mmi UVCut – User Manual
Contents
1. Software description 6 1 Overview of functions The MMI CELLTOOLS together with the MMI UVCUT plug in provides all necessary controls for e displaying live video e saving images e motorized xy stage e laser adjusting the power and laser focus e storing the preferred camera settings e manipulating the automatic cap lift This results in an easy and user friendly method for e scanning the sample e documenting the sample e marking the path for the laser cutting around single cells or cell clusters e marking several areas to be cut in one operation e storing the marked cutting paths for later cutting e dissecting the marked sample areas automatically e collecting the dissected areas without radiation radiation or risking contamination of the isolated material s The operation of all modules is controlled by the comfortable and easy to use MMI CELLTOOLS The main application gives all the tools necessary for displaying the video saving the im age adjusting the camera control and basic xy stage move ment The MMI UVCUT plug in adds the specific MMI CELLCUT functions module Additional plugins for MMI CELLMANIPULATOR and MMI CELLEXPLORER are available 01 05 MMI MMI CELLTOOLS software with UVCUT plug in Additional plugins 10 MMI UVCUT 6 2 Quick Start 6 2 1 System start up To start MMI CELLCUT proceed the following steps as follows Start up the PC and allow the boot pro2. sample and chosen objective Typically fluorochromes will differ in emitted intensity For fine tuning use the scrollbar Gain This setting is used until you change it again As standard the camera control is set on Remote In this mode Camera control MMI CELLTOOLS can switch the exposure time between stan dard and fluorescence Other camera setups are part of the installation of the system If you want to change to camera set tings which are not accessible through the camera menu of mmi CellTools choose Manual The recommended and installed settings for the ccd camera are listed in the annex of this manual If you change the illumination devices of your microscope you White balance should run the automatic White balance If the button is pressed the white balance is reset If the button is released color settings will be used To run the white balance you have to observe a white object white sheet of paper with the microscope If the observed object is not white the white balance will fail and the standard settings will continue to be used 6 3 5 Fluorescence To define settings for a fluorescence experiment it is recom mended to start with defining a new setup see chapter 6 3 2 For a set up name use Fluorescence dye1 for example Now you can set the ccd camera on fluorescence exposure Long term integra time by clicking the Fluorescence button A The fluores tion CCD camera cence camera settings and vid
3. the E button and get the Objective Editor To use this editor follow the method of Slide Editor described above If you create a new setup all objectives will automatically been copied from the active slide The distance between two points in the visible sample area Choose and con depends of the magnification of the active objective Therefore firm magnification the program needs information on the chosen magnification for the table control From the menu choose the appropriate ob jective for this magnification If the xy stage is still not following the mouse movement the objective has to be recalibrated See Objective calibration 01 05 14 MMI UVCUT 6 3 3 Video settings In the standard setup the MMI CELLTOOLS are using a specific frame grabber board Only with this frame grabber board is the fluorescence mode fully supported In this case the user has no video setting available In special cases the MMI CELLTOOLS also support Microsoft Windows vfw video drivers If the standard board is not in stalled the user can select between different video drivers supported by Windows Depending on the video driver se lected specific video setting can be available 6 3 4 Camera configuration For different illumination setups like bright field phase contrast or fluorescence the ccd camera needs different parameters for integration time and gain to produce good images The camera settings can be changed in the m
4. 50 pieces 50103 Boxed not individually wrapped Box of 50 pieces 50104 Membrane A4 sheet 50105 Glue Fixogum 125 g 7 2 Caps 50201 tube with adhesive lid with diffusor 500 ul standard size 50202 tube with adhesive lid with diffusor 500 ul standard size pack of 50 pieces 50203 tube with adhesive lid without diffusor 500 ul standard size 50204 tube with adhesive lid without diffusor 500 ul standard size pack of 50 pieces tube with adhesive lid with diffusor 1 5 ml 7 3 Cell Chamber 50301 cell chamber with membrane complete with Petri dish coated with sili cone pack of 10 pieces 01 05 33 MMI UVCUT MMI 8 Maintenance Maintenance and service should be only performed by qualified Maintenance and MMI personnel or our designated representative s service The MMI CELLCuT contains no user serviceable parts 01 05 34 MMI UVCUT 9 Trouble shooting This Section provides help for problems that can occur when working with UV Cur Most of the problems can be easily solved When needed please refer to the problem description and the trouble shooting tips 9 1 View Image not clear too dark too bright or no image on monitor e Check the camera set up and compare it with the camera manual e Check that there is sufficient illumination from the white light and that the light is not obstructed e For improved image quality use the diffuser e The microscope port button should b
