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mmi CellEctor User Manual

1. New o 17 Current i Current a Standard colors b Custom colors Figure 8 17 Advanced color selection will be handled separately for each color set If multimode is not selected the set of shape parameters will be applied to all color sets 8 2 3 5 Mark tool panel After selection of the colors the image will appear as a black and white image The darker the pixels are the better will the color match the selected color Mark Sensitivity J 0 43 Background Suppression J E Figure 8 18 Mark tool panel In the Mark tool panel Fig 8 18 objects can be separated from the surround ings of similar color by adjusting Sensitivity 0 01 is the lowest intensity 1 00 is the strongest intensity The Background suppression filters the background 1 means low filter suppression 20 means high filter suppression For small objects high filter suppression is recommended while large objects should be selected with a low filter suppression 116 MMI CellEctor By applying the Mark function the candidate pixels for an object will be labeled red Important Please try to mark all objects in red with a white back ground 8 2 3 6 Split tool panel The mmi CellExplorer contains a mode for automatic splitting of objects In microscopic images of cells or structures the relevant objects are often located next to each other in a way that the computer detects those objects as one single structure The
2. b Results Figure 8 21 Example for calculating the roundness of different objects independent from size 8 2 3 8 Shapes toolbar With the shape toolbar the contours of the selected objects can be optimized Shapes Factor J 1 00 Constant J 2 02 Smoothina J 23 Figure 8 22 Shapes tool panel The Factor defines a percentage correction of the shape size e Factor lt 1 00 reduction of the shape size Min 0 8 e Factor 1 00 no correction e Factor gt 1 00 increase of the shape size Max 1 2 119 8 IMAGE ANALYSIS USING MMI CELLEXPLORER 8 2 4 The Constant parameter adds a pixel correction to the shape size e Constant 0 00 no pixel correction e Constant gt 0 00 shape size increases by constant pixels Max 50 The pixel correction can be used to compensate for the laser cutting width independent from the object size With the Smoothing feature the contours of the selected objects can be smoothed e Smoothing 0 no smoothing e Smoothing gt 0 increase smoothing of object contours Max 100 Project panel The mmi CellExplorer project panel shows all images contained in the current session You can choose whether the image is displayed in the work image panel using the checkboxes on the left of the images You can also delete an image from the current session by marking it with the mouse and clicking the Delete button 120 MMI CellEctor Service Service should only be perf
3. Encoding method Image quality Recommended for Uncompressed Lossless Short videos Extracting frames in full quality Compressed Windows Lossy General use Media Video default Playback primarily on Windows systems Compressed XVID Lossy Color video not usable for black and white cameras Playback on any system with XVID codec installed To create basic time lapse movies only adjust the Time lapse factor as de sired This factor defines the speed at which the movie will play back Enter 1x to turn off time lapse standard time The Playback rate specifies at how many frames per second the final video should be played back This is identical to the recording rate unless using time lapse The default playback rate is 10 Hz and should be suitable for general use The Recording interval specifies the minimum time interval between two recorded video frames The recording interval is computed from time lapse factor and playback rate and is for information only To play back Windows Media Video files on Mac OS X install Windows Media Player for Mac OS Flip4Mac which is downloadable from the Microsoft website 18 5 4 5 4 1 5 4 2 MMI CellEctor Depending on the current camera settings and available computer hardware it may not be possible to achieve very short recording intervals To control the rates at which frames are recorded and played back change the Playback frame rate If time lapse
4. Capillary 3D positioning t t y y Speed max V medium min 1 um 3D Positioning Wizard SINGLE CAP Step 2 of 6 Move XY table so that the anticipated capillary landing point in the cap is placed in the screen center The landing point should not be far away from the left hand wall of the cap Cap f a The control panel b The capillary landing point within the cap is placed in the screen center and located at the left hand side of the cap Figure 6 35 Step2 of capillary vertical positioning wizard with a cap 70 MMI CellEctor 3 If necessary adjust the height of the inner wall of the cap Then change to the objective you anticipate to work with during collection of cells Then press Next itch to advanced Speed max V medium min 1pm 3D Positioning Wizard SINGLE CAP Step3 of 6 4 E mm Cap al height 1 Adjust the height of the cap inner wall if necessary 2 Change to the working objective Cos a The control panel b Left side of the cap un der high magnification work ing objective Figure 6 36 Step3 of capillary vertical positioning wizard with a cap 71 6 MMI CELLECTOR 4 Focus the microscope on the cap bottom Be sure to focus on the upper not on the lower surface of the cap bottom otherwise the capillary may break in the next steps Then press Next ed Speed max V medium
