor must have been aligned with the laser foci and if the magnification is changed any deviations through the objectives must be compensated Objective Offset Calibrating with the software establishes a rela tionship between the dots in the image and the ac tual positions of the stage This ensures that automatic laser functions are performed at pre cisely the required locations The Laser Markers must mark the points where the laser beams strike the sample To check the cor rect setting of the Laser Markers you must release the laser Hold a sample that you no longer need for examination or one containing areas that are no longer needed for examination ready A slide that has been colored with a black felt pen is par ticularly helpful for setting and checking the Trap ping Laser Marker You must check and if necessary correct the posi tion of the Laser Marker before you start after a relaunch and after a change in the magnification 415109 2614 101 e 09 09 Preliminary remarks 4 2 Setting the magnification Before you start work you must set the micro scope magnification in PALM RoboSoftware To set the required magnification Microscope Common Reflected Light Hw Settings Objective alolalal la e Select the required magnification in the Micr
236. nt 75 Position stage 45 center 52 center elements 76 centering tool 47 in Stage Mode 45 joystick 46 keyboard 46 return from CapCheck 51 return from Load Position 52 scroll 46 Scroll Bar 46 set speed 48 stop movement 48 target CapCheck 51 target Load Position 52 with mouse in Stage Mode 45 with mouse scroll 46 Position wells 143 Print elements 112 415109 2614 101 e 09 09 Program exit 12 functionality 9 general help 21 installation 11 layout 15 operation 20 PALMRobo Instructor 21 software modes 20 standard configuration 15 start 12 Q Quadratic Attribute 73 74 90 R Recorder function 117 make settings 117 Reference 195 Reference Position trapping laser movement define 149 move to 149 significance 195 Reference Position trapping laser define 149 Reference the stage 50 Reflector Offset adjust 164 reset 164 Restore 83 changed element 33 deleted element 79 Retain morphology grid rectangle 74 operating mode 99 Ruler 90 S Save elements 107 Save images 115 116 automatic during laser function 116 Copyright Note 113 create folder 114 manually 115 116 select folder 114 201 specify file name 115 time lapse Recorder 117 Save video sequence time lapse Recorder 117 Scale Bar switch display on off 18 Scanning interrupt scan 59 load scanned image 61 save scanned image 61 section 60 show elements 62 Screen Center show hide 18 Scroll Bar
237. ntrols on the scan tab 415109 2614 101 e 09 09 Analyzing images using the Definiens software You can display separate tiles as on the Scan tab e Select the Stored tiles radio button Buttons appear for navigating between the tiles see chapter 6 8 Info In Live mode the image is re recorded using the current camera settings and then analyzed while in Stored tiles mode the saved images from a previous scan are analyzed 6 11 Analyzing images using the Definiens software The Definiens software enables images of sam ples to be analyzed using sets of rules that have been defined in advance and also allows elements to be generated This helps with routine operations to automate the selection of the areas of interest Info You can only utilize this analytical facility if your system includes the Definiens software Please contact Carl Zeiss MicroImaging GmbH if you wish to upgrade your system e Open the Navigator window e Select the Navigation tab and define the area that is to be analyzed as described in chapter 6 2 e Select the Elements checkbox on the Scan tab Navigator Navigation Scan Definiens Analyze AxioVision Analyze VisDat Parameter Job Status Discriminate nuclei by red ratio e Click on the Test button 415109 2614 101 e 09 09 e Select the Definiens Analyze tab e From the Analyze Ruleset selection list choose the set of rules you wish
238. o show or hide To move the individual tools or icon bars e Click on the white vertical bar at the left end of the icon bar or the horizontal double bar beside the name of the tool that you want to move and keep the mouse button pressed e Drag the bar or the tool to the required posi tion Release the mouse button Info You can also move tools to positions outside the program window e g to a second moni tor if you have one connected 18 To restore the default configuration for all bars and tools View Default Bar Configuration e Click on menu item Default Bar Configuration in the View menu To switch the Scroll Rectangle or the screen center display on or off e Inthe View menu click on Scroll Rectangle or Screen Center to display or hide the Scroll Rectangle To show or hide the Scale Bar e Click on menu item Yardstick in the View menu to display or hide the Scale Bar The size of the Scale Bar is dictated by PALM RoboSoftware depending on the magnification set ting this cannot be modified Note The scale is dependent on the calibration refer to chapter 4 To show or hide the Copyright Note e Click on menu item Copyright in the View menu to display or hide the Copyright Note Info Don t forget that the Copyright Note can only be displayed if you have defined it in advance refer to chapter 10 1 415109 2614 101 e 09 09 Customizing the user interface
239. of the PALM RoboSoftware with the Advanced setting The Basic setting provides a simplified user interface 1 4 InformationCenter The program PALM InformationCenter is a com ponent of PALM MicroLaser Systems The program is to display and post process the sample data generated using PALM RoboSoftware The InformationCenter recognizes two operating modes File Mode and Database Mode refer also to chapter 1 7 Database Mode is available only when an appropriate software license has been purchased The standard version of the Information Center program file based File Mode can be used for or ganizing the stored microscope images You can thus view the stored images display them in the form of a slideshow creating a video sequence from the slideshow save them in a different for mat as well as display edit or export information that was saved with the images In the upgraded version of the program file based and database based File Mode and Database Mode you can access even more powerful func tions for documenting your work than those in cluded in the standard version If you are working with PALM RoboSoftware in Database Mode refer to chapter 1 8 Launching and exiting the program not only the images you are saving are stored in a database but also the elements you draw information on samples experiments and microdissection operations You can also enter ad ditional information to be attached to the images
240. ollection ves sels over your sample Various collectors with one collection vessel each are available for PALM CapMover II PALM RoboMover offers considerably more op tions Collectors with one or more collection ves sels are available up to 96 wells with PALM CapturePlate Collector 96 If you use collectors with more than one collection vessel you can have elements of your sample automatically catapulted into different collection vessels You only need to start the laser function once for this purpose Info The illustrations descriptions and instruc tions below relate to PALM CapturePlate Coll ector 96 PALM RoboMover and the Single Tube Collector 500 CM II PALM CapMover II All other collectors with more than one collec tion vessel are used similarly On the use of PALM CapMover II and with collectors with only one collection vessel none of the func tions for collectors with several collection ves sels are available All functions for the control of PALM RoboMover or PALM CapMover II are in the RoboMover and CapMover II windows respectively 415109 2614 101 e 09 09 12 1 Opening the RoboMover or CapMover II window To open the RoboMover or CapMover II window Devices Capture Device Fl e On the Devices menu click on Capture Device The RoboMover or CapMover II window opens On the left it displays an image of the collector used and on the right various settings and items of inf
241. omatically switches to Freeze Mode Additional options appear in the Multi channel fluorescence menu which you can use to call up the following functions Save the recording as a multi dimensional image Display individual channels or a superimposed overall image in the PALM RoboSoftware main window Display individual channels or a superimposed overall image in the picture viewer see 16 5 415109 2614 101 e 09 09 To save your images and display them on the monitor E Save multidimensional image as zwiimage e Click on the Save multi dimensional image as zvi image option special Zeiss format if you want to save the images save the image in jpg bmp or tif format as described in chapter 10 The images are saved in a file as a multi dimensional image Channel 1 Channel 2 Overlay e Click on the Channel or Overlay button to display the individual images or the super imposed image on the monitor Info You can use the F and B keys to scroll for wards and backwards through the individual channels Note The multi channel fluorescence recordings are not live images of your sample PALM RoboSoftware therefore automatically Switches to Freeze Mode as soon as you start a multi channel fluorescence experiment You can exit Freeze Mode at any time to see the live image from the camera on the monitor Using the commands described above you can return to the fluorescence recordings
242. on filter necessary when using a filter wheel only refer to chapter 14 7 swivel in the desired reflector position in the microscope refer to User Manual switch the microscope light on or off as neces sary activate the timer if you want to preset a time for which the excitation light should illuminate your sample refer to chapter 14 11 and open the shutter chapter 14 6 Note Close the shutter chapter 14 6 before switching on the fluorescence lamp and before finishing or interrupting your work This will prevent unintentional exposure of your sample to fluorescence light 14 3 Settings for working with flu orescence motorized microscope To use the fluorescence illumination you must make the following settings switch on the fluorescence illumination refer to User Manual Select the desired fluorescence filter unit chap ter 14 4 With this the functions Excita tion filter Reflector position Microscope light Timer and Shutter are performed via a single command The basic settings of these functions has been made during installation of your PALM system PALM RoboSoftware provides you with the capability of saving various combinations of these parameters and retrieving them when they are needed chapter 14 9 157 Activating a fluorescence settings record 14 4 Activating a fluorescence settings record You can activate a set of fluorescence settings refer to chap
243. or GSE 202s R Slide Slide2 Slide3 Summary Show Types al v Color Nr Name Type Laser function Objective Well Area um Grp 1 Freehand RoboLPC 5x Fluar 5x 0 25 349332 Grp04 2 Freehand RoboLPC 5x Fluar 5x 0 25 324515 Grp04 E 3 Freehand RoboLPC 5x Fluar 5x 0 25 282382 Gip04 4 Grp04 Group 5x Fluar 5x 0 25 The selected elements are merged into a group A new entry for the group appears in the Element List window For the elements belonging to the group the name of the group is entered in the Grp column The name of the group is automat ically assigned by the PALM RoboSoftware in the figure above Grp04 you can change the name as described in chapter 7 8 5 Grp04 Grp04 1 rpo4 3 To the element number of each element contained in a group the group number is prefixed All ele ments of the group are displayed with a surround ing dashed rectangle 415109 2614 101 e 09 09 To add other elements to a group Edit Add to Group e Select the elements you want to add to a group either on the monitor on in the Element List window e Click on the menu item Add to Group in the Edit menu in the main window or in the Element List window If you have created only one element group so far the elements will be added to this group If you have created more than one group the Groups window will open e Click on the desired group to which the ele ments should be added and close the window
244. or for which you want to change the color information contained in the Color column e Enter the desired name Repeat for all reflectors which you want to rename To define whether the laser is allowed to be used with a reflector e In the table double click in the row of the reflector for which you want to change the set ting contained in the Laser column Yes the laser can be used with this reflector setting No the laser cannot be used with this reflector setting To select a reflector e Open the drop down list Actual Position and select the desired position The reflector block moves automatically to the selected position 161 Editing a fluorescence settings record 14 9 Editing a fluorescence settings record Fluorescence adjustments Fluorescence Fluorescence Filter Reflector Fluorescence Shutter Fluorescence Names and used Functions Ho Name Filter Shutter Reflector 1 Laser 1 Laser CLOSE DAPI 2 DAFI OFEN o ae 4 Rhodamine 4 Rhodamine OPEN a Texas Red A Texas Red OPEN new Select an Entry and DoubleClick to Change If you need certain settings and sequences for flu orescence more frequently you can save them as a settings record You can create and save as many of these records as you wish You can assign a name to each setting These names then appear on buttons in the Microscope Tools on the Reflected light tab You can activate the related set of settings by click