5. E The cursor appears as a symbol of the chosen form Move the cursor pressed left button over the length of the area you want to mark If you right click on these buttons you can choose to draw a circle or ellipse or respec tively a rectangle or line You can also give a defined dimen sion to your mark by typing the value in the corresponding in put box The drawing mode can be activated through the buttons in the MMI UV CUT plug in or in the context menu The context menu can be activating by pressing the right mouse button with the cursor in the video window and choose Paint The last used drawing mode will be activated A quick switching between moving and drawing mode is possi ble by pressing the Space key on the keyboard You can delete the current contour by pressing the lt del gt key If you want to delete any object choose the delete mode with _ button and click on the contour you want to delete With the context menu you also can delete one or all contours Of a group or all contours on your slide 01 05 MMI Drawing modes Freehand drawing Preset form Contours Switching to draw ing mode Delete contours 26 MMI UVCUT MMI 6 4 2 Using the MMI lsolationCap The MMI IsolationCap for a Single Step Collection contains a Image improve diffuser insert in the lid Position the lid over the area of tissue ment using the you want to review for maximum image quality during micro mmi Isolation d
6. If liquid gets inside the system do not attempt to use it Contact MMI Unplug all electrical supply before cleaning the system Do not use cleaning fluids or sprays but only smooth and dry cloth If the stage control is not calibrated table movements can be sudden and fast As sure that the work area around the table is free of clutter and material Read the manual of your microscope for specific microscope precautions If you do not have the manual contact your microscope provider or MMI 2 installation of the system The system is installed by MMI Service personnel or our des Installation by ignated representative in the laboratory of the customer After MMI the installation training will be provided in the use and opera tion of the system The customer should not change the instal lation of the equipment see precautions in Section 1 3 The MMI CELLTOOLS instrumentation family The MMI Cell Tools are a fully modular instrumentation family including the following components MMI CELLCUT laser microdissection to isolate single cells or areas of tissue MMI CELLMANIPULATOR optical tweezing to manipulate cells or beads with an optical trap MMI CELLECTOR automated micro pipetting to me chanically manipulate cells or beads with a capillary and mechanical micromanipulator MMI CELLEXPLORER pattern recognition software for PC based image analysis Fluorescence microscopy extension for both fluores cence and
7. chosen detail With this navigation method you always see the position on the slide You can also move to the position of interest by double clicking into the overview area with the left mouse button 01 05 MMI EW Area of inter est B start scan D Stop scan a WINDOWS image viewer 3 CODy to clip board save image on hard disk Navigation 18 MMI UVCUT MMI Deceleration of 6 3 8 Additional Settings for the xy stage the xy stage If you press in the menu bar item Settings gt X Ytable gt XYmotionparam it takes following window 10 x A Table lt YMotion Velocity KO Accelerate 40 Laser Ston mpu millisecond 50 J OK Cancel In this window you can limit the speed and acceleration of your motorized xy stage This could be necessary if you mount a heavy load on your xy stage or if you re working with liquid cells otherwise no changes are recommended from the factory settings Additionally you can define the time for a single laser shot only Single Laser pulse for MMI UVCUT by default is 50 msec A range of 50 100 duration msec is typical 6 3 9 Calibration procedures The MMI CELLTOOLS are calibrated by MMI during the in strument installation When you remove or add objectives or if the original camera position has been changed you have to repeat the calibration as described below 6 3 9 1 Camera orientation To check the camera orie
8. is available to process this command for overcome these difficulties right mouse click under overview window Set inspection position Ctrl I Overview Image Quality and choose Overview Image Quality item Because of smart and fast algorithm you will see nearly no visibly differences for 10 or yet more percents but size will be match less Overview Image Quality Form x Overview Image Qualit Height OF Cancel by default mage Quality is 50 01 05 24 MMI UVCUT MMI 6 4 MMI CELLTOOLS UV CUT Plug in The MMI UV CuT plug in appears as a separate sheet on the The MMI UVCuT right side of the program window plug in To switch from one plug in to the other you only have to click on the appropriate sheet 6 4 1 Drawing the cutting contour J KE n kw l Ji A 7 F na a E i j a a T a a z mn mY h j n 7h n 25 MMI UVCUT The laser cuts along the contour you have drawn around the interesting area you want to microdissect You can choose between freehand drawing and preset forms such as circles ellipses lines and rectangles respectively To activate the freehand drawing mode press The cursor appears as a pencil Press the left mouse button to draw the contour around the area you are interested in The program closes the contour automatically if you have selected the option AutoEnclose Shape in the menu UVCut gt Draw and Cut Choose O or
9. press the button Calibrate The Calibration window will be minimized and you will find two Space key marked areas in the top left and bottom right of the screen calibration areas Move one small and remarkable point of the sample in the upper left calibration area Mark it with a left mouse button click Move this point to the bottom right calibration area 01 05 20 MMI UVCUT MMI Mark this point again with a left button click Confirm calibration with a right mouse click Now the calibration window comes back Accept the calibration by pressing OK or discard your changes by pressing Cancel Remark You always can switch between the move mode and the mark mode by pressing the Space key on your keyboard In the move mode you see the hand courser in the mark mode the marker cursor 01 05 21 MMI UVCUT 6 3 9 3 Lens OffSet calibration When you install a new objective into your microscope you have to calibrate Lens OffSet Over the time of operation it may be necessary to recalibrate the control when contours and cutting lines are no longer exactly fitting Calibration 1 Take a slide with sample 2 Make an objective calibration if necessary See Objective calibration 3 Choose an objective in the objective revolver and select the corresponding objective in the program Make a shape around some noticeable object Move a noticeable object into center of field of view 4 Change an objective phy