5. Exit service slide calibration SERVICE SLIDE Calibration steps Place the center of the well 1 in the view field by moving the stage Then press lt Done gt Done Figure 6 47 Service slide calibration window 82 6 10 6 10 1 MMI CellEctor Using mmi CellEctor mmi CellEctor can be used in a variety of modes starting with the manual mode via the automatic mode ending with workflows running fully automatic after a single click In the manual mode the basic steps of acquisition and deposition like moving the XY stage moving the capillary or pumping are not combined in sequence of actions Thus the user can perform a single step separately using the corresponding buttons in the action panel Manual mode is used mainly for the tuning of the system to new experimental conditions type of cells and buffer solution Once the optimum conditions for the movement of the capillary and pumping have been found one of the buttons in either automatic or workflow mode can be used In the automatic mode the work is divided in three steps acquisition deposi tion and rinse In the workflow mode these three steps are combined in a series of actions workflow The simplest workflow is Acquire and deposit the most advanced is Scan for cells If required the rinse step can be included in the workflows by checking the check box With rinse A workflow can be interrupted by pressing Esc While working in
6. While moving up out of the sample the capillary moves with the normal speed 48 MMI CellEctor Both immerse as well as the slow down distance can be set for the sample and the deposit independently The numbers at the right hand side of the dialog in mm are calculated from a given capillary tilt and a given immerse distance They help to adjust the immerse distance if the capillary has to be immersed in liquids confined to container walls 6 8 3 Pump configuration 3 Configuration Pump a Current capillary diameter um 40 in oul Volume nL 30 150 Y 6 fp Pre pumping volume nL 0 Over pumping volume nL 0 oA aA aw Relaxation time ms 0 Speed 50 or Acceleration 10 or save this parameter set Cancel OK Figure 6 19 Pump configuration dialog All the pump parameters can be set in the pump configuration dialog that can be opened either from the main menu CellEctor gt Configuration Pump or by clicking any pump relevant button with the right mouse button The fol lowing pump parameters can be changed by the user Volume in main volume that is pumped in if the user presses the button Pump in or Acquire in the action panel Volume out main volume that is pumped out if the user presses the button Pump out or Deposit in the action panel Pre pumping volume volume that is added to the main volume when ever the pump
7. Large spacing values may conflict with the upper limit for the microscope Z drive if automatic refocusing on the capillary tip is selected or the user toggles the focus manually using the Refocus on capillary slide button in the action panel The above calibration procedure sets not only the capillary position within the cap but also the cap position with respect to the XY coordinates of the screen Thus contrary to grids as deposit targets there is no separate calibration procedure for the deposit position for the cap 15 6 MMI CELLECTOR Capillary vertical contact position advanced method In the advanced method one has to be aware of two independent 3D coordinate systems used by mmi Cel lEctor e that of the capillary manipulator that of the microscope with its stage Their relative orientation is shown in the Fig 6 41a and Fig 6 41b The XY 0 X z Microscope Z drive Y Capillary z v aa a 3d manipulator b stage and microscope fo cus Figure 6 41 Coordinate systems used in mmi CellEctor planes of the capillary and of the stage work fully independently On the other hand the relative position of the Z axes of the capillary and the microscope must be determined whenever a new capillary is inserted or its position is changed For this purpose the capillary is moved down to the contact position with the slide and the microscope is focused on both the capillary tip and the slide surface at the same t
8. Set IDLE position sets the idle position of the capillary e Set CONTACT position sets the contact position of the capillary on a sample or a grid slide CAP position is visible only for a single cap as deposit target and shows the current settings values for the capillary in the cap as determined in the simple method e Calibrate motors recalibrates the capillary motors X Y and Z Initialize motors initializes the capillary motors X Y and Z Exit closes the dialog and moves the capillary up Once the capillary orientation and the tilt have been selected proceed as fol lows Procedure 1 Ensure that the Z drive control is ON Microscope gt Z Drive Control must be checked 77 6 MMI CELLECTOR Activate the joystick Ensure that the capillary is high enough above the stage Move the capillary within the XY plane towards the optical axis of the microscope Lower the capillary down to approx 2 3 mm above the slide plane Focus the microscope on the capillary tip either manually or by using the Z drive slider Center the capillary tip on the screen using the joystick Focus the microscope on the slide plane Using the joystick lower the capillary down to the slide until it touches the latter The contact position can be recognized by the onset of the sideward movement of the capillary as shown in Fig 6 43 Figure 6 43 Capillary starts moving sidewards when touching the slide