245. ormation As long as you have not yet used a collector or have not yet made a scan for the coll ector used refer to chapter 12 2 1 Preparing a collector a black cross appears in place of the il lustration 3 RoboMover Dperation Adjust NIDA dO Subtype Configuration optional Information Current field Target not initialized No valid Target found 137 Working with PALM RoboMover or PALM CapMover II 12 2 Working with PALM RoboMover or PALM CapMover II 12 2 1 Preparinga collector Info Only for PALM RoboMover It is recommen ded to find and select all interesting areas pri or to fitting the liquid filled PALM CapturePlate Collectors 96 To avoid the eva poration of the liquid filling e Prepare the collector system with which you want to work refer to PALM User Manual Change Collector e Insert the collector and click on the Change Collector button PALM RoboMover or PALM CapMover II is moved to the Load Position As soon as PALM RoboMover or PALM CapMover II has reached the Load Position the Load collector window opens e Fit the collector as described in PALM User Manual in PALM RoboMover or PALM CapMover II To work with the collector you must tell PALM RoboSoftware which type of collector you have fit ted PALM RoboMover Change collector Now you can change the insert or the collector type Scan new collector type Cancel S
246. orting elements To import your elements e Click on the menu item Import Elements in the File menu in the PALM RoboSoftware main window or in the Element List window The Import Elements window will open e Ifnecessary open the folder in which you have saved the file e Fromthe displayed list select the file you want to load and click on the Open button The imported elements are displayed on your monitor Any elements that were already on the monitor will be deleted 111 Printing elements 9 5 Printing elements Select Attributes and Define Columns The Print Elements function can be used to Current used columns either print your elements on a printer or to gen Nr Color Type erate an Excel import file Drag amp drop a Column to the right place Click in Column Header to remove Column To print your elements Add a new Column Select Attribute v File e Select the desired columns and column order Print Elements and click on OK to close the window e Inthe list Sorted by select the element prop e Click on the menu item Print Elements in erty to which the list should be sorted the File menu in PALM RoboSoftware main You can select whether the file should contain ele window or in the Element List window ment summaries The Print Elements window will open print Summary records the number of the elements and the sum of the sizes of their areas P
247. oscope After catapulting the sample into the cap it can be observed with the microscope through this open ing Check Position Freely definable focus setting of the objective to set by a menu command motorized microscope only The setting should be defined so that the catapulted sample in the cap is displayed sharply on the monitor or in the ocular Close amp Cut Closing and Cutting An incompletely closed ele ment is automatically closed and cut completely according to the given conditions 415109 2614 101 e 09 09 Close amp Cut AutoLPC Closing the marking line and cutting before cata pulting With critical samples the marked area must first be detached from its immediate sur roundings by a clear cut line Only in this way can pure sample preparation be assured when cata pulting This function can only be used with glass mounted samples Cut Cutting along a selected line The laser cuts pre cisely along the pre drawn line so that a clear line of detachment is created between required and unrequired material In the cut area all biological material is destroyed by ablation Thus a pure sample preparation can be carried out without fear of contamination Double Beam System PALM MicroTweezers is available as a Single Beam System and as a Double Beam System In the Double Beam System the trapping laser beam is split into two beams Depending on the configura tion one or both of the beams DuoFlex or T
248. oscope Tools Common tab Info The available selection depends on which ob jectives are fitted to your microscope and whether they have been configured in the MTB 2004 e Adjust the microscope to the desired magnifi cation only necessary for a manual micro scope on motorized microscopes adjustment is automatic Note Note that the trapping laser can only be used in conjunction with 63x and 100x lenses Note To obtain a correct display of the drawn ele ments and to achieve a correct laser function the setting in the Microscope Tools must match the magnification that is selected on your mi croscope If the settings do not correspond to each other the actual cut lines and catapult points of the laser will not be correctly dis played on the monitor 39 Setting the laser marker 4 3 Setting the laser marker A Y O The Laser Markers are shown as follows with the default program configuration blue triangle Cutting Laser Marker red Y Laser Marker for trapping laser beam 1 green square Laser Marker for trapping laser beam 2 only DuoFlex TwinFlex system The apex of the triangle or the center of the Y Squares should indicate the point at which the respective laser beam will strike the sample upon release Note PALM RoboSoftware calculates the cutting functions of the cutting laser from the position of the Laser Marker The position of the Laser Marker should therefore always mat
249. ou will find the settings for the following pa rameters you will find detailed information on these parameters in the AxioVision User s Guide and in the AxioVision online help Digital Gain Image Orientation Black Reference Shading Correction Sharpening Shutter 34 The Shading Correction function You can optimize your live image using the Shading Correction function recommended for all camera types In this way you can compensate for a reduction in brightness at the edges of the image You must have already set the lamp brightness and also have performed a white balance e Ensure the camera is looking at a completely empty light image move the stage such that the image is pointing at an area outside of your Sample or remove the slide e Click on the Shading Correction button 415109 2614 101 e 09 09 Set image parameters 3 11 Set image parameters You can modify the way in which your microscope image is displayed in order to make any areas that are of particular interest for your work stand out more clearly You may for instance want to run an analysis under AxioVision or Definiens see chapter 1 9 2 because of the clearer results they produce Info Any changes made here will only affect the image on the screen and not the original camera image in other words you are not reconfiguring any camera settings Info You should bear in mind that one conse quence of changing
250. ovement During an automatic laser operation of type Cut Ifyouarein the Stage Mode The Reference Position of the stage 5 4 The Reference Position of the stage In the drawing plane PALM RoboSoftware requires a reference position for calculating the coordinates of your elements This reference point with its co ordinates 0 0 is stipulated by PALM RoboSoftware With each start of the system the stage is moved to the end positions in x and y direction This po sition is defined as Reference Position The Reference Position is retained for as long as the controller unit remains switched on and even if you shut down and restart PALM RoboSoftware The point of origin is particularly important when you have drawn elements and have saved them for later use The automatic positioning of the stage to the Reference Position ensures that on re loading your elements they will appear at the same positions at which you drew them To work with saved elements you need only insert the respec tive sample into the slideholder and reload your el ements Note Make sure you reinsert the slides in the cor rect order and that you insert each slide ex actly as you had it inserted during previous processing i e not tilted or even rotated 180 Info It may also happen that the drawn elements shift after you change objectives If the optical axes of both objectives do not match exactly the microscope image on the monitor will
251. perms are isolated fast and easily simply by clicking on the cell of in terest refer to chapter 11 10 11 1 3 Three dimensional cutting To ensure that also thick samples can be cut with out problems the PALM RoboSoftware provides a special setting Here you can define how often an element is to be cut If more than one cutting pass is defined you can set that the focus is to be changed by a specific amount for each cutting pass refer to chapter 11 4 2 Info The function is only available in systems with microscopes with a motorized z focus 11 1 4 Non automatic laser function Apart from the automatic laser functions you also have the option of releasing the laser with the footswitch refer also to chapter 11 10 This func tion is only available in Stage Mode In this case you must move the stage with the mouse or the arrow keys 415109 2614 101 e 09 09 Procedure 11 2 Procedure Before you can use automatic laser functions you must perform various settings You must also have made settings for the laser energy focus if necessary Auto change and LPC Distances refer to chapter 11 4 It is recommended that you experiment in order to ascertain which settings best suit your working requirements Chapter 11 5 gives you instructions on finding the optimal values Youmusthave adjusted the speed of the move ment of the stage during the execution of the laser functions refer to chapter 5 You mu
252. position stage 46 Scroll Rectangle show hide 18 Section scan 60 go to overview scan 60 Select elements element list all 78 single or multiple 78 within a group 90 Select elements monitor 77 all 77 deselect selection 78 multiple 77 single 77 within a group 90 Serial Sections edit elements 103 prepare samples 101 reference elements of original sample 101 reference elements of target sample 103 transfer elements automatically 104 transfer elements manually 105 Set camera parameters 31 Set color 84 Draw Preferences window 84 85 129 current elements 87 for new elements 87 Set image parameters 35 Set Laser Marker 40 Set magnification 39 167 Set RoboLPC joint 130 Set speed keyboard commands 49 menu commands 49 PALM CapMover II 142 202 PALM RoboMover 142 set speed stage 48 Speed Tools 49 stage 48 units 49 Set speed stage keyboard commands 49 menu commands 49 Speed Tools 49 units 49 Set speed trapping laser movement 152 keyboard commands 154 menu commands 154 Speed Tools 153 units 153 Settings 25 adjust Reflector Offset 164 automatic saving 30 camera parameters 31 fluorescence manual microscope 157 fluorescence motorized microscope 157 histogram 36 image parameters 35 Laser Marker 40 magnification 39 167 Objective Offset 43 PALM CapMover 142 PALM RoboMover 142 reset calibration 43 reset Objective Offset 44 reset Reflector Offset 164 restore fac
253. purchased a system with motorized microscope the objective is moved automati cally To move the stage to the CapCheck Motion Q Goto CapCheck Cerler e If you do not have a motorized microscope Move the objective down as far as possible e Click on the menu item Goto CapCheck in the Motion menu Note To avoid damage to the objective or to the sample Move the stage back again to the original position after observing the collec tion vessel For this proceed solely as described below To return the stage to its original position Motion Q Goto CapCheck Ctrl k e If you do not have a motorized microscope Move the objective down as far as possible e Click on the menu item Goto CapCheck in the Motion menu 51 Load Position 5 6 Load Position When the stage is in the Load Position you can easily change the slide and the slideholder Caution Set the objective to the lowest possible posi tion before you move the stage to or from the Load Position If the objective is set too high the stage will collide with it This can cause se rious damage to the objective and to your sam ple if you have purchased a system with motorized microscope the objective is moved automatically To move the stage to the Load Position Motion Fa a Goto Loadposition e If you do not have a motorized microscope Move the objective down as far as possible e Click on the menu item Goto Loadposition