10. radiation is used The membrane and the adhesive lid are chemically inert and have no influence on further molecular biological processing 01 05 MMI In the figure change micro scopic objective to microscope objec tive Separation of cells from the slide Single Step Collection Protective membrane No radiation directly on dissectates MMI UVCUT The membrane material is a thin PET membrane The mem brane is transparent and does not perturb the light beam The lid of the reaction tube also contains a diffuser insert The lid improves the image quality remarkably and can be placed directly on top of tissue and membrane during all operations 5 2 Preparation of the slides The Single Step Collection of the dissection requires special slides The slides are provided with a 1 4um thick clear PET membrane Intermediate slide tissu Dissection slide r 01 05 membrane tissue slide membrane slide The tissue is mounted on the membrane as on an ordinary slide Paraffin sections cryo sections or smears can be used After the usual processing deparaffinize staining etc the membrane with the tissue is inverted and placed onto a new glass slide and fixed in position on the microscope stage Thus the tissue is now under the membrane and protected against con tamination Image quality Protective membrane MMI MMI UVCUT 6 MMI CELLTOOLS
11. usual microscope features are available The MMI CELLTOOLS software controls the laser image capture and scanning stage actions without blocking any other microscope action 01 05 MMI Isolation of cells Advantages of the system MMI ISOLATIONCAP Scan and cutting time shifted opera tion possible Components MMI UVCUT high precision xy stage ee us __________ microscopic objectiv a High resolution olor CCD camera 5 Handling of samples 5 1 Single Step collection using the MMI Isolation Cap Microdissection for the isolation of cells is only useful when you can remove the parts of the tissue you are interested in from the tissue surroundings and from the slide The single step collection makes sampling of one or several isolated areas easy and contamination free The Single Step Collection uses a protective membrane and a reaction tube with a special adherent lid The purpose of the protective membrane is to avoid contamination of the sample and to facilitate easy removal and collection of the cut area lid of tube EB sample membrane tissue slide With the laser beam originating from below the stage the laser cuts through the tissue and the membrane The separated tis sue and membrane are collected with the lid of the reaction tube The laser cuts around the cells to be isolated For the collection step no additional
12. 0 go left screen 90 go right screen 90 go up screen 90 go down Emergency stop motion Shift the contours relative to the sample Press ALT while moving the sample 01 05 MMI ENTER key ESC key ESC key SPACEBAR key HOME key END key key key PAGE UP key PAGE DOWN key UP ARROW key DOWN ARROW key LEFT ARROW key RIGHT ARROW key MOUSE WHEEL UP MOUSE WHEEL DOWN NUMPUD 6 key NUMPUD 4 key NUMPUD 8 key NUMPUD 2 key NUMPUD 5 key ALT Move mode 41
13. MMI UVCUT MMI UVCut Software manual v2 0 Molecular Machines amp Industries AG MMI www molecular machines com 01 05 1 MMI UVCUT MMI CELLTOOLS MMI UVCUT User Manual 1 SECURITY ADVICE 1 1 Laser safety 1 2 General safety 2 INSTALLATION OF THE SYSTEM 3 THE MMI CELLTOOLS INSTRUMENTATION FAMILY 4 PRINCIPLES OF MMI CELLCUT 4 1 System set up 5 HANDLING OF SAMPLES 5 1 Single Step collection using the MMI Isolation Cap 5 2 Preparation of the slides 6 MMI CELLTOOLS SOFTWARE DESCRIPTION 6 1 Overview of functions 6 2 Quick Start 6 2 1 System start up 6 2 2 System preparation 6 2 3 Handling a new slide 6 2 4 System turn off 6 3 MMI CELLTOOLS Main Application 6 3 1 Main Window and plugins 6 3 2 User specific database 6 3 3 Video settings 6 3 4 Camera configuration 6 3 5 Fluorescence 6 3 6 XY stage motion 6 3 7 Slide overview for navigation 6 3 8 Additional Settings for the xy stage 6 3 9 Calibration procedures 6 3 9 1 Camera orientation 6 3 9 2 Objective calibration 6 3 9 3 Lens OffSet calibration 6 3 10 Multi User Report 01 05 MMI 10 10 11 11 11 11 L2 13 13 13 15 15 16 17 17 19 19 19 20 22 23 MMI UVCUT 6 3 11 Hardware self monitoring 6 3 12 Overview Image Quality 6 4 MMI CELLTOOLS UV CUT Plug in 6 4 1 Drawing the cutting contour 6 4 2 Using the MMI IsolationCap 6 4 3 Cutting of objects 6 4 4 Group handling 6 4 5 Dista
14. UVCUT MMI 11 Customer support For questions about the MMI CELLCUT contact technical consumables warranties please contact Europe and Asia _ Za service molecular machines com Molecular Machines amp Industries AG MMI Flughofstrasse 37 CH 8152 Glattbrugg Switzerland 41 0 1 8 09 10 10 SI 441 0 1 8 09 10 1 North America lmm Molecular Machines amp Industries Inc North America Don Armstrong Ph D P O Box 23991 Knoxville TN 37933 1991 USA 1 865 988 7500 1 865 988 6666 MMI via the Internet www molecular machines com 01 05 38 MMI UVCUT MMI 12 System data overview Required minimum workspace The table for the microscope laser optical equipment com puter monitor and keyboard requires a minimum dimension of 1 20m x 0 90m The computer should be positioned under or near the table The camera computer connection cable is 2m long to ensure reliable data transfer Components Name Description Specifications microscope research microscope inverted with fluorescence port Zeiss Nikon standard con figuration Olympus Leica xy scanning table scanning table with stepper scanning area 120x100 mm2 motors accuracy lt 1um laser system wavelength 355nm line voltage 200 240 VAC or 100 110 VAC 50 60 Hz 1 0A Optical and elec Laser control unit tronical coupling MMI unit ccd camera with DXC 390P 3CCD Color Video Camer