9. min 1pm 3D Positioning Wizard SINGLE CAP Step 4 of 6 Focus the microscope on the cap bottom Ensure to focus on the upper not the lower bottom ace Caos a The control panel b Left side of the cap un der high magnification work ing objective focused on up per surface of teh cap bottom Figure 6 37 Step4 of capillary vertical positioning wizard with a cap 72 MMI CellEctor 5 The system moved the capillary to the position closely above the cap The capillary is not seen under the microscope yet At this moment it is recommended to verify the position of the capillary by visual inspection of the XY stage The capillary body should be above the upper right edge of the cap in a distance exceeding the cap wall height otherwise it would break in the next step Then press Next Capillary 3D positioning Switch to advanced t t a y y Speed max Y medium min 1 um 3D Positioning Wizard SINGLE CAP Step 5 of 6 The capillary will now be lowered into the cap approximately by the value of the cap wall Click lt Cancel if there is not enough distance between the capillary body and the edge of the right hand wall of the cap Caos Figure 6 38 Step5 of capillary vertical positioning wizard with a cap 73 6 MMI CELLECTOR 6 The system moved the capillary into the cap so that the capillary tip stays approx 200 umabove the cap bottom The capillary tip may be
10. vii CONTENTS 6 9 6 Capillary horizontal position on the screen 80 6 9 7 Deposit calibration o o e 80 6 9 8 Service slide calibration 82 6 10 Using mmi CellEctor o o 83 6 10 1 Manual pump use 0000 e as 83 6 10 2 Moving the capillary Up Or down o 84 6 10 3 Automatic modes Acquire deposit and rinse 85 6 10 4 Acquire and deposit workflow o 86 6 10 5 Scan for cells workflow coca ra al Ww x 86 6 10 6 Automatic inspection and reporting of deposit grids 87 6 11 Optimization of the system for collecting cells 88 6 11 1 Pump parameters o 88 Dale SPACINGS 4 a a AE nina 90 6 11 3 Capillary bevel parallel to the slide surface 90 6 11 4 Capillary bevel vertical to the slide surface and cell scratching 91 6 11 5 Collecting cells to a matrix of spots grid 91 6 11 6 Standard settings for beads o o 92 6 11 7 Collecting cells to a single Cap 92 7 AUTOMATED MICROSCOPE CONTROL 95 7 1 Activating microscope control 95 7 2 Observation methods 95 7 3 Objective control 96 7 4 Z drive control o o 96 7 4 1 Required calibrations 0 97 7 4 2 Sample plane definition o o 97
11. mmi CellEctor User Manual Molecular Machines amp Industries AG www molecular machines com User Manual MMI CellEctor Version 4 3 Copyright 2013 by MMI AG All rights reserved for patents or trademark registration No part of this document may be distributed or reproduced utilized or imparted to a third party without the prior written permission of MMI AG Non observance will result in liability for damages MMI is a registered trademark of MMI AG MMI AG Flughofstrasse 37 8152 Glattbrugg Switzerland Technical features are subject to change without notice MMI CellEctor Contents 1 SECURITY ADVICE 1 1 1 Generalsafety 20 0002 eee eee 1 1 2 mmiCellEctor safety o o ooo 1 2 INSTALLATION 3 3 THE MMI CELLTOOLS INSTRUMENTATION FAMILY 5 4 GETTING STARTED 7 4 1 System Startup e 7 4 2 System preparation aoaaa aa 7 4 3 System turn off 2 2 2 2 0 00208 8 5 MMI CELLTOOLS MAIN APPLICATION 9 5 1 Main window and plug ins 9 5 2 User specific database 10 5 2 1 Slide editor ae ocd se whe RES Rg ee oe 11 52 2 SCP o waa oe Oh ace se Ek ae OR EOS a 11 5 2 3 Objective editor 6 6 bee eee ee ee ee 12 5 2 4 Default database reconstruction 13 5 3 Camera operati0N eee 14 5 3 1 Multiple cameras e o 14 5 3 2 Camera settings ei deal a dc
12. 6 5 Use 3D manipulator with joystick 39 6 6 Typical workflow in collecting cells 39 6 6 1 Checking the microscope o e 40 6 6 2 Setting up the slides the capillary and the pump 40 6 6 3 Calibrating the capillary 41 6 6 4 Finding the optimum conditions for collecting cells 41 6 6 5 Performing cells collection 42 6 7 mmiCellEctor user interface 42 61 Men ri a eee toe a ada de 42 AS A A A 44 6 13 Atin panel se p o ad a 44 6 7 4 Distance measurements o 46 6 8 Configuration routines 47 6 8 1 Choosing the position of sample target and service slide 47 6 8 2 Capillary movement configuration 47 6 8 3 Pump configuration 4 2 42 Aho he Sew a ed BAS 49 6 8 4 Acquisition configuration o e 50 6 8 5 Deposition configuration 52 6 8 6 Workflows editor 2 a e Se ie SAS 53 6 8 7 Depositinspecti0N 0 e e 55 6 9 Calibration routines o 57 6 9 1 Objectives magnification 0 57 6 9 2 Partocal lens offset 2a wee a pe Se 58 6 9 3 Paraxial lens offset a ee ia ele ee Bare 59 6 9 4 Capillary vertical contact position automated microscope 61 6 9 5 Capillary vertical contact position manual microscope 79
13. 7 4 3 Automatic Z drive control wos see te A eH Gd et 99 7 4 4 Z drive Oscos IS uo noe e Whe es eS Goad BR 99 7 5 Microscope safety parameters 100 vill MMI CellEctor 7 5 1 Objective collision prevention 100 7 5 2 Objective sample collision 101 7 6 Fluorescence shutter control 102 8 IMAGE ANALYSIS USING MMI CELLEXPLORER 103 8 1 Getting Started o 103 8 1 1 Encircling objects of one uniform color 103 8 1 2 Encircling Objects with different uniform colors 105 8 1 3 Encircling Objects with color gradients 105 8 1 4 Transfer to other mmi CellTools modules 107 8 1 5 Optimization of results a y o aa 107 8 1 6 R s lt lists 4 DE a aa E bow a ine ea 107 8 2 Reference soi ros ana Bt Bo Sel Be Me e 108 8 2 1 Menu reference cu inca cd bee bd gob ee ee de Sepa 108 32 2 LOOM UNCION ds we Bs Rings E E eed Geet Bd 109 8 2 3 Control Panel oc ose ws a Be ee ee ees 110 6 2 4 Proje t panel seitura dhe ase ne oe ee aad 120 A SERVICE 121 B LIST OF KEYBOARD SHORTCUTS 123 C TECHNICAL DATA 125 C 1 Required minimum workspace 125 C 2 Systemcomponents 125 C 2 1 Microscope 2 2 6 oo etek Ge we ee Re Se ee eae a 125 G2 2 IOS n o Gta eee NS NA 125 C23 Digital CAMELA ip Sere ak Glee eal be eH He Se 126 6 24 Computer 4 eats and dart ar