254. r 11 5 415109 2614 101 e 09 09 11 4 1 Setting energy and focus There are several options available for setting the laser energy and laser focus with the Laser Tools on the left side of the program window in default configuration using menu commands in the Laser menu using keyboard shortcuts You can combine the settings for Cut and LPC If you change the setting for one laser function then the other laser function is automatically changed to compensate How you can find the optimal settings is described in chapter 11 5 Note The optimal setting for energy and focus de pend on the objective in use on the micro scope The settings that you enter always apply to the magnification that you have set in the program As soon as you have selected another magnification the values that were last used or the factory defaults are automatically applied This also applies to combining the settings for Cut and LPC Therefore prior to setting laser energy and la ser focus first select the magnification that you want to work with 125 Specifying laser settings To set the laser energy and laser focus with the Laser Tools e Click on the MicroBeam tab in the Laser Tools Laser 3 Laser A esi le A MicroBeam Tweezers MicroBeam Tweezers Cut LPC Cut LPC Energy Energy Energy Energy 100 100 100 lt I Delta 1 1 1 4 q 4 Focus Focus Focus Focus 100 100 100 1
255. rance changes ac cording to the current status e In the Microscope Tools click on the Reflected light tab refer to figure on page 158 e Click on the Shutter icon button The shutter will be opened or closed depending on its position before the command was sent 415109 2614 101 e 09 09 To open and close the shutter on the filter wheel via a menu command Adjustments Fluorescence e Inthe Adjustments menu click on the menu item Fluorescence The Fluorescence adjustments window will open e Select the Fluorescence Shutter tab The window contains three fields displaying a number of buttons Shutter Type field information about the manufacturer Shutter Error field if there are problems with the shutter messages will be displayed here Actual state field this indicates whether the shutter is currently open or closed Buttons for opening and closing the shutter and for closing the window not shown in the figure above e Click on the Close button to close the shutter or on the Open button to open the shutter e Close the window by clicking on OK 159 Selecting fluorescence excitation filter and assigning filter names 14 7 Selecting fluorescence excitation filter and assigning filter names Fluorescence adjustments Fluorescence Fluorescence Filter Reflector Fluorescence Shutter Filter Colors and Names Ho Color L ABI Filter Error Filter
256. ranged horizontally or diagonally AutoLPC Center shot With this parameter you define if a laser shot should be set into the center of an element This makes sense when working with very small or narrow elements for example If the distance of the AutoLPC shots to each other and or their distance from the border line of the element is approximately as big as the element or bigger possibly no LPCshot is set into the element without this function ie the element would not be catapulted Monitor display during an automatic laser func tion You can determine whether the elements are to be displayed or hidden on the monitor while the laser function is in operation The default set ting is that the elements are not to be dis played Position of the stage after completing a laser function You can determine whether the stage is to be returned to start position on completion of the laser function The default setting is that the position is maintained as it was during the last laser function Info When comparing microscope images in the case of serial images before and after cutting or catapulting it is advisable to return to the orig inal position The set values can be saved and reloaded You can reset all values to the factory default settings refer to chapter 3 7 or chapter 3 8 Info It is recommended that you experiment in or der to ascertain which values best suit your working requirements refer to chapte
257. re visi ble 80 To show or hide the anchor points of the elements View Show Points e Click on the menu item Show Points in the View menu The menu command works as a toggle The anchor points are displayed as white squares and are numbered To move elements You can move elements inside the scroll rectangle or move elements at the same time as positioning the stage using the Scroll function refer to chap ter 5 1 2 By this method you can move elements or groups of elements over greater distances quickly R Pointer Tool e Select the element you want to move e Position the Pointer on the element you want to move The cursor changes to a fourfold headed arrow e With the Pointer Tool click anywhere in the ele ment or in the Selection Rectangle if one exists and drag the element to its new position with the mouse button held down If you drag the Pointer out of the scroll rectangle during this process the PALM RoboSoftware changes to the Scroll function and moves the stage The selected element is moved at the same time Info If you have selected multiple elements or an el ement group all selected elements or all ele ments of the groups respectively are moved 415109 2614 101 e 09 09 Processing elements To continue elements lines rectangles ellipses Note Select the laser function Cut before continu ing the element refer to chapter 11 3 for in structions
258. re working in File Mode see chapter 1 7 the images are saved in the specified folder see chapter 10 2 If you are working in Database Mode the images will be stored in the database You can either activate the recorder function man ually or define an event to activate the function automatically The required settings are made in the Recorder and Time Lapse Configuration window or on the Recorder configuration tab in the Preferences and Configuration window The available settings are identical in the two windows To open a window for setting the Recorder parameters nd Recorder Adjustments PALMA obo e Click on the Recorder icon button The Recorder and Time Lapse Configuration window opens Or e Inthe Adjustments menu click on PALMRobo The Preferences and Configuration window opens e Click on the Recorder configuration tab 415109 2614 101 e 09 09 gt Recorder and Time Lapse Configuration Recorder configuration Experiment Mame Description File prefix test_ State finished Show Log File Time Lapse Definition data _ alltime data intervals and durations in minutes Interval In sec Duration in sec endless Mode Video 30 Time Lapse 1 5 10 EA Je C Use auto focus C Switch off transm light cloze tranem light shutter between picture recording Close reflected light shutter between picture recording Trigger Point Defini
259. recommended that the samples are processed in the same sequence as when they were cut out of the tissue block and to transfer each of the elements from one sample to the next 1 gt 2 2 gt 3 if necessary after manual cor rection The sample for which you draw the elements is re ferred to below as the source sample The sample to which the elements are transferred is referred to as the target sample You can carry out the transfer and adaptation with PALM RoboSoftware the adaptation if required also manually 415109 2614 101 e 09 09 To carry out automatic adaptation proceed as fol lows 1 Prepare the samples create three markings in your block before cutting the wafers e g three pinpricks These markings must be clearly visible under the microscope Then cut the wafers and mount them on the slides If required treat one sample the source sam ple in order to make the structures visible 2 Creating reference elements on the source sample Insert the first sample your source sample into the microscope and define three reference points on the markings made before cutting 3 Drawing and processing elements Now draw the elements for all the samples which are to be processed with the laser and join them to gether with the reference points into a group 4a Preparing the target sample insert the target sample in the microscope also define three reference points here at the markings in the same order
260. rint Elements Ed for each type and color to the file print Totals records the total number of ele ments and their total area to the file IR e If you wish check the print Summary and from Screen or Element List pri nt Totals boxes e Inthe Print to section you should specify whether you want to output the elements on a printer or save them in a file i e Click on OK print Summary print Totals Filter criteria Elements Types Freehands Lines Rectangles Circles Dots 4utoCircles Attributes Me Color Type e If you have chosen File the Print Elements to File window will open es e Enter a name for your file select a folder if nec Printer output to default print device essary and click on OK Eile co output Eo File csv formati Your elements will be printed to a file e Choose Elements if you want to print all ele ments or the selected ones only the option selected is available if only certain elements are selected only e In the Types list select the element types that should be contained in your file You can select which kind of element properties the file should contain amp ttributes Mr Color Type Select Atributes and Define Columns default like Element List like Export File e Inthe Attributes list select the desired entry As soon as you select the first entry the Select Attributes and D
261. rties to be archived as a printout To help you with this three functions are available that generate the different output formats Save Elements generates a binary file That binary file can be reloaded or reimported into PALM RoboSoftware refer to chapter 9 2 Info In Database Mode refer to chapter 1 7 your elements are automatically saved in the data base The menu commands Save Elements and Open Elements are therefore not available if you are working in Database Mode Chapter 9 3 explains how you can reload elements from the database Export Elements generates a text file That text file can be reloaded or reimported into PALM RoboSoftware and opened with any text editor You can then choose to view print or edit the data refer to chapter 9 4 The file contains all the properties of elements that are displayed in the Element List window display color number element type laser function objective well area group member ship number of laser operations performed comment length x width position as well as the coordinates of their anchor points Print Elements prints your elements on a printer or generates an Excel import file You can open such a file with Microsoft Excel or with another corresponding program The file con tains all element properties as you have defined with printing of the elements refer to chapter 9 5 Info When you save images you can specify tha
262. s Info Note that PALM RoboMover must be configured before you can define wells refer to chapter 12 2 1 Note Ensure that a well is assigned to each element before you trigger the automatic laser func tions If a well is not allocated to an element the PALM RoboMover remains stationary at the current position and the element is cata pulted into the well last used The allocation of the elements to the wells is also output using the Export Elements refer to chapter 9 4 and Print Elements refer to chapter 9 5 functions In this way you can log which element is in which well 415109 2614 101 e 09 09 8 6 1 Defining wells manually e Selectthe element for which you want to define a well for the next laser operation e In the well drop down list in the Element List window click on the coordinates of the well into which you want to catapult the selected ele ment Info You can also select several elements simulta neously and allocate these all to one well Info The selection manual means that a specific element is not allocated to a specific well If this element is processed using the laser PALM RoboMover will not be moved The element will be catapulted into the last well used Info You can also allocate a different well to an indi vidual element in the Element properties win dow Select the element and open the Element properties window using the Edit gt Change menu command