15. a power supply CCD Chip 1 3 inch 3 Chip with Power HAD technology horizontal resolution 800 TV lines Sensitivity 2000 lux at F8 single to noise ratio 61dB computer Pentium PC with Win XP Pro With RGB frame grabber control software MMICELLTOOLS with 32 bit Software MMIUVCUT plug in 01 05 39 MMI UVCUT A Software and hot key List A 1 Shortcuts MMI CELLTOOLS application run MMI CELLTOOLS Main Window Main Menu File New Slide Save Image Start Record Movie Stop Record Movie Exit Edit Copy Image for Move mode Copy Shape for Paint mode Insert Shape for Paint mode Video Driver non Flashpoint card Video Type non Flashpoint card Help Help Topics Info MMI UVCUT only Cap lift up down Select next marker Select prev marker 01 05 MMI CtritAlt F12 Alt F Ctrl N Ctrl S Ctri Alt S Ctrl Alt D Alt F4 AItt E Ctrl C Ctrl C Ctri V Alt V Ctrl D Ctri T AIt H F1 Alt l F2 Ctrl Tab Shift Tab 40 MMI UVCUT A 2 Hotkeys For any window Accept changes amp close window Close current opened window Stop current procedure or cancel last command Main Window Changing type of cursor screen to first marker go screen to last marker go Screen to next marker go Screen to prev marker go Screen to next marker go screen to prev marker go Screen 20 go up Screen 20 go down Screen 20 go left screen 20 go right Screen 20 go up screen 20 go down screen 9
16. cess to complete reaching the Windows desktop Turn on the microscope white light power supply Energize the electronic controller with the key A yellow LED will illuminate Start MMI CELLTOOLS software and wait until the soft ware has finished the load procedure Power the laser by pressing the button on the electronic box controller A green LED will illuminate 6 2 2 System preparation For the handling of the MMI UVCUT start with the definition of a new setup For simplicity we call the first setup Default In this setup the laser power and focus settings the on screen laser position the xy stage calibration and the camera settings for each ob jective will be stored To reach the setup explorer using the PC mouse press E on the left side of the setup list If the objective you want to use does not appear in the objec tive list define a new objective For each objective you prefer to use follow the calibration pro cedure for laser position laser focus laser power settings xy stage calibration Chapter 6 3 9 2 and camera settings Chap ter 6 3 4 6 2 3 Handling a new slide Prepare your sample as described in Chapter 5 or following the detailed application notes provided by MMI Define a new slide by using the menu item File gt New Slide or press E on the left side of the setup list The slide editor will open and you can add a new slide by pressing the button All docum
17. e set on side 9 2 Movement The movement is not accurate and repeatable the stored posi tions cannot be found again e Check if the sample is fixed correctly on the stage and cannot move 9 3 Drawing The drawing line is difficult to see e Choose dark line colors for bright samples and bright line colors for dark samples Increase the thickness of the line if needed 9 4 Cutting No cutting can be observed e The laser has not been switched on key and button e The magnification chosen not correspond to the objective in use e The laser power is too low e The laser focus is not well adjusted e The cutting is not perfect bridges remain in the cutting path Repeat the cut if necessary e The cutting speed is too high e The cutting path does not correspond to the drawn contour Check the camera orientation 6 3 9 1 The laser position 01 05 View Movement Drawing Cutting MMI 35 MMI UVCUT may need to be adjusted Incorrect magnification may be selected e The calibration may have changed Recalibrate when nec essary 9 5 Collection The dissected objects are not collected e Make sure that the lid is placed directly over the treated area and lowered down onto the membrane Ensure that it is not on the metal frame e Assure that the cutting action has been complete and suc cessful Adjust laser settings or repetitions to achieve full penetration of the beam through the m
18. embrane and tissue 01 05 Collection MMI 36 MMI UVCUT 10 Warranty In Europe and Asia The optical system and the control system are protected against defects in materials and workmanship for a period of one year from the date of installation This warranty is limited to repair or replacement of system components which returned to MMI within the given one 1 year period Unauthorized repairs replacements and modification will cancel all warranties The microscope is not included in the warranty given by MMI AG The microscope warranty depends on the manufacturer of the microscope the country of location and the responsible distributor Consult Service at MMI AG for details In North America For the standard CellCut instrument configuration the optical system including the microscope and the control system are protected against defects in materials and workmanship for a period of one year from the date of installation Any failure resulting from unauthorized system modification abuse ne glect misuse or acts of nature are not covered under this or any warranty Custom instrument configurations may be covered under the terms of the standard warranty via written acknowledgement by MMI Inc Unauthorized instrument repairs replacements or modifica tions cancel all warranties expressed or implied Consult Service at MMI Inc for details 01 05 MMI software optical system microscope 3 MMI