14. MMI CellEctor Leave capillary down after acquisition check this option if you want to verify that the cell has indeed been caught in the capillary during the acquisition process If it has you can click Deposit button to move the capillary up and continue the process If it has not you have the op portunity to retry pumping the cell into the capillary by pressing Pump IN Whenever the capillary is down movement of the XY stage is still possible However please be aware that large movements ex ceeding the size of the field of view may damage the capillary when the slide plane is significantly tilted These options are especially useful during the fine tuning of the acquisition process for a given application In the workflow mode Scan for cells acqui sition is preceded by making a snapshot of the current field of view and auto matic cell recognition and selection setting markers The sample is scanned horizontally and vertically and the user can define the number of screenshots that have to be made scan a number of screens horizontally and a number of screens vertically The recognition of cells can be repeated a number of times at each stage location if there are several cells of interest that can be acquired there Minimize fluorescence excitation option is relevant only for the case when the automatic cell recognition takes place in the fluorescence 51 6 MMI CELLECTOR observation mode and one wants to prevent bleach
15. the highest magnification and or click lt Start gt to confirm it e 07 Figure 6 26 The mmi CellEctor plug in panel in objective paraxial calibration mode 60 freehand drawing tool drawing mover tool Move the selected object to the center of the video panel click the free hand drawing tool and draw the contour of the object with the mouse Press Del on the keyboard if you want to redraw the contour Change to the objective with the next smaller magnification After the change the previously drawn contour will move away from the object Re position it using the drawing mover tool so that it again over laps with the object Click the button Set offset and confirm the message that shows up Repeat steps 3 through 5 for the remaining objectives Change from the objective the smallest to the objective with the highest magnification without deleting the drawn shape If the procedure suc ceeded the shape should overlap with the object Terminate the procedure by clicking the link Exit objective calibration on the panel MMI CellEctor 6 9 4 Capillary vertical contact position automated microscope The procedure for the determination of the vertical contact position described in the first part of this chapter applies to the automated microscope only A simplified method for manual microscopes is described at the end of the chapter Whenever a new capillary is inserted one has to calibrate the capill
16. 106 8 1 4 8 1 5 8 1 6 8 1 6 1 8 1 6 2 MMI CellEctor Transfer to other mmi CellTools modules After selecting your colors click on the calculate button green arrow in the process toolbar The software will now search and mark your objects Please wait until the process has been completed This can take a while for large images To return to the main application click the Return to application button at the top of the Cel Explorer panel After switching back all detected cells are lined out and stored in the group statistics Now you can start the cutting or collecting process of all marked specimens Optimization of results If your result is not totally satisfactory you can optimize each step of the object detection Please refer to section 8 2 The settings for optimizing the object detection procedure described herein can be executed directly in the respective section of the Settings panel Result lists Object list The following information is available via the menu item View Object list after each object detection It displays the calculated parameters like diameter total area circumference and roundness for each detected object Count list Under the menu View Count list the count list shows a summary of the detection results The table lists the number of objects as well as relative area sample information and respective calculated objects per unit in your sample 107 8 IMAGE AN
17. Ee ees 14 CONTENTS 5 3 3 Freeze video live video 16 5 3 4 Saving images 2 sn 2 6 be o e ee ae 16 5 3 5 Recording movies coa Ge Gok ade ete ee eee ee ad 17 5 4 Motorized stage Control 19 5 4 1 Mouse movement o e eee 19 5 4 2 Keyboard movement o 19 5 4 3 Stage movement settings 20 5 5 Slide navigation and scanning 21 5 5 1 Mechanical limit calibration 21 5 52 Overview Scan aa a ds hr G 22 5 5 3 Multi slide handling 2 a he a Aes 23 5 5 4 Pin positions Da A tae BM tag 24 5 6 Distance measurement 25 5 7 Calibration 0003 Y 08 408 reo cal a es E 25 5 7 1 Camera orientation 2 aa a 25 5 8 Multi user report 2 0 200004 26 5 9 Help a has be hg E 26 5 9 1 Help topics e o 26 592 MMLONING s a ae Boos E Be ee ed Geel a 26 JS VISION IMO ct laa beech ls ed a 27 6 MMI CELLECTOR 29 6 1 Principles Dosis FAIRS ar doe eo Os 29 6 2 Slide Types is 5 il Bae Bl NR le HS as 32 6 2 1 Sample SSS Lava A Re ae Ro ee 33 6 2 2 Target slides 02 0002 eee ees 33 6 2 3 Service slide oS aah eee aoe a OE Se 33 63 Capillaries ery was a as DG a 34 6 3 1 Capillary types ir Gece Sate ce Dat hg stare te Sa 34 vi MMI CellEctor 6 3 2 Capillary handling ER A PY SS 34 6 4 The mmi CellPump sonnan anaana 38