263. s 3 User Copy of DAPI zwhs User Copy of FITC 2hs a User Copy of Fluoreszenz_Shutker_zu 2vhs a User Copy of Normal 10 Durchlicht zwhs User Copy of Rhodamine zwhs a User Copy of Texas red zvhs a User Copy of TexRed vhs lt Ill Selected Setting User Alles aus z vhs Device E Component Component settings b Microscope CI Extra Shutter 1 Closed ST Microscope Peer Transmitted Light HAL Manual Pcro 00w Microscope Reflector 1 100 Mirror Reflector 4255 Show all Available Components E E PAra H Meteorzhc El Tj Microscope Remove all gt The rest of the procedure is the same in all three cases If you want to add individual components e Select the components in the Available Com ponents list and click on the Add button If you want to add all components e Open the drop down list for the Add button and select Add all If you want to remove a component from your configuration e Select this component in the Selected Setting field and click on the Remove button If you want to remove all components e Click on the Remove all button sd e Click on the button on the Edit column to change the parameters for the related compo nents A window opens in which you can make the related settings Save 415109 2614 101 e 09 09 e Clickon the Save button to save your changes To change the name of a configuration Rename e Select the configuration t
264. s text references etc Show Types Freehands Lines Rectangles Circles Dots AutoCircles e Open the selection list Show Types and select the desired entry You will find the following information on the indi vidual elements in the columns of the table see figure above Color color in which the element is displayed on the monitor Nr sequential number of the element Name name of the Elements you can enter and edit the name via the menu command Edit gt Change Type element type line freehand dot ruler text reference etc Laser function laser function selected for the element you can change the laser function re fer to chapter 8 4 For elements that are not processed with the laser text ruler reference group this cell remains empty 91 Items shown on the summary tab Objective objective that is used to process the element with the laser as a rule the objective that was used when the element was drawn you can select a different objective refer to chapter 8 5 Well coordinates of the well into which the el ement is catapulted when a laser function with LPC is triggered refer to chapter 7 3 2 Defin ing well for catapulting and chapter 8 6 Defin ing wells for the next laser operation Area um area of the elements of type Figure Freehand Line Rectangle Circle Grp name of the group if the element belongs to an element group cut
265. s you can call up the function repeatedly 79 Processing elements 7 8 Processing elements You can process and change drawn elements in to tal or in part in various ways You can edit elements refer to chapter 7 8 1 You can copy elements refer to chapter 7 8 2 You can update the numbering of the elements if you have deleted some elements refer to chapter 7 8 4 You can add comments to the elements or edit existing comments refer to chapter 7 8 5 You can change the color in which the elements are displayed on the monitor refer to chapter 7 9 3 7 8 1 Editing elements There are a number of ways for you to change drawn elements at a later time The options avail able to you depend on the type of the element Figure elements lines rectangles ellipses You can move these elements You can change their shape You can extend lines You can merge two elements into one Dot elements Ruler elements measuring lines and Reference elements You can move these elements Text elements You can move these elements You can change the text You can restore elements changed refer to chap ter 7 7 Elements are treated by PALM RoboSoftware as polygons they consist of straight lines and dots the anchor points Each element consists of at least two anchor points and a line With some functions for editing elements you can work more precisely if the anchor points a
266. scan displayed in the Navigator window either the overview image only or the overview image and the individual tiles Info The overview image of the scan has a maxi mum resolution of 900 x 900 pixels this is significantly lower than the resolution of the in dividual tiles In the second case when you load a saved scan you have the same options as with a scan that has just been created For example if you click on a tile the corresponding section of your sample is displayed in the PALM RoboSoftware main window You can perform an analysis using the AxioVision program and generate elements see chapter 6 10 How to save a scan Thumbnail e On the Scan tab click on the Save button The PALMRobo Input Request window opens e Click on Yes if you want to save the individual tiles in addition to the overview image Click on No if you only want to save the over view image The Save scanned picture window opens The overview image and tiles are saved If you only want to save the overview image several file formats are available e Select the file format in which you want to save the image JPEG TIFF or Windows bitmap e Choose the folder in which you want to save your image e Enter a name for the image e Click on the Save button Info For multi channel fluorescence recordings only the superimposed image made up from the separate channels is displayed in the over view image Info
267. sed well s 4 cer 2 Number of elements to process 4 Start with Field 14 Number of elements per well until Field Fo Remaining elements Cancel e Enter the area that is to be catapulted into each well in the Area field e Select the co ordinates of the first and last well into which the elements are to be catapulted on the Start with Field and until Field drop down lists Info The order of the wells is defined by the setting in the Subtype Configuration field in the RoboMover window line by line or column by column refer to chapter 12 2 2 Note Elements are catapulted into the individual wells until the area you have defined is ex ceeded Then PALM RoboMover is moved to the next well I e the actual area catapulted into a well is in general somewhat larger than the value you have specified Note that the area catapulted into each well can vary if your elements are of varying size 98 Operating Mode sequence area area delta Using this mode a defined area is catapulted into the first well the defined area a delta is cata pulted into the second well the defined area 2 delta is catapulted into the third well etc The area and delta can be selected as required Distribution Calculator Operational Configuration Distribution calculation Operating Mode sequence area area delta area 2xdelta Ww Number of selected well s Number of used well s PR
268. ser Then you can trap a further object and move it to the same place by triggering the function again To move the objects along a defined path e Drawthe element e g a freehand line that is to be used as the path for the laser beam Tweezers Functions Track path Target 3 Freehand e O Trap 1 Trap 2 Track with Trap XY e Select this element in the Laser Tools in the Track path Target field e Select the laser beam along which you want this element to move Trap 1 or Trap 2 The movement can be made in two ways Track with stage in this case the stage is moved under the laser beam in such a way that the laser beam covers the figure required The laser beam itself is not moved during this pro cess Track with Trap XY the laser beam is moved along the selected element The stage is not moved during this process e Click on the button for the required movement type After the movement the stage or the laser beam stops at the end dot for the movement e Click on the Back to start position button if the laser beam is to be moved back to its start position 155 156 415109 2614 101 e 09 09 General introductory remarks 14 Working with Fluorescence Option A supplementary fluorescence unit is available as an option According to the configuration this is suitable for the following applications Configuration Basic non simultaneous appli cation of fluorescence and laser
269. ser beam to a particular pO0i Mt lt lt o oo 150 Settings tor Ene TOO TS WIEN Eus ss 28 be daa a aa Fe wa 150 Activating and deactivating the trapping laser ooo ooo oo 151 Indications of the operating state of the laSer ooo ee es 151 MOVINO OBJects Mela cs 2 ce eS hes ES E E fa ca ap DA UE ee ee E 152 TMemMovINd tabs dra rasa de ae ie ad ae dl li 152 Setting Speed OF THE MOVING trap nadia a A aa 152 Moving the trapping laser beam with the mouse TrapXY 1 TrapXY 2 Mode 154 Moving the trapping laser beam with the keyboard 0 oo eee o 155 Moving the trapping laser beam with the joystick eee eee ee o 155 Moving objects along defined paths 000o oo ooo 155 Working with Fluorescence Option 0 00080 eee eee 157 General Introduetory FEMAarKS ss be dace ac Ap a e a a 157 Settings for working with fluorescence manual microscope a 157 Settings for working with fluorescence motorized microscope 00 00 eee ees 157 Activating a fluorescence settings record 158 Freeze Mod sr Es 6S aca dara iii abeti A 158 Opening ana ClOSING the SHULCCR arma dee a E 159 415109 2614 101 e 09 09 Contents 14 7 Selecting fluorescence excitation filter and assigning filter names 160 14 8 Edita reflector SCCUIN GS diia ana aaa 161 14 9 Editing a fluorescence settings record iio ria e aa A AEE ea eS 162 14
270. shot number of cut and shot laser functions performed 8 2 Items shown on the summary tab Al Element List File Edit Motion Laser Collection Device 268 UcaRO Slidel Slide2 Slide3 Summary Show summary for over all v Color Type of Elements Number of Elements Areas um Remarks 1 Freehand 1 Line 2 167351 1 Freehand 1 Line 412641 1 Rectangle 1 Circle 288911 2 Dots 0 2 Freehands 199446 4 Rectangles 196771 E MM rr Total 14 1265121 pm If you are using a multiple slideholder e g SlideHolder 3x1 0 you can use the field Show summary for to specify whether total values are to be shown for only one slide or for all the slides combined The elements are grouped in the table by color and element type Elements of the types Ruler Text Reference and Group are not listed on the Summary tab Color color in which the elements are dis played on the monitor Type of Elements element type 92 Comment a comment entered by you You can edit this entry via the menu command Edit gt Change For element types Reference Reference is automatically entered here For elements that you have drawn using the Grid Rectangle tool appropriate information is entered automatically morph followed by the number of elements number of rows x number of columns orientation Hx W height and width of the elements Position coordinates of the element Selecte
271. sible 43 Resetting the Objective Offset Click on any dot within the selection rectangle and hold the mouse button down Note If you click inside the rectangle and inadvert ently do not hold the mouse button down the selected elements will not be moved to the cor rect position In this case reset the Objective Offset as explained in chapter 4 8 and start the operation again Align the element you cut previously using the laser with the mouse button pressed with the cut line and release the mouse button Then even elements that are outside the current monitor section will be moved 44 4 8 Resetting the Objective Offset You can restore the default factory settings for the Objective Offset at any time To reset the Objective Offset Adjustments Calibration k Factory Objective Uffset e Click on the menu item Factory Objective Offset in the Adjustments gt Calibration menu Note The Objective Offset will also be reset if you re store the factory default settings refer also to chapter 3 8 415109 2614 101 e 09 09 5 Positioning the stage PALM RoboSoftware offers various methods of moving the stage with the mouse Stage Mode and Scroll with the scroll bars below and to the right of the microscope image with the keyboard arrow keys with the joystick via commands that target predefined positions or elements positioning with the PALM Navigator refer to chapter 6 9
272. speed separately for every type of stage movement Stage movement speed in Stage Mode move using mouse Arrow keys movement speed using arrow keys For movement with the arrow keys as was described for Position you can set higher speeds than for the other movement types This movement type is therefore particularly Suitable for rapid movements over larger dis tances Scroll speed of the movement during scrolling movement of the stage by pressing the left mouse button outside the Scroll Rectangle as well as maximum speed for the movement of the stage using the joystick Position speed for automatic targeting of ele ments and defined positions CapCheck Load Position and speed for automatic laser function LPC the defined dots for this function are tar geted sequentially at the set speed For this movement type as was described for Arrow you can set much higher speeds than for other movement types Cut speed for automatic laser function Cut LPC speed for automatic laser function AutoLPC the defined dots for this function are targeted sequentially at the set speed You can save the speed settings together with oth er parameters in a settings file refer to chapter 3 7 You have two options for setting the movement speed of the stage with the Speed Tools tool with keyboard or menu commands Speed slower Speed faster These setting options are intended particularly to al