19. entation will be stored in a 01 05 MMI GetUp El default T Define new setup Objective E Ay Choose objective Prepare sam ple Define new sam ple slide New Slide Ctrl M 11 MMI UVCUT folder with the same name which you used for the slide Define the membrane slide work area by moving to the upper left corner of the membrane window and pressing F Limit1 and then by moving to the bottom right corner for the membrane Limit2 Use the objective with the lowest magnification standard is 4x By pressing the scan button the software creates an over view of sample e g a roadmap window and pressing By double clicking into the overview you can navigate into the area of interest You can also use the cursor keys to move the xy stage or drag the blinking rectangle with the mouse Move the Caplift down by pressing the corresponding button After lowering the Caplift you should adjust the microscope stage fine focus as needed Activate any button in the toolbar with a mouse click For ex ample the freehand drawing tool Z and enables the user to draw a line around the object to be microdissected Press the cut button to microdissect the marked object Lift the cap holder to collect the microdissected sample Now everything is clear to take out the cap and proceed with application 6 2 4 System turn off Shut down MMI CELLTOOLS by using the Exit command
20. enu Settings gt Camera RC FA Camera Configuration E Camera Control bianual Il Remote Exposure time sec r Gain dB Shor Long Gain Gain Limit FL ee Ne Gis Eee 0 ie 6 t 1 125 Coa 1 250 r 05 1 500 z i a Soo o ceo e 25 A 1 4000 m 30 110000 jj Boy 6 1 20000 E f 6 0 f 1740000 Coon Kev ev Manual Auto Electronic shutter OFF __Manual lize balance Auto no AB DK Cancel Default For the gain the automatic adjustment by the camera can be selected In this case the camera measures the light intensity and attempts to choose appropriate settings With the gain limit you can define the maximum gain which is used by the camera in automatic mode Note that a high gain induces a lot 01 05 MMI Auto illumination 15 MMI UVCUT MMI of more noise and the image quality may become poorer For standard i e white light choose an exposure time from Standard light 1 125 to 1 100000 according to light conditions sample and chosen objective In the FL mode the camera corrects for the fluctuation in the illumination 50 Hz Europe 60 Hz North America The video screen shows you immediately the effects of the camera setting For fine tuning use the scrollbar Gain These settings are used until you change it again or switch to another objective or setup For fluorescence mode choose an exposure from 0 1 up to 8 Fluorescence according to light conditions
21. eo settings will automatically be started To avoid photobleaching of the sample by the lamp intensity Frozen image MMI CELLTOOLS offers you a frozen image to draw the cutting path To freeze the image click the right mouse button with the cursor over the video screen Choose Freeze from the context menu The video will be frozen so that you can close the fluo rescence illumination shutter All procedures still work in the 01 05 16 MMI UVCUT frozen image but you will see no results To check the results you have to switch back to LTE long term integration mode by using the context related menu right mouse button After go ing back one step you have to open the fluorescence illumina tion shutter and check the results 6 3 6 XY stage motion The movement of the stage is controlled by the MMI CELLTOOLS Three different modes allow fast scanning for overview or slow scanning for details Choose Yl or open the context menu by pressing the right mouse button with the cursor in the video window and choose Move with a left mouse click By pressing the left mouse button the table follows directly the mouse movement With the keyboard cursor keys the table can be moved slowly and continuous in the four directions The arrow keys in the number block on the PC keyboard move the sample step by step with a 10 overlap from one viewing area to the next This allows searching for a particular location in the sample without missi
22. er absorption filter is inside the microscope and tilted backwards the lasing action is interrupted and provides the necessary eye protection for those times when the oculars may be used To ensure full safety of the system please follow the steps below e Turn off the laser with the key switch to prevent unauthorized operation of the system e Do not remove any objective while the laser is operating e Never stare into the objective turret while the laser is operating e Use only the provided objectives for the laser microdissection e Never place reflecting objects in the beam path e The laser source and the optical equipment are enclosed within the blue hous ing To avoid electrical or laser hazards do not open the housing By design the laser beam is contained in a well defined beam path which is not serviceable by the user 1 2 General safety Do not disassemble the system The installation of the system is provided by MMI service personnel or MMI designated representative Repairs removal or exchange of components beyond the operations described in this manual may only by carried out by MMI service personnel or persons expressly authorized by MMI to do so If you have any problems with the instrument contact MMI The power supply is installed by MMI MMI assures that the system is provided with the appropriate voltage Do not change the power cords 01 05 5 MMI UVCUT MMI Avoid wet or dusty conditions near the system
23. he laser beam position be exactly marked on the viewing screen Over time and due largely to mild vibration to temperature extremes in the laboratory envi ronment or to accidental moving or jarring of the instrument recalibration of the laser position may be required If the cutting action no longer follows the marked line you should adjust the laser position It should be done for each magnification separately 01 05 Laser position MMI 31 MMI UVCUT Cut a hole in the sample with A red arrow will illuminate on the button when the laser fires or is on constantly Open UVCut from the menu bar and choose Set Laser Posi tion in the menu Calibration Fit the cursor cross with the hole which had been cut and confirm this position with a right mouse button click Continue with the next objective until the beam position has been defined for each one 6 5 2 Correction of slide offset If you have removed the slide from the xy stage that you have drawn may not fit exactly when viewed on the monitor if the slide is returned to the xy stage for further work 0 For correc tion you may reposition the contours by pressing and holding the Alt key as you move the stage with the mouse When the contours once again fit properly to the image release the Alt key 01 05 MMI 32 MMI UVCUT MMI 7 Disposables 7 1 Slides 50101 RNase free individually wrapped 50102 RNase free individually wrapped Box of