18. PM Img__1 Figure 8 6 Object list MES Objects Relative rea Colonies ml SampleVolume DilutionFactor DateTime ImageFile SettingsProfile 1 1 6649 22000 0000 1 0000 1 0000 9 17 2009 1 35 52 PM Ima Figure 8 7 Count list 8 2 Reference Once the CellExplorer tab is selected in the component control panel of the mmi CellTools main window CellExplorer becomes the active component It adds its own menu items group in the main menu and shows its own control panel 8 2 1 Menu reference The CellExplorer menus consists of four main menu groups File Edit e View Tools The main menu Tools is not used yet lt is reserved for future use 8 2 1 1 File Menu Open Opens the window for opening a saved counting session 108 8 2 1 2 8 2 1 3 8 2 1 4 8 2 2 MMI CellEctor Save Quick save of the current counting session when it has already been saved before Save as Opens the window for saving a counting session Printer Setup Opens the window for printer setup Open Image file Opens the window for loading a photo Edit Menu Copy Copies the current photo to the clipboard of your PC Undo Cancels the last change of settings Reset Resets the current photo to its initial state Calculate Starts the analysis View Menu Image View Shows the current photo Object List Shows the object list Count List Shows the count list Panel Settings Switches the panel between settings and projects
19. Z y y Speed max medium min V 1 pm 3D Positioning Wizard SAMPLE SLIDE Step 5 of 5 The capillary tip should now be in contact with the slide You may prove it by gently moving the XY stage with the mouse If necessary re djust the vertical position of the capillary Set default idle position too a The control panel b At the contact position both the slide and the capil lary tip should be in focus Figure 6 32 Step5 of capillary vertical positioning wizard point of the capillary tip with the slide and to complete the capillary 3D positioning It is recommended to leave the check box Set default idle position too checked The default idle position is defined as the maximum XZ distance from the sample while leaving the Y coordinate unchanged Setting user defined idle position of the capillary is possible in the advanced 3D positioning only 67 6 MMI CELLECTOR Once Finish is clicked and the wizard completed the following dialog Fig 6 33 If the contact position of the capillary on the other slide is still valid i e you Information request Please select O the contact position on the DEPOSIT is still valid will set the contact position on the DEPOSIT in the next step Figure 6 33 Dialog at the end of the capillary vertical position calibration wizard have replaced neither the capillary nor the other slide select the option still valid Otherwise select
20. by moving the capillary down 10 Click Set CONTACT position in the 3D positioning dialog The current X 78 Y and Z values are stored as the coordinates for the down position of the capillary MMI CellEctor 6 9 5 Capillary vertical contact position manual microscope Defining the vertical reference position of the capillary at the contact with the slide is done in the 3D positioning dialog accessible through the link Switch to system or the menu Cellector gt Capillary and than click on the button Contact Point Calibration of the vertical contact position of the capillary for manual microscopes or if the plane tilt is not active is done in a single step Use one of the dialogs Fig 6 44a of Fig 6 44b The capillary has to be manually moved down to the contact position using either the XYZ arrows or the joystick Once the contact position is reached the button Set DOWN position has to be clicked The idle up position is set by moving the capillary to this position and pressing Set UP position tm Capillary manual movement ima Capillary manual movement AMPLIGRID Switch to joystick AMPLIGRID Switch to arrows Current position am rt t x 25000 3 Y 12000 S zo 8 a mo gt B E Set DOWN position Set UP position Set DOWN position Set UP position Speed V max medium min 1um more gt gt Exit more gt gt Exit a using but
21. clicking on the name Use the button to remove the active slide Setup editor The setup selection box contains all parameters defined for the active slide To add remove or change the database press the edit button To use this editor follow the same steps as for the slide editor described above If you create a new slide all setups will automatically be copied from the active slide a SetUp Editor 14 ll 4 gt Pl Setup Name Filter Condenser Optical path Camera T BF single0 5 trans 1 1 1 lt default camera gt BF single0 5 trans New as copy of selected EPR Figure 5 3 Setup editor 11 5 MMI CELLTOOLS MAIN APPLICATION 5 2 3 If you run different types of experiments e g microdissection with bright field microdissection with fluorescence or optical tweezers it is recommended to define one setup for each of these experiments To rename the defined setups click on the setup name and type in a different name The Filterblock Condenser and Opticalpath settings are only used with mo torized microscopes The use of these parameters is explained in section 7 2 If your system is equipped with more than one camera different setups can be associated with a certain camera You can then switch to a specific camera by selecting a setup defined for that camera The camera associated with a setup is displayed in the setup editor column Camera and can be changed by clicking on the name The camer