273. st and close the window by clicking on the OK but ton Info You can only change the color of one element at a time 87 Working with element groups To change the color using an icon button and the Color Palette Info For text elements the color of the flag can be changed as described below How to change the color of the text background is described further below ng Change Figure Color e Click on the Change Figure Color icon button e Click on the desired color in the Color Palette e Click on the element whose color you want to change e Click on any other tool on the graphics toolbar to turn off the Change Color function again To change the color of the comment back ground for text elements Info If you follow the procedure described below you will change the background color of all the existing text elements It is not possible to change the background color of one individual text element e Select the Text Tool in the Graphic Toolbar e Choose the desired background color from the Color Palette The background color will be changed 88 7 10 Working with element groups You can merge multiple elements into a group For example you need element groups for the Serial Sections function refer to chapter 8 7 An element group can contain any element types In the Element List window groups are processed as elements for each group a separate entry is displayed You can e
274. st have drawn the areas to be cut and catapulted elements refer to chapter 7 If you want to release the laser with the foot switch you do not need to draw any elements You must have selected the required laser func tion for each element refer to chapter 11 3 You must have made the settings for multiple cutting and for three dimensional cutting if you want to use these functions refer to chapter 11 4 2 You must have selected the elements to be pro cessed with the laser refer to chapter 11 6 You must have defined the collection vessel for elements that you want to catapult cap or well refer to chapter 8 6 Note Do not process your sample or at least the ar eas important for your work until you have as certained your optimal setting by experiment Application of the laser with unfavorable set tings can cause irreparable destruction to your sample 123 Selection of automatic laser functions 11 3 Selection of automatic laser functions As a rule you select the required laser function for an element before you draw the element refer to chapter 7 3 1 You can however also change the laser function later For instructions on how to do this refer to chapter 8 4 11 4 Specifying laser settings In order to achieve correct results with automatic laser functions you must set a series of parame ters When relaunching PALM RoboSoftware the set tings apply that were in use when the program
275. stage 41 reset 43 trapping laser movement 42 Camera settings AxioCam 33 exposure time 31 white balance 31 CapCheck 193 definition 51 return to original position 51 targeting 51 Catapult 121 Catapult elements 140 PALM CapMover II 140 PALM RoboMover 140 Catapulted samples view 140 Cellenger 13 Center 193 Centering tool 47 Centric Attribute 73 74 Change color elements 87 Check Position define 169 meaning 168 set 169 415109 2614 101 e 09 09 Collector prepare 138 Color Palette change colors 86 Color value microscope image 37 Combination trapping laser focus 146 Comment add 84 change 84 Context Menu 20 Control microscope 167 Copyright Note specify 113 switch display on off 18 Crosshairs show hide 18 Cursor Mode select 45 Customize User Interface 17 Cut 121 D Database Mode change database 12 change to File Mode 12 InformationCenter 10 load elements 110 PALM RoboSoftware 11 save elements 110 save image 116 Definiens 65 Definiens Cellenger 13 Delete elements all 78 all on a slide 79 any 79 last drawn 79 Deselect elements element list 78 Dot thickness change definition 86 Double Beam System 193 197 Draw elements circle 74 dot 75 grid rectangle 74 line 73 line freehand 73 measuring line 90 rectangle 73 Reference Point 73 resume 81 text 75 Element allocate laser function 94 allocate objective 94 Element List al
276. t If you want to work with a different wafer of the same tissue sample you must enter the re quired data in the Specimen section If you would like to work with a different tissue sample you must describe it in the Source Data section 415109 2614 101 e 09 09 Creating a new set of elements To prepare a new sample for processing in Database Mode File S Enter Select Data e Click on Enter Select Data in the File menu in either the main window or the Element List window Click on the Enter Select Data icon button The Please select or enter data window opens Info If you are using a multiple slideholder a sepa rate tab will be displayed for each slideholder position as well as a tab for entering data for the catapulted samples If you wish to draw elements for more than one slide you must enter data for each slide to be processed 4 Please select or enter data Slide1 Slidez Collection Device Data Source Data Label Liver Type Description Specimen Data Label Description Holder Label Holder Type Experiment Data Label Description Blue labeled fields are mandatory You must enter all blue fields to enable a position for working e Enterthe details for your sample Start with the uppermost field and move down one field at a time Info If you would like to resume work on an existing experiment or with an existing sample you can specif
277. t ed These rectangles are individually cut and cata pulted when an laser function is started Using the morphology conserving operating mode the indi vidual rectangles are catapulted into neighboring wells such that the arrangement of the rectangles is represented by the wells in the collection device Distribution Calculator Operational Configuration Distribution calculation Operating Mode morphology conserving Orientation Column oriented Rows 7 Columns rg E 4 Number of elements to process 8 Start with Field 12E Note Note that you must enter the same values in the Orientation Rows and Columns fields that you defined when drawing the Grid rectangle element If you cannot remember your settings you can view them in the Comment column in the Element List window e Inthe Orientation drop down list select the order in which the wells are to be used e Enter the number of rows and columns in your Grid rectangle in the Rows and Columns fields e Select the coordinates of the first well into which the elements are to be catapulted in the Start with Field drop down list Info The order of the wells is defined by the setting in the Subtype Configuration field in the RoboMover window line by line or column by column refer to chapter 12 2 2 415109 2614 101 e 09 09 8 6 3 Automatic calculation of the distribution of the elements If the parameters for the s
278. t they are to be saved along with superimposed elements refer to chapter 10 However itis always only an image of the elements that is stored and you cannot load or import it back into PALM RoboSoftware window again 415109 2614 101 e 09 09 9 1 Settings for saving elements in File Mode To save elements you can specify a folder When you save your settings later this folder will be se lected automatically However you do not have to accept this specification You can also save your elements later in another folder You can also cre ate a new folder when saving You can determine whether an element file which has already been saved but subsequently changed is to be saved automatically when you exit the program or that the changes made are to be rejected The settings for saving elements can be carried out in this way Adjustments PALMA obo e In the Adjustment menu click on the menu item PALMRobo The Preferences and Configuration window opens e Click on the Elements tab Elements Saving Elements Folder C Documents and Settings drministratori br y Save Elementtiles automatically The name of the currently set folder appears in the Folder field of the Saving Elements section e Click on the button ee The Element folder window will open e Select the desired folder and click on OK to close the window You are back in the Elements register in the Pre
279. t colors and thickness Color Names LPC Distances _ _ E E E Mie Mmnb6n Ta E All colors that can be added to the Color Palette are displayed on the tab All colors with a name appear in the Color Palette You can enter color names by hand or select one of the presettings 415109 2614 101 e 09 09 Changing colors and line thickness You can delete all color names using the Clear All button Info Note that no colors are displayed in the Color Palette if you have deleted all color names and not selected any new colors Select the default color set using the Default button black white red green and blue You can add all the colors to the Color Palette using the Set all button e Click on one of the buttons mentioned to select a presetting or click on the name field for a color that you want to use and enter a name for this color Info You can select any term as a color name that is not just the name of the color In this way you can e g allocate specific colors to certain structures in your sample for instance cancer or liver If you move the mouse pointer onto a color field in the Color Palette the name of the color is displayed as a tool tip To select a color for new elements to be drawn e Select the Graphic Tool with which you want to draw the next element e Left click on the desired color in the Color Palette to select it e Draw the required e
280. t it can be used to acti vate the cutting function e Setthe value for the microscope stage move ment speed so that you can easily follow the movement with the naked eye when in Stage Mode enter Stage Mode and try moving the microscope stage with the mouse Change the speed during movement in order to find an appropriate value Remain in Stage Mode e Release the laser with the footswitch and at the same time move the stage The laser remains in operation as long as the foot switch is depressed e If necessary increase the energy until the laser achieves a cutting effect You will get a cut line in the sample This line will initially be unclear and wide or the sample will be cut incompletely as a result of an inadequate set ting of the laser focus and energy e Continue cutting and change the laser focus Aim for a thinner cut line e If you have achieved that then reduce the energy also with the intention of getting a thin ner cut line e Nowtry to continue optimizing your results by repeatedly changing both focus and energy e Once you have attained the best possible result you should either save or note the appropriate settings refer to chapter 3 7 132 11 6 Selecting elements for the next laser action It depends on your selection which elements are processed using the laser You can make the selec tion in the Element List window If you wish to process all elements that have not yet been processed wi
281. t visible Note If you click inside the rectangle and inadvert ently do not hold the mouse button down the selected elements will not be moved to the cor rect position In this case reset the Reflector Offset as explained below and start the opera tion again e Move the elements holding down the mouse button so that your reference element matches the respective structure of your sam ple When you have moved the window to the desired position release the mouse button Then even elements that are outside the current monitor section will be moved To reset the Reflector Offset Adjustments Calibration k Factory Rellector Offset e Click on the menu item Factory Reflector Offset in the Adjustments gt Calibration menu Note The Reflector Offset will also be reset if you re store the factory default settings refer also to chapter 3 8 415109 2614 101 e 09 09 14 11 Activating and deactivating the PALM RoboSoftware includes a timer for your use timer Use the timer to set the shutter to close again automatically after a period defined by you when you have opened it Note In the Basic configuration with the manual microscope the function Timer is not active Timeout Close Shutter automatically after E a Seconds Select the Reflected light tab in the Microscope Tools Activate the Close Shutter automatically after checkbox to enable or disable the timer En
282. ter 14 9 as follows Microscope Common Reflected Light Hw Settings Objective lalalala Focus Fluorescence Laser DAPI FITE Rhodamine Texas Red e Select the Reflected light tab in the Micro scope Tools e Inthe Fluorescence area select the required set of settings The selected settings record is activated ie all in this record defined settings are entered For changing the settings refer to chapter 14 9 Note When using a manual microscope only the set tings of the filter wheel and the shutter are ac tivated The position of the reflector revolver and the microscope illumination has to be switched on manually on the microscope 158 14 5 Freeze Mode Using Freeze Mode of PALM RoboSoftware you can display the actual fluorescence image on the mon itor and edit it for example by drawing a freehand line or marking objects while the fluorescence light is switched off again In this way sensible flu orescence samples are protected from fading To switch into and out of Freeze Mode Motion se Freeze mode Alt F e Click on the menu item Freeze Mode in the Motion menu With this menu command you can toggle between Freeze Mode and Cursor Mode Note In Freeze Mode all functions for moving the Stage or functions that cause a movement of the stage e g all positioning functions as well as all laser functions are blocked In this way an unintentional movement of the sta