24. in the File drop down menu File gt Exit Or press the cross on the right upper corner of the program window Shut down the laser controller by pressing the button to deactivate the laser and then rotating the key to the off ver tical position to deenergize the electronics controller Shutdown the PC as needed by using the keyboard command 01 05 MMI E Create over Navigate into the area of interest J Caplift down Nolo Aa Ba Cut t Caplift Up 12 MMI UVCUT MMI 6 3 MMI CELLTOOLS Main Application 6 3 1 Main Window and plugins Extended tool area Pe wer rte see ev Extended tool O O _z JEJ Pido F lt lt 0 area MA At Sn The MMI CELLTOOLS provides a live video screen of the actual Live video and field of view of the microscope To get a fast overview of the overview scan sample scan functions are available in the overview panel on the right upper corner of the program window On the upper function bar the program gives access to the user User specific da specific database This database handles and stores all the tabase parameters defined by user Each plug in or in other words module appears as a separate Switching between sheet on the right side of the program window To switch from plugins one plug in to the other user only has to click on the appropri ate sheet 6 3 2 User specific database All settings saved in the MMI CELLTOOLS are unique fo
25. issection operations Cap To mount the tube simply slide the lid into the cap holder and open the tube The tube holder can easily be fixed to the auto matic cap lift The lift has a small magnet and two positioning pins to hold the Inverted tube You can cut an object with the Caplift up or down But the MMI f IsolationCap works most reliably if microdissection is done with the cap lowered onto the membrane surface Caplift Up Down button The xy stage can also be moved when the cap is down The Automatic cap maximum moveable distance without lifting the cap should be up down function limited to avoid tearing or other damage to the sample mem brane The Caplift free distance is 1000 microns 1mm as the default Under specific conditions it could be helpful to increase the free distance to be able to cut bigger objects with the cap down The Caplift free distance can changed by using the menu item UVCut gt Caplift free distance The Caplift free distance window appears E Caplift Free Distance n r j Free Distance IY Free Distance micron Nu If the cap is down on the tissue the cap will automatically be lifted whenever you leave the free distance area by moving the xy stage Then it will be repositioned repositioned automati 01 05 2 MMI UVCUT cally If the Caplift is down you can cut only objects fitting into the free distance area If you work with a 0 5ml cap it is recom mended to set a dista
26. most important parameters for the user to master Before dissecting the tissue of interest test the laser perform ance in another area of the sample or on a sacrificial slide with the same tissue Draw a line or a circle and start cutting this figure with a low speed Change the focus and power parame ters during cutting to observe the effects by the laser The sharpest line widths can be reached with power as low as pos sible and exact focus To adjust the parameters start with high power value The fo cus can be adjusted easier with high power Make several cuts with different focus values and compare the results With a reduced power the focus will be in a more defined area De crease the power step by step and repeat focusing until the cutting line will be continuous fine and clear 01 05 MMI Restriction for ob ject size if cap lift is down Automated cutting Objective E z0 Magnification Parameters amp A K 100 41 5 Laser set up Test of cutting Adjustment proce dure 28 MMI UVCUT A cutting timer is selectable in the menu bar under UVCut gt Draw and Cut gt Time left window This timer gives you an estimate of the cutting time To get access to the sliders click on the timer with the left mouse but ton The timer will disappear and comes back if you do not use the sliders for more than three seconds You can store as many of the laser parameter settings that you wan
27. nce measurement 6 4 6 Pin positions 6 4 7 Autodocumentation 6 5 Calibration 6 5 1 Laser position 6 5 2 Correction of slide offset 7 DISPOSABLES 7 1 Slides 7 2 Caps 7 3 Cell Chamber 8 MAINTENANCE 9 TROUBLE SHOOTING 9 1 View 9 2 Movement 9 3 Drawing 9 4 Cutting 9 5 Collection 10 WARRANTY 11 CUSTOMER SUPPORT 12 SYSTEM DATA OVERVIEW A SOFTWARE AND HOT KEY LIST A 1 Shortcuts 01 05 MMI 24 24 25 25 2 28 29 30 30 30 31 31 32 33 33 33 33 34 35 35 35 35 35 36 37 38 39 40 40 MMI UVCUT MMI A 2 Hotkeys 41 01 05 4 MMI UVCUT MMI 1 Security advice The system should only be used for microdissection as described in the manual Do not use the system for any other purpose Damage due to unauthorized use is not subject to warranties Persons who have been properly trained should only use the system Read completely the Security Advice in Section 1 and the Manual before operation 1 1 Laser safety This system contains a laser for microdissection The system includes safety de vices to prevent laser interference with the user Due to the power losses incurred as the beam passes through the microscope objectives the average power is nomi nal at the microscope work surface i e at the membrane slide tissue surface If the illumination arm is and tilted backwards the lasing action is interrupted by an electrical interlock A blocking las