22. immunomagnetic enrichment In the second step the mmi CellEctor technology is used for individual cell picking 6 2 Slide Types The slides carrying the suspension of cells sample or serving as target for cells have to fit the standard slide size of approx 76 x 26 mm Within this constraint a variety of slides can be used On request MMI can also provide custom made slides tailored to you particular experiment tion of target molecules of diagnostic and therapeutic value Clin Exp Metastasis 5 24 317 2007 32 6 2 1 6 2 2 6 2 3 MMI CellEctor Sample slides Putting a droplet of a liquid with cells on a standard flat glass slide is the easiest way to prepare the source of cells for the capillary The disadvantage is that rather sizeable and thin liquid samples are formed which evaporate relatively quickly There is a range of commercially available slides e g IBIDI slides that offer some advantages as compared to this simplest solution Target slides Target slides can either be matrices of spots hereafter called grids or just a single spot Advalytix AmpliGrid shown below is one of the standard grids predefined in the mmi CellEctor software In addition custom grids can be 75 6 0 2 a oO o la i sal u N pu o pad E wo th 3 e y o 1 wo N w lay on edge Figure 6 5 The mmi CellEctor Advalytix AmpliGrid defined with up to 24 spots in arow or column 24 x 24 576
23. items grouped as CellEctor to the main menu and shows its own control panel either System or Action panel Menu The CellEctor menu consists of two sub menu groups Configure and Calibrate The menu item Export grid data shows up only if AmpliGrid or custom grid is selected as the deposition target 42 CellEctor CellCut CellExplorer MMI CellEctor Switch to system w g n Automatic Acquire Deposit Workflow Acquire and deposit Full auto Scan for cells With rinse Inspect deposit Capillary spacing um to sample deposit o S o or Figure 6 13 The mmi CellEctor plug in panel in action mode Configure Calibrate Export grid data Acquisition Capillary movement Deposition i Deposit Pump Deposit scan Slides Workflow Figure 6 14 The mmi CellEctor menu 43 6 MMI CELLECTOR 6 7 2 System panel 6 7 3 There are two major panels in the mmi CellEctor control panel System panel and Action panel You can switch between the panels by clicking on the links in the top right corner The System panel shows the readiness of the major system components The figure below shows the system ready for use If one of the major system components requires calibration the corresponding button is highlighted red Clicking the button with the left mouse key launches pen CellEctor CeliCut CellExplorer Switch to action System ready for use Object
24. out of focus at the moment Fig 6 39b Now focus the microscope on the x D y Speed max medium Y min 1 um 3D Positioning Wizard SINGLE CAP Step 6 of 6 1 Fine tune the XY position of the capillary in the cap 2 Re focus the microscope on the capillary tip a The control panel b Left side of the cap un c Left side of the cap un der high magnification work der high magnification work ing objective with unfocused ing objective with focused capillary capillary Figure 6 39 Step6 of capillary vertical positioning wizard with a cap capillary tip and fine tune the XY position of the capillary if necessary Fig 6 39c The capillary tip does not need to be placed in the screen cen ter At the moment you press the Finish button the system calculates the spacing between the capillary tip and the cap bottom 206 umin the example shown 74 MMI CellEctor here You can change this value later in the range from O contact with the cap bottom to 4000 um CellE ctor CellCut CellE xplorer Exit capillary calibration Horizontal Position Move capillary down then mark its position with the left mouse key tanec Done Vertical Position Capillary spacing pum to sample 20 E Automatically refocus on O capillary capillary slide slide Contact Point Figure 6 40 System panel after finishing capillary vertical positioning wizard with a cap
25. pumped out to each spot 87 6 MMI CELLECTOR 6 11 6 11 1 The file can be generated from the main menu item CellEctor Export Grid Data Optimization of the system for collecting cells There are two key steps in the process of collecting rare cells getting a cell into the capillary at the sample getting the cell out of it at the target Since there is a large variety of cells and of buffer solutions the working pa rameters of mmi CellEctor have to be optimized for the given experimental conditions The major optimization parameters are pump parameters spacing between the capillary and the slide surface e size tilt and orientation of the capillary Please note that prior to the tuning of the system one has to determine the tilt of the slide plane s Pump parameters In order to understand the meaning of the pump parameters let us go through an example of the following series of the user s actions Procedure 1 Acquisition of a cell 2 Deposition of the cell 3 Acquisition of another cell however the cell could not be pumped in the first attempt 4 Retrial to pump the cell in The Fig 6 54 shows how the pump behaves during these four actions under assumption that the following pump parameters have been set 88 MMI CellEctor Acquisition Depo Acquisition Acquisit Figure 6 54 Panel representing the workflow functions of mmi CellEctor e Main volume 10