283. ter 5 5 When the microscope stage is at the CapCheck you can observe catapulted samples in the collection device under the microscope Centering the stage refer to chapter 5 7 Navigating with PALM Navigator With one mouse click you can freely center positions on the monitor refer to chapter 6 9 47 Stopping the stage in an emergency 5 2 Stopping the stage in an emergency You can stop the movement of the stage at any time for example in an emergency or in the event of a malfunction To stop all current movement The quickest way to stop all movements is to use the following commands Stop e Click on the Stop icon button in the Tool Bar in PALM RoboSoftware main window or in the Element List window see chapter 8 OF e Press Esc on the keyboard you can only use these commands if PALM RoboSoftware main window is the active window In addition you can also stop the movements us ing a menu command Motion gt Stop ESC e Click on the menu item Stop in the Motion menu in PALM RoboSoftware main window or in the Element List window All current operations are stopped immediately Repair the malfunction if necessary Info The Stop function ends all ongoing actions including any ongoing cutting laser functions and movements of the capture device The Stop function also switches off the trap ping laser if necessary 48 5 3 Setting speed You can set the
284. ter the required time the shutter is to be open in Seconds 415109 2614 101 e 09 09 Activating and deactivating the timer 14 12 Colibri fluorescence illumination with LEDs As an alternative to the standard fluorescence illu mination a module for fluorescence illumination with several light emitting diodes is available The module includes the following features The excitation filters are built into the module You therefore no longer need separate excita tion filters With this module it is possible to switch on LEDs with different wavelengths at the same time and in this way perform multiple channel fluorescence experiments without a fil ter change The switch on switch off times for the illumina tion are very short The fluorescence illumina tion can therefore be limited to the time during which the camera takes the image As a result the sample does not fade as badly The Colibri fluorescence illumination module can be equipped with various LEDs By combining different settings for the various LEDs numerous fluorescence illumination settings are possible You can combine sets of settings as a hardware configuration and save them and assign them to a button in the Microscope Tools Reflected Light tab refer to chapter 15 5 It is then possible to quickly change between different settings using these buttons 165 Colibri fluorescence illumination with LEDS To make settings for the LED fluorescenc
285. th the laser e Deselectall elements If you want to process all elements using the laser including those that have already been processed using the laser e Select all elements If you wish to process individual elements with the laser e Select the desired elements If you want to process all elements of a type and a color using the laser e Click on the related row in the Element List window on the Summary tab 415109 2614 101 e 09 09 11 7 Starting the automatic laser functions Note Take care that you have fully completed all set tings prior to carrying out any of the laser func tions Incorrectly set parameters can cause your sample to be destroyed The laser must be activated to be able to start it Cut A O e Click on the Activate button The laser is activated Instead of the Activate button a Deactivate button appears Cut e Click on the Deactivate button The laser is deactivated Instead of the Deacti vate button a Activate button appears There are several options available for releasing the laser Start Laser Ra Start Lazer function Fi In PALM RoboSoftware main window e click on Start Laser icon button below Speed Tools or click on the Start Laser function entry in the Grafic Tools or click on the Start Laser function entry in the Laser menu In the Element List window e click on the Start Laser icon button or click on the
286. the first or last element drawn the element drawn immediately before or after the element currently centered In the Element List window you can center any element To center defined elements in the main window Motion KK Goto First Element lt q Goto Prey Element gt Goto Next Element PI Goto Last Element e Click on one of these entries in the Motion menu Goto First Element Goto Prev Element Goto Next Element or Goto Last Element 76 To center a defined element using the Element List e Inthe Element List window select the element that you want to center click in the row that contains the element you want to select a Goto selected element Motion Go to Element e Click on the menu item Go to Element in the Motion menu Info You can change the movement speed of the stage when targeting the desired element Refer to chapter 5 3 for instructions on how to proceed speed for the Position movement type 415109 2614 101 e 09 09 7 6 Selecting and deselecting elements You can select elements either on the monitor in the microscope image or in the Element List win dow The result of the two methods is the same elements selected on the monitor are automatical ly also selected in the Element List and vice versa Which method you should choose for your purpose depends on the next operation you want to do with the selected elements If for instance you want
287. the insert or the collector type Select Collector Type SingleTube Collector 500 CM Il DI Click on OK if you want to continue to use the same collector type Use this selection if e g you have only replaced the insert Click on Cancel if you want to continue to use all the configuration data already known to the system including all information on the wells already used e Select the required collector type e Click on the button with the required function An illustration of the collector fitted appears in the CapMover II window The name of the collector used is displayed in the field at the top right 3 Cap Mover II Operation Adjust K IDP 99 SingleT ube Collector 500 CM II Subtype Configuration optional SingleTube 500 CM Il Information f Current field O so Close 415109 2614 101 e 09 09 12 2 2 Defining the sequence of the wells for the next laser action only PALM RoboMover If you are using a collector with more than one well there are various ways you can define the number of wells and into which wells your ele ments are catapulted The PALM RoboSoftware provides various operating modes for this purpose In the RoboMover window you can define whether the wells are to be filled row by row or co lumn by column Make all other settings in the Element List window refer to chapter 8 6 Defi ning wells for the next laser operation Subtype Configuration
288. the operating state of the laser The appearance and color of the icons changes de pending on the operating state of the laser The displays are as follows A The laser is switched off A The laser is switched on The laser interlock has been tripped dla please check microscope support The laser is ready for use but inhibited The laser is not ready for use this indica tion appears during the laser warm up phase or if the interlock is tripped The laser is ready for use and enabled 151 Moving objects in the z axis 13 10 Moving objects in the Z axis Regardless of the configuration of your PALM MicroTweezers you can lift trapped objects in the z axis away from the plane of the slide by altering the focus of the trapping laser beam The object will remain trapped in the plane of the trapping laser focus It is simpler to alter the trapping laser focus with the scroll wheel on the mouse Chapter 13 2 includes an explanation of your options for displac ing the trapping laser focus If you have purchase PALM MicroTweezers with Laser Beam Positioning you can also move trapped objects in the x and y axes i e parallel to the plane of the slide please contact Carl Zeiss MicroImaging GmbH if you wish to upgrade your system 152 13 11 The moving trap With the moving trap movable trapping laser beam you can move trapped objects in the x and y direction i e parallel to the plane of the slide
289. the qualities of the tissue Close amp Cut AutoLPC This function combines the functions Close amp Cut with AutoLPC refer to appropriate sections Line elements are closed and cut completely along the border lines the cut area contents are then catapulted to the cap using multiple laser pulses 122 With this function defined structures are cut out and catapulted intact into the cap When cutting automatically a straight line between the start and end dots of the border line is left as a joint You can determine the length of this with the parameter Distance of RoboLPC joint A laser pulse is then set exactly into the center of the RoboLPC joint which catapults the entire cut area into the cap This function is only possible when there is an LPC membrane between your sample and the slide As soon as you release one of these functions the stage is moved automatically along the defined cut lines or to the defined dots and the laser is activat ed and deactivated as necessary Center RoboLPC tures are cut out and catapulted intact into the cap Unlike with the RoboLPC function the laser pulse is placed in the center of the figure for cata pulting This function is only possible with membrane coat ed samples 415109 2614 101 e 09 09 11 1 2 Laser function Onegjjeg LPC With one click a designated element will be locat ed in the screen center and lifted up by a single la ser pulse Thus single cells or s
290. thickness settings will also be lost The settings in the Draw Preferences window can be saved with your other settings in a file to be loaded later refer to chapter 3 7 415109 2614 101 e 09 09 Changing colors and line thickness 7 9 1 Presetting colors and line thickness Ez Colors e Click on Colors in the Graphic Toolbar The Draw Preferences window will open e Select the Element colors and thickness tab Draw Preferences Cub rea LPC Dot AutoLPC Dot FioboLPC Line RoboLPC Dat Buler Line Text Field Number Field 000000000 BESE E Select values for all values are in pixel 1 to 20 1 to 50 E 1 to 50 Line thickness Dot thickness Ruler thickness E In the Elements section you will find a list of the element properties for which you can change the color the colors available are given in the Colors section In the Select values for section you will find set tings for the dash thickness and dot thickness 415109 2614 101 e 09 09 To change the colors defined for the display of the elements on the monitor e From the Elements list in the left window pane select the element the color of which you want to change Cut Line color of the lines along which the laser is to cut Cut Area color of the element areas if you activate the function for showing or calculat ing areas refer to chapter 7 11 LPC Dot color of th
291. thousandths of the brightest pixels in the image will be set to completely white and the dark est pixels in the image will be set to completely black To set the gamma value to 0 45 al G 0 45 e Click on the G 0 45 button At a gamma value of 0 45 you will obtain realistic color reproduction with the AxioCams 415109 2614 101 e 09 09 To set the brightness contrast and gamma using the sliders only in the Live image window Cy P la 1 Brightness 2 Contrast 3 Gamma LI e Drag the slider to the required value To display the brightest and darkest areas of your image e Click on the Min Max button To save and load a setting Save e Click on the Save button The current settings are saved A further Restore button appears e If you have the changed the settings after sav ing and want to return to the previous settings click on the Restore button 415109 2614 101 e 09 09 Displaying color and brightness values for the micro 3 12 Displaying color and brightness values for the microscope image Advanced Settings e Click on the Advanced Settings button in the Display Tools Camera tab The Live Image window is displayed e Select the Measurement tools tab Live Image Camera selection Measurement tools Display Axio Pixeldata Value Ratio Red 0 3421 Green 0 3346 Blue 0 3233 Brightness Imagedata Minimum Maximum Brightness 29 197