28. nce of about 1 2mm The free distance can be adjusted as needed when working with the 0 2ml or 1 5ml tubes 6 4 3 Cutting of objects The automated cutting along the contours makes your work easy and fast You can decide if you want to cut one area or one group of areas For most applications the best cutting results are observed with the 20x or the 40x objective The user must select the appro priated objective in the objective selection box The laser set up includes the stage s parameters speed fo cus and power It is essential for the cutting performance to properly set these settings for each objective and tissue type The speed slider defines how fast the xy stage moves during the cutting procedure itself It does not affect any other move ment parameters of the xy stage The focus slider adjusts the focus position of the laser beam in z direction within the tissue sample The cutting performance is very sensitive to this parameter For example the user would expect to use different focus settings for soft tissue vs bone The power slider defines the laser power The power needed at the sample normally will be proportional to sample thickness Choose the power setting which enables clean laser cutting In laser microdissection the laser is used as a light scalpel The correct combination of focus and power define the sharp ness of the scalpel tip i e the overall cutting line width for a given speed These are the
29. ng any part of the sample 6 3 7 Slide overview for navigation In the right lower corner of the MMI CELLTOOLS you will find a panel representing the area of the sample defined by Piimit1 and sbdlimit2 See Handling a new slide After selection of the area of interest with the si button and scanning the slide by pressing the start scan button EB this window shows you an overview over your sample the roadmap image The position of the detail of the large video image on the slide is indicated with a red blinking point The maximum area of the scan is limited by the computer memory and the magnification of the microscope As standard a scan is executed with the 4x objective In this case you can define the left upper corner of the inner window of the mem brane slide as limit1 and the right lower corner as limit2 To do so follow these steps move the xy stage to the left upper corner of the mem brane press the limit1 button and observe the small blink ing red rectangle in the overview move the xy stage to the right lower corner of the mem brane press the limit2 button Ld and observe the small blink 01 05 MMI Three table move ment modes Direct mouse con trol Keyboard control Keyboard control step by step movement Overview Groups Statistic EETENGE l Scan And Prosess Image Limit definition Limit1 wed Limit2 17 MMI UVCUT ing red rectangle in
30. ntation Using the 40x objective move one small and remark able point of the sample to the left upper corner of the video panel Now press the right arrow key in the number pad If you move the object horizontally to the right upper corner and the object is still on the same level the camera orientation is useable If the result shows a shift between the two corners loosen the hex screws on the camera port and rotate the cam era carefully Repeat this move and observe operation again until 01 05 19 MMI UVCUT MMI the object moves on the same level across the monitor view When the object moves in a straight line the camera orientation is correct Retighten screws 6 3 9 2 Objective calibration When you add a new objective to your microscope you have to Calibration for calibrate the xy stage control Over the time of operation it may objectives be necessary to recalibrate the control when contours and cutting lines are no longer exactly fitting Take a slide with sample Choose an objective in the objective revolver and select the corresponding objective in the program Elle r If the objective does not exist create it as described in Section Confirm objective 6 3 2 Open in the menu bar item UVCut gt Calibration gt Objective calibration The objective calibration window appears A Objective Calibration i Calibrate this OE Cancel To start the calibration of the active objective you can
31. quantum dot applications Any or all of these modules can be combined in one micro scopic environment 01 05 6 MMI UVCUT 4 Principles of MMI CELLCUT The MMI CELLCUT is a method to isolate under microscopic view small areas or single cells from histological sections for further microbiological analysis Only the cell s wanted for further investigation are cut out DNA RNA as well as proteins from undisturbed pure samples can be investigated No me chanical contact is necessary for the laser microdissection of the samples Thus the method avoids contamination of the samples MMI CELLCUT permits comfortable working with high precision and a large throughput without contamination Several areas of interest can be microdissected in one auto mated operation and collected in the MMI ISOLATIONCAP One MMI ISOLATIONCAP used for Single Step Collection can collect several dissections even from different slides Review of the sample and selection of areas to cut do not have to be directly followed by the cutting operation The automated cutting can be carried out later and by another person 4 1 System set up The MMI CELLCUT system consists of a high performance research microscope with motorized scanning stage an elec tronically controlled solid state laser requisite laser beam de livery and transfer optics and a high end Pentium computer with Windows and the sophisticated control software MMI CELLTOOLS All
32. r the Full support of Microsoft Windows user actually logged in MMI CELLTOOLS Microsoft Win fully supports the Microsoft Windows user management dows user man During program start the last settings saved by the active user agement are loaded In the slide selection box you find all samples you defined in rewar I the past All documentation is saved under this name To El reneiate el 01 05 13 MMI UVCUT MMI change the database you press the E button and get the slide Editor B Slide Editor el Es r Mouse brain M New as copy of selected OF Cancel L Define new slide all parameters from the active slide will be copied You rename the slide by clicking on the name E Remove the active slide HEL EREN HEN HEN Navigate through the slides In the SetUp selection box you find all setups you defined for ear the active slide To change the database you press the E E defaut zi button and get the SetUp Editor To use this Editor see Slide Editor described above If you create a new slide all setups will automatically be copied from the active slide If you run different experimental settings e g microdissection with bright field microdissection with fluorescence of FITC or tweezers it is recommended to define one setup for each of these situations In the Objective selection box you find all objectives you de lt a fined for the active setup To change the database you press Elf E