26. see sec tion 8 2 4 Tools Menu The main menu Tools is not used yet It is reserved for future use Zoom function The mmi CellExplorer contains a comfortable zoom algorithm Moving the slider on the right hand side of the displayed image zooms the image in or out With the zoom in function the mmi CellExplorer zooms into a specific part of your image Draw a rectangle over the respective area with the mouse button held down The mouse pointer will change to a cross shape With the zoom out function the mmi CellExplorer zooms out to the original image size 109 8 IMAGE ANALYSIS USING MMI CELLEXPLORER zoom out gt zoom in 77 0 Return to application Process Settings Profile Advanced Imaging Color Mark Split zoom slider 3 Find Figure 8 8 Zoom controls 8 2 3 Control panel The mmi CellExplorer controls are subdivided into control panels that can be opened and closed on demand by clicking on the button to the left of the panel s title Fig 8 9 Return to application Process Xx Ku Stopped 0 Obj Settings Profile Advanced Imaging Smooth radius J 0 0 Color ToT i Power J 50 C Multimode Mark Split Find Shapes Figure 8 9 The mmi CellExplorer control panel for cell recognition Most panels contain a preview button When clicked CellExplorer will perform all calculations defined on the res
27. select the target slide button 23 Y a 5 MMI CELLTOOLS MAIN APPLICATION 5 5 4 A aai Figure 5 13 1x3 Multi Slide Alternatively you can move to another slide with the keyboard arrow keys The slide number indicator will automatically adjust to the current slide If the number in the slide number indicator does not fit with the position of the slide holder move the stage to slide position 1 with the keyboard arrow keys and redefine the slide origin as described in chapter 5 5 1 Pin positions With the pin button you can save the current stage position By clicking on the arrow to the right you obtain the pin positions menu Fig 5 14 Overview Slide Scanning Navigation HSH PS ea Y Pini Pin 2 N Delete current Delete all 61 62 Es TTT Figure 5 14 Pin positions You can select a pin which moves the stage to the respective position The last two entries enable you to delete either the currently selected or all pin positions 24 MMI CellEctor 5 6 Distance measurement Select the measurement tool to measure distances on the sample dl Figure 5 15 Distance measurement Press the left mouse button and drag the mouse After releasing the mouse button you can see the measured distance Fig 5 15 5 7 Calibration 5 7 1 Camera orientation To check the camera orientation e Using the 40x objective move one small and obvious point of the sample to the upper left corner of the video
28. spots A single spot as a target can be just a selected spot on a grid MMI also offers a slide capable to hold a single transparent cap of a standard 0 2 ml PCR tube This kind of the target slide is called single cap Service slide On the service slide the capillary can be treated with various solutions for cleaning filling with additional reagents or diluting the reagents already present in the capillary MMI offers IBIDI y Slide 8 well as the service slide The slide can be purchased with different coatings hydrophilic hydrophobic biopolymer or protein coating 33 6 MMI CELLECTOR Figure 6 6 The mmi CellEctor 8 well service slide 6 3 Capillaries 6 3 1 6 3 2 Capillary types mmi CellEctor standard glass capillaries are straight with the outer diameter of 1 mm and the tip inner diameter 10 15 20 30 or 40 um The capillary tip is beveled at 45 deg Any other capillary shapes and sizes that fit the capillary holder may also be used however in such a case the handling the calibration and the standard usage procedures might differ from those described in this manual Capillary handling Please observe the safety precautions described in the first chapter of this manual mmi CellEctor is delivered with a ready for use capillary placed in the capillary holder of the 3D manipulator illustrated below The following capillary replacement procedure is recommended 34 MMI CellEctor Figure 6 7 The cap
29. the object to the bottom right rectangle 4 Toggle the cursor to the target shape and mark the object again 5 Click Done on the objective calibration panel to finish or Cancel to restart the procedure After a successful calibration any object dragged to any location within the video panel should be exactly under the mouse cursor at the end of dragging Whenever an objective is re calibrated re calibration of the XY position of the capillary as well as of the deposit is necessary This is indicated by marking the Capillary and the Deposition buttons red in the CellEctor system panel Parfocal lens offset This procedure applies to the automated microscope only When one changes an objective and the microscope was in focus the new objective requires a slightly different Z drive position to get in the new focus 58 6 9 3 MMI CellEctor The difference is called parfocal lens offset The mmi CellTools software can automatically refocus after a change of the objective if the offset has been determined for all the objectives in use Proceed as follows in order to calibrate the parfocal lens offset Procedure 1 Change to the objective with the highest magnification Focus the microscope Change to the objective with the next smaller magnification 2 3 4 Focus the microscope 5 Select menu item Microscope gt Z Drive Offset Objective parfocal 6 Repeat steps 3 and 4 for the remaining objectives After the