292. tion Laser AAA MicroBeam Tweezers Trap 1 Trap 2 Cut Power Balance Energy 100 CIl In Te 100 44 o 1 e ola 4 4 4 Focus 1 Focus 2 Focus 100 100 Delta 1 F Simultaneous Focus Simultaneous Focus activated e If coupling is activated enter the value for the difference between the values for the focus in the Delta field Info Negative values are also possible 415109 2614 101 e 09 09 To set the power and focus for the trapping laser using the Laser Tools e Click on the Tweezers tab in the Laser Tools Laser po A kicroBeam Tweezers Trap 1 Trap Cut Power Balance Energy 100 Il T2 100 0 4 4 oF 4 Focus 1 Focus 2 Focus 100 100 100 1 1441 4 F 4 4 gt Wheel C Simultaneous Focus Simultaneous Focus deactivated If you have installed a Mono MonoFlex system the only settings you can choose are Power and Focus 1 e Set the required values for Power and Focus e g using the sliders If you have installed a DuoFlex TwinFlex system you can choose between the following settings Power this is used for controlling the entire laser output for both trapping laser beams Balance Tr1 Tr2 this is used to distribute the laser output between the two beams Focus1 Focus2 this is used to control the focus of laser beams Trap 1 and Trap 2 Info If you have purchased a DuoFlex system Doubl
293. tion 2 Manually Foot switch Cut LPC LPC laser function Info As soon as the Recorder and Time Lapse Configuration window or the Recorder configuration tab in the Preferences and Configuration window is open the InformationCenter is launched if it is not already running The InformationCenter is responsible for generating the video sequence Therefore do not close this program as this will mean that no video sequence can be generated To enter the settings proceed as follows e Activate the desired functions using the check boxes and or radio buttons and enter relevant information in the fields All the settings are described in detail below You should enter various items of information on your sample in the Experiment section Enter a name for your series of images or video sequence in the Name field this field MUST be filled in You can enter a description in the Description field The File Prefix field contains the prefix for the name of your video or image files The content of the Name field is entered automatically as a default it can however be modified or added to although the field must not be left blank PALM RoboSoftware automatically adds date 117 Saving images and videos using the Recorder function and time to the prefix entered in the field Every video and image file thus receives a unique name If you are working in File Mode refer to chapter 1 7
294. tion if you wish to work with more than one slide 70 e If you want to catapult samples select the Collection Device Data tab YE 4 Please select or enter data Slide1 Slidez Collection Device Data Collection Device Data Label Collector Type Collector config Blue labeled fields are mandatory You must enter all blue fields to enable RoboMover for collecting e Enter details of your collection device CapMover II RoboMover Info If you wish to resume work on an existing ex periment or with an existing sample you can do this using the selection list for the fields The more button provides access to further fields for entering supplementary details e Click on the more button The Collection Device Data Details window opens Collection Device Data Details Label of Collector Description of collector Label of collector type Description of collector type Cancel e Enter the details you wish to record then close the window by clicking on the OK button Info Slide positions for which you have entered in formation are represented in the Navigator as white areas Positions for which no data has been entered are shown shaded see illustra tion in chapter 6 1 415109 2614 101 e 09 09 7 3 Drawing new elements PALM RoboSoftware provides a range of tools for drawing elements Freehand Line Rectangle and Circle Tool With these tools you ca
295. tive does not collide with the slide refer to chapter 5 5 for instructions on moving the stage to the CapCheck and back 415109 2614 101 e 09 09 To define the Work Position e Setthe focus so that your sample is displayed sharply e Click on the Set Workpos button To define the Check Position SetCheckPos e Setthe focus at the Load Position on a manual microscope e Move the stage to the CapCheck refer to chap ter 5 5 Instead of the SetWorkpos button a Set Check Pos button appears e Setthe focus so that a catapulted sample in the cap is displayed sharply e Click on the Set CheckPos button e Resetthe focus to the load position on a manual microscope e Move the stage back from CapCheck To set the focus to one of the default settings Focus to Load Position Focus to Focus to Work Position Check Position e Click on the desired button 415109 2614 101 e 09 09 The Microscope Tools 15 1 4 Making illumination settings To switch the microscope lamp on and off Q Lamp lamp is switched on Q Lamp lamp is switched off e Click on the Lamp button to switch the lamp on or off To regulate the brightness of the microscope lamp e Drag the slider in the Light area to the required value or click on the left or right arrow button The current brightness is indicated in percent in the field to the left of the arrow buttons To adjust the color temperature of the m
296. tive on the microscope For this purpose you must move the stage to the CapCheck Note Set the objective to the lowest possible posi tion before you move the stage to or from the CapCheck If the objective is set too high the stage will collide with it This can cause serious damage to the objective and to your sample if you have purchased a system with motorized microscope the objective is moved automatically e If you do not have a motorized microscope Move the objective down as far as possible Q CapCheck e Click on the CapCheck icon button The stage is moved to the CapCheck refer also to chapter 5 5 CapCheck You can position the well with the content you want to view in two ways gt Last well K First well gt Next well lt Previous well e Click one of the icon buttons shown above until the required well is selected or In the image of the collector click on the well with the content you want to view bb d sao 000000000000 00 00000000000 EO 00000000000 FO00000000000 co 0 o o EEE o HO 0 0000000000 The well is positioned e If you do not have a motorized microscope manually adjust the image until it is sharp Info The focus is set to the pre set value for view ing catapulted samples Chapter 15 1 3 Defining and setting fixed positions for the focus describes how you can change the set ting 415109 2614 101 e 09 09 Even with the smallest magnification you will not
297. tor and Light one after another and select the desired menu items Fluorescence Entry Specity the Fluorescence Entry Mr Mame Filter don t care Shutter don t care Reflector don t care Laser No Light don t care You can define the entries of the drop down list Filter on your own refer to chapter 14 7 The entries of the drop down list Reflector are prescribed in the control program for the micro scope The three setting options for the shutter have the following effect if you activate one of the saved flu orescence settings records as described in chapter 14 4 OPEN the selected filter will be targeted then the shutter will be opened CLOSE the shutter will be closed then the selected filter will be targeted The shutter then remains closed CLOSE OPEN the shutter will be closed then the selected filter will be targeted The shutter is then opened again The setting for Light controls the microscope illu mination not the fluorescence lamp available with the motorized microscope only e Close this window by clicking on OK e Repeatin the same way if you want to create more settings records 415109 2614 101 e 09 09 To change a set of settings e Click on the row in the table containing the set tings record that you want to edit e Click on the Change button This opens the Fluorescence Entry window e Make your changes as descri
298. tors available each of which contain one vessel All of PALM CapMover II functions can be controlled by PALM RoboSoftware Fluorescence Unit With the addition of a fluorescence unit PALM MicroBeam System can be used as a fluorescence microscope According to the equipment basic or advanced this module can be used for non simul taneous application of fluorescence and laser func tion or even for simultaneous laser function under fluorescence illumination PALM RoboSoftware 1 2 PALM RoboSoftware PALM RoboSoftware is designed to facilitate oper ation and control of PALM MicroLaser Systems PALM RoboSoftware places at your disposal all the functions you require for precise control of the fol lowing functions Display of the microscope image recorded by a camera on the monitor Software controlled movement of the stage Definition and processing of lines and areas elements for marking and further processing with the laser Measurement of dimensions of the sample parts length and area The measuring func tion offers an approximate value only and must be calibrated by the operator with the help for example of an ocular micrometer Saving and loading of the geometry and posi tion of defined elements Exporting and importing of the elements and their properties sequential number type number of laser operations performed size of area position etc Saving the portion of the microscope image
299. tory defaults 30 save elements File Mode 107 save images automatic 116 slideholder 27 Shading correction 34 Single Beam System 195 Slideholder settings 27 Software modes 20 Cursor Mode 45 Stage Mode 45 Trap Mode 154 155 Speed Tools 49 stage 49 trapping laser movement 153 Stage reference 50 195 Stage Mode exit 45 invert movement 45 select 45 set speed 48 415109 2614 101 e 09 09 Status Bar 16 Status display 24 T Three dimensional cutting 127 Time Lapse 181 Time Lapse function cancel 119 Timer alarm 31 Timer fluorescence 165 Tool Bars show hide 18 Tools Laser Tools cutting laser 126 Laser Tools trapping laser 146 147 Microscope Tools 158 167 show hide 18 Speed Tools stage 49 Speed Tools trapping laser movement 153 Track path 155 Trap Mode exit 154 select 154 155 set speed 152 toggle between Trap 1 and Trap 2 154 Trapping laser movement 152 define Reference Position 149 function 193 move to Reference Position 149 set speed 152 Trap Mode 154 155 U User Interface customize 17 415109 2614 101 e 09 09 W Wells define 95 defined sequence 139 operating mode 96 White balance 32 Window Draw Preferences 84 85 86 129 Element List 91 Laser Marker layout 19 microscope 171 states 24 System Info 22 Work Position define 169 meaning 168 set 169 Z Z Focus 29 203 204 415109 2614 101 e 09 09 Carl Zeiss MicroImaging GmbH
300. tware components purchased later setting a short term alarm All other settings are explained in detail else where Settings for moving the stage chapter 5 3 Settings for drawing elements chapter 7 9 Settings for the laser functions Chapter 4 Calibrating the system Chapter 11 4 Specifying laser settings and Chapter 13 MicroTweezers Option Settings for catapulting elements chapter 8 6 and chapter 12 Settings for saving elements chapter 9 1 Settings for saving images chapter 10 415109 2614 101 e 09 09 3 1 Launching settings If your system does not include database function ality you will always be working in File Mode refer to chapter 1 7 In this case there is no need to configure the launch settings If your system does include database functionality you can choose whether to work in Database Mode or File Mode PALM RoboSoftware allows you to specify which mode the program should start in File Mode the program will start up in File Mode database functionality is not available Database Mode with selectable database when the program is launched you can select which database you would like to use or whether you wish to work in File Mode Database Mode with pre selected database you can specify a database for PALM RoboSoftware to open automatically when it is launched Info The procedure for creating a database is described in the InformationCenter manual
301. u The last changed element will be restored Note If you have joined two elements and if you use the function Restore for the new developed element the original element with the lower number is restored The original element with the higher number cannot be restored 415109 2614 101 e 09 09 Processing elements 7 8 2 Copying elements You can copy any element and insert the copy in your image To copy elements Edit Copy Ctrl C Paste Ctrl e Select the element you want to copy either in the main window or in the Element List win dow refer to chapter 7 6 e Click on the menu item Copy in the Edit menu in the main window or in the Element List window e Click on the menu item Paste in the Edit menu in the main window or in the Element List window Info If you want to use the keyboard commands Ctrl C and Ctrl V the main window must be the active window A copy of the element will be pasted the copy is selected e Move the copy to the required position Info You can also select and copy multiple ele ments at the same time Info You can also copy elements using the Stamp Tool refer to chapter 7 3 11 7 8 3 Enabling or disabling the element numbering display PALM RoboSoftware numbers all the elements that you draw As soon as you have drawn a new ele ment it is assigned a number automatically This number is increased by one for each additional el em