33. re cisely see the measured distance 6 4 6 Pin positions With the x button you can save the current xy stage position With the x button you can delete it again You can save as many positions as you want You can move the xy stage from one pin position to the next by moving the pin position slider 6 4 7 Autodocumentation To set the auto documentation features open the auto docu mentation window from the menu bar UVCut gt Autodocumentation gt Set Autodocumentation 01 05 MMI Distance meas urement tool ajx create and delete pin positions Auto documenta tion for images before and after each cut 30 MMI UVCUT Autodocumentation Form x Report Folder Name 2004 07 08 1 BUS jv Text Report I First Image lw Image tor Each Shape of Group fv Last Image File Extensio f JPEG Guality 2 70 _ oot O BMP C TGA In the window you can activate the auto documentation and define the parameters of the function The program creates automatically Text Report htm file First Image Image for each shape of a group if not marked only an image before and after cutting a whole group will be saved Last image Note that you should work with a jpeg image in order to avoid accumulation of many large TIFF or BMP files on the PC hard drive The images in tiff format don t appear in the report 6 5 Calibration 6 5 1 Laser position Precise cutting requires that t
34. sically and programmatically Please note the Lens offset calibration procedure should go from objective with smaller magnification to objective with higher one for example from 4x to10x and should have no gap like 4x to 20x instead of 4x to10x or 10x to 20x etc Press the menu item Settings gt Lens OffSet calibration Right mouse click to set up a difference between a shape and corresponding object on the screen Repeat steps 1 4 for each objective 01 05 Lens OffSet calibration MMI 22 MMI UVCUT MMI 6 3 10 Multi User Report Multi User Report User can get information about time spend for mmi Cell Tools for each Windows user account Press the menu item Settings gt MultiUser Report Or start it from Start gt Progreamms gt mmiCellTools gt MultiUser Report E mmi MultiUser Report Form _ j Tools idl a LES 0000 07 28 ananuna eran hardware 0002 22 11 a time format is hhhh mm ss 01 05 23 MMI UVCUT MMI 6 3 11 Hardware self monitoring nagar self monitoring When system has a problem with some hardware there is a red blinker The detailing of problem or problems see in that combo box Camera hot connected 6 3 12 Overview Image Quality Overview Image Quality For some special cases for example when you scanning a huge image in overview window under 20x or more for full slide you can get a follow message mmi CellTools a x X Mot enough storage
35. t for each objective To define new setups see Chapter 6 3 2 Cut Laser microdissection can only be started with 4 Repeat field does number of times of cutting 6 4 4 Group handling If you have several types of cells to cut you can define an unlimited number of groups If you draw an object it will always be assigned to the actual group shown in the group selection Dox The most common reason for using more than one group is to permit the collection of different types of cells in different MMI lsolationCaps i e Group A with Cap A Group B with Cap B etc All drawn areas belonging to one group are marked with the same contour color You can adjust the drawing attributes such as group name color and line thickness in the editor by pressing the El button in the group name box The group editor window appears 01 05 MMI Timer and access to cutting parame ters during cutting SetUp E setups Laser parameter settings Cut Start cutting Repeat j j Number of times Group name aroun J Several dissec tates in one operation Contour color and thickness 29 MMI UVCUT GroupList Form Remove Rename Pen Color Pen Width 3 OF Cancel 6 4 5 Distance measurement Click E3 to measure different distances on the screen The cursor appears as a ruler Press the left mouse button and drag the mouse After releasing the mouse button you p
36. the overview For higher magnifying objectives the limits must be selected closer together to get a good visible overview and not overload the PC memory If the scan area is too big the white red blinking rectangle will be change to big red dot Define the actual area of the scan quickly with the u area tool After pressing the button you can select the area of interest in the overview window using the PC mouse Only the area of interest will be scanned With a right click on the area of interest you can define the im age quality of your scan The highest possible quality is de pending depends on the PC resources Begin the scan by pressing E the Start Scan button With the D stop scan button you can always interrupt the scan Also pressing the ESC key will interrupt the scan With al you can open the overview image in the standard Windows image viewer The time that Windows needs to open the application is dependent on the image size and PC performance With the Ee copy button you can copy the overview image into the Windows clipboard Following this step the image is available for all other standard Windows applications With the j save button you can save the overview image on your hard disk or other drive A white red blinking rectangle represents the position of the detail visible in the large video window You can move this red frame with the left mouse button The motorized xy table moves automatically to the
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