302. u are using chapter 3 3 explains how you should notify the software of which insert you are using The illustrations below show the windows for the PetriDish Holder and the SlideHolder 3x1 0 Save elements to file ES C Documents and Settings dministratori My Doc DishHolder 50 Save elements to file EJ Slide1 TestSlide1 palm Slide TestSlidez palm m Slides TestSlide3 palm al Test m all active If you are using a slideholder that can accommo date more than one slide this window can be used to save the elements for one or more slides at the same time The elements for each slide are saved in a separate file so that they can be reloaded sep arately see section To load an elements file in this chapter The window also enables you to en 415109 2614 101 e 09 09 Saving and loading elements in File Mode ter a common name that is used automatically for all files before a suffix is added to produce a unique file name e If you wish to save the elements for all slides Tick the check box all active enter a common file name in the adjacent field to the right Test in the example shown above and then click on the OK button e If you wish to save the elements for one or more slides Tick the corresponding check boxes Slidel Slide2 and or Slide3 in the example enter a file name for each and then click on the OK button Your elements will be saved in the specified folder refer
303. unction a designated element will be located in the screen center and lifted up by a single laser pulse by one click Thus single cells or sperms are isolated fast and easily simply by clicking on the cell of interest Sr with element detection without element detection This tool has different functionalities depending on the selected menu entry Menu entry with element detection If an element was not lifted up properly e g because of too less energy you can use this tool to lift up the element again Select the tool and then click with the mouse pointer on the point within the element laser shot The PALM System recognizes the element automatically positions the capture device correctly if a well has been assigned performs the laser shot and increases the shot counter of this element Info If the PALM System cannot identify an element e g you click on a position where no element is located no laser shot will be released The stage will be directed to this point only Menu entry without element detection If this menu entry is activated the stage will be centered on the clicked point and the laser shot will be released independently of drawn ele ments or the position of the capture device You can use this functionality to collect small pieces from a glass slide on a fast way e Open the Onegqick LPC menu on the Grafic Tool bar e Click on the required menu item e Click on the elements resp
304. win Flex system can be moved within the field of view moving trap This means that you can trap two objects and move them relatively towards each other You can select different power and focus values for each of the two split beams Three dimensional cutting The element is cut several times During each cut ting process the focus is changed by a specific amount such that each cutting pass is made in a different plane on the sample In this way it is pos sible to cut even thick samples without problems Element An element is a drawing on your monitor Ele ments serve to define selected areas for the appli cation of laser functions to annotate selected points of the microscope image to measure length and areas and to define reference points Accord ingly the program differentiates between five types of elements Figure freehand line rectan gle and circle dot ruler text and reference point 193 Extended focus The Extended focus option is particularly suitable for thicker samples or samples that do not lie ex actly on a plane Such samples can normally not be displayed with full sharpness the focus must be changed for different areas of the sample Us ing the Extended focus function several images are taken of the sample with different focus set tings The program analyzes these images and cal culates from the sharp portions of each image a sharp overall image that is then displayed on the monitor Fi
305. witch between Cursor Mode and Stage Mode or Freeze Mode and between Cursor Mode and Trap 1 or Trap 2 Mode manually All other modes are activated or deacti vated automatically by the program when the cor responding functions are selected Cursor Mode This is the standard mode which opens by default when the program is launched In Cursor Mode you can open menus select functions click on icon buttons or draw elements Stage Mode In the Stage Mode you can syn chronize the movement of the stage with that of the mouse movement refer to chapter 5 1 1 In Stage Mode you can only access commands and functions that can be operated with key board commands Freeze Mode In Freeze Mode the movement of the stage and the microscope image on the screen is frozen In Freeze Mode all functions that cause or require movement of the stage such as scroll ing positioning of elements targeting specific points positioning a Laser Marker laser func tions are blocked This function is particularly useful when work ing with fluorescence refer to chapter 14 5 Trap 1 Mode and Trap 2 Mode only if you have purchased a system with one or two movable trapping laser beams In Trap 1 Trap 2 Mode you move the trapping laser beam syn chronously with the mouse refer to chapter 13 11 415109 2614 101 e 09 09 2 4 Help and information about PALM RoboSoftware PALM RoboSoftware includes a general Help Function PALMRobo
306. with keyboard commands The advantage of this method is that you can cut and test various settings at will within cer tain limits You should try to move the stage more slowly if possible so that you can evalu ate the cutting effect during the movement As the cutting action is also dependent on the speed of the movement you should move the stage at the speed that you want to use subse quently for the automatic cutting function refer to chapter 5 3 415109 2614 101 e 09 09 Finding suitable laser settings with the automatic laser functions You draw an element cut along this line with the automatic Cut function and change energy focus and speed during the cutting operation by using keyboard settings The advantage of this method is that you do not have to work with the footswitch mouse and keyboard at the same time but with the key board alone However you will probably have to draw and cut several elements until you have found your optimal settings With this method you can also set higher speeds but only evaluate the cut after conclud ing the automatic laser function Apart from that you can also find settings for multiple cutting processes with this method since the laser cuts along exactly the same line during each operation In order to find the optimal values for the catapult ing functions you must draw appropriate ele ments for the required automatic catapult functions LPC AutoLPC RoboL
307. y time If you have purchased a system with two movable trapping laser beams you can define a separate Reference Position for each beam To define a reference position for a trapping laser beam e Insert the slide that you colored with the black felt pen into the stage you can also use a sam ple that you no longer need for your work e Switch the trapping laser on refer to chapter 13 8 e Setthe power so that you can see the effect of the trapping laser beam e g an eroded site on the colored slide e Using one of the methods described in chapter 13 11 move the trapping laser beam to the location where you want to mark the new Reference Position and switch the trapping laser off Motion Set Reference 1 e Trapl 2 Trap ee la e Drag the cursor on item Set Reference in the Motion menu e Click on the name of the trapping laser beam for which you want to re define the Reference Position Trap 1 or if available Trap 2 To move the movable trapping laser beam to its Reference Position Motion Goto Reference k Group 1 ENA Trap 1 2 Trap ns fap e Drag the cursor on item Goto Reference in the Motion menu e Click on the item Trap 1 or Trap 2 The selected movable beam will be moved to its Reference Position 415109 2614 101 e 09 09 13 5 Moving trapping laser beam to the center Using a menu command you can move the trap ping laser beam to the center of the
308. y scanned tiles are shown in the slide image in the Navigator win dow When the scan is complete you can work on them as described in chapters 6 5 to 6 11 Info When using automatic focusing make sure that the thickness of the samples on the differ ent slides is approximately the same 415109 2614 101 e 09 09 How to scan a single specified area e Make sure that you have made all settings as described in chapter 6 2 Slide Navigation QD Selected slide 2 e On the Navigation tab select the desired slide Click on the slide or click on one of the Slide Navigation arrow keys in the top right to switch from one slide to another e Select the Scan tab The tab displays a single colored area with the same proportions as the rectangle you drew with the mouse see Figure on next page 57 Scanning a sample Navigator Navigation Scan AxioVision Analyze VisDat Section Navigation Objective Fluar 10x 0 50 M2 Information Slide Section O 110 tiles Not scanned yet Thumbnail Navigation Mode Live Focuscorrection O None O Suspend scan for focus Use Auto Focus every 5 tiles Draw Elements C Marker elements The following details can be found in the When the scan is complete the Information Information section some of them depend on section shows the magnification at which the scan the slideholder fitted those specified here relate has bee
309. y this using the selection lists for the Label fields The more buttons bring up further fields for you to enter additional details of your source your sample and or your experiment 69 Creating a new set of elements e Click on the more button in the area for which you have supplementary information to enter One of the windows Source Data Details Specimen Data Details or Experiment Data Details will open Source Data Details Label of source Description of source Type of source FE Archived flag Archive name o Archive location o Additional Information o Cancel Specimen Data Details Ra liver oon Specimen staining hone Description of specimen fite Label of holder slide 5647 Description of holder o Cancel Experiment Data Details Label of experiment o Name of experimenter o Description of experiment o Video file name of Time lapse video o Log of experiment o Cancel The first field in each of these windows is pre populated with the Label entries from the win dow Please select or enter data If you have added your own additional fields to the database these will also appear in the Data De tails windows the InformationCenter manual explains how to create additional fields e Enterthe details you wish to record then close the window by clicking on the OK button e Proceedin the same way for each slide posi
310. y to the mag nification that you have set in the program As soon as you have selected another magnifica tion the values that were last used or the fac tory defaults are automatically applied Therefore before setting the speed first select the magnification that you want to work with 415109 2614 101 e 09 09 Specifying laser settings To enter these settings proceed as follows You can make the settings in the Preferences and Configuration window or in the Draw Preferenc es window Open the Preferences and Configuration window as follows Adjustments PALMA obo e In the Adjustments menu click on the menu item PALMRobo The Preferences and Configuration window opens TO Oon eEZsgErR _ 5 Laser LPC Distances um for Objectree LD Plan Meofluar 40x 0 6 Korr e Distance of AutoLPC shots Distance of AutoLPC shots from line A AutoLPC shots diagonal AutoLPC Center shot Distance of RoboLPC joint lt x e Select tab Laser Open the Draw Preferences window as follows Colors e Click on Colors in the Graphic Toolbar The Draw Preferences window will open e Select the LPC Distances tab Draw Preferences Element colors and thickness Color Names LPC Distances LPC Distances um for Objective Fluar 5x 0 25 v EM Distance of AutoLPC shots o 320 Distance of AutoLPC shots from line F AutoLPC shots dia

September 2009 Operating Instructions PALM RoboSoftware 4.3 SP1

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