30. the second option move to the other slide in this case to the deposit and go through the calibration procedure once again 68 MMI CellEctor Capillary vertical contact position Single cap simple method The wizard for a sin gle cap is very similar to that for the flat slide However before you start with the single cap calibration you have to ensure that the capillary contact point on the sample slide has correctly been determined Also it is essential that the plane tilt is defined for the single cap If it is not the case the capillary may break during one of the following 6 steps Procedure 1 Change to the objective with the smallest magnification and then move the XY stage so that the cap is visible on the microscope Fig 6 34b Then press Next t a y y Speed max Y medium min 1 pm 3D Positioning Wizard SINGLE CAP Step 1 of 6 Change to the objective with the smallest magnification x4 Warning capillary position on the sample slide Y must already be known MED ES di a The control panel b The cap is visible on the microscope and centered to the marker Figure 6 34 Step1 of capillary vertical positioning wizard with a cap 69 6 MMI CELLECTOR 2 Move the XY stage so that the future capillary landing point within the cap is placed in the screen center This should be a location at the left hand side of the cap as shown in Fig 6 35b Then press Next
31. tip the slower is the dynamics of the fluid movement through the tip and the larger is the under over pressure necessary to pump in out If the dynamics is slow one has to increase the pre pumping volume the relaxation time as well as the speed and the acceleration of the pump Light cells can be moved into the capillary with a small main volume and no over pumping Heavy cell objects e g cells that are labeled with a number of param agnetic beads tend to quickly penetrate the capillary even at small main volume values In order to catch such cells reduce the main pump in volume to nearly zero and work with over pumping volume solely The pump speed and acceleration should also be reduced in this case Whenever possible use capillaries with a large tip diameter even if the cells are relatively small The optimum pump parameters can be much easier found for such capillaries and the pumping process is faster 6 11 2 Spacing 6 11 3 The distance between the capillary tip and the surface of the slide affects the efficacy of the cell acquisition and deposition in a quite significant way There are different practical rules of thumb for setting the spacing value depending on the orientation of the capillary bevel with respect to the slide surface Capillary bevel parallel to the slide surface If the capillary bevel is oriented parallel to the slide surface the cells get into the capillary by the vertical flow forces Following rul
32. with an arrow with which you can measure the size of the objects in the video panel Pump IN and Pump OUT Pressing these buttons launches the relevant pumping action Volume IN OUT Two spin boxes that allow to adjust the amount of the liquid pumped in or out respectively Acquire Launches the acquisition action that consists of the following steps 1 Moving a marked cell to the capillary down position 2 Moving the capillary down 3 Pumping in 4 Moving the capillary up Deposit Launches the deposition action that consists of the following steps 1 Moving the stage to the deposit slide 2 Moving the capillary down 3 Pumping out 4 Moving the capillary up Rinse Launches the workflow to be done solely on the service slide Acquire and deposit Acquisition and then deposition are launched The user has to mark a cell before pressing this button If the check box With rinse is checked the capillary treatment sequence on the service slide is included It means that the entire workflow as defined in the workflow editor table will be done see chapter 6 8 6 if the check box Full auto is checked the workflow starts immediately after selecting a cell with a marker 45 6 MMI CELLECTOR p 6 7 4 WAAI e Scan for cells starts scanning of the predefined sample area cells recognition acquisition deposition and rinse with the latter being per formed only if the With rinse check box is checked Capillary spacin
33. ALYSIS USING MMI CELLEXPLORER ObjectNo ColorNo Unit Diameter Total rea _ Circumference Roundness RelativeArea DateTime 1 120 5660 10306 7551 409 4428 E 6 1333 17 2009 1 35 52 PM Img__1 106 9294 7978 2835 350 6021 4 7477 17 2009 1 35 52 PM Img__1 95 8472 6349 4134 305 8837 3 7784 117 2009 1 35 52 PM Img__1 158 0753 14469 0305 474 1817 8 6102 17 2009 1 35 52 PM Img__1 80 8497 4353 0759 253 4891 2 5904 17 2009 1 35 52 PM Img__1 124 2890 8529 4847 370 8822 5 0757 17 2009 1 35 52 PM Imq__1 141 2918 11550 4910 414 7095 6 8734 17 2009 1 35 52 PM Img__1 67 9304 3141 1400 208 7576 1 8692 17 2009 1 35 52 PM Img__1 105 5324 6529 6138 311 5219 3 8856 17 2009 1 35 52 PM Img__1 94 0611 4886 6103 267 0753 2 9079 17 2009 1 35 52 PM Img__1 71 5282 2982 1396 206 1013 1 7746 17 2009 1 35 52 PM Img__1 72 5581 3077 5398 222 2597 1 8314 17 2009 1 35 52 PM Img__1 73 8612 3063 4065 222 3631 1 8230 17 2009 1 35 52 PM Img__1 93 1931 4964 3438 294 6811 2 9542 17 2009 1 35 52 PM Img__1 94 3985 4890 1437 286 6229 2 9100 17 2009 1 35 52 PM Img__1 108 5039 6815 8144 345 5217 4 0559 17 2009 1 35 52 PM Img__1 127 6272 9317 4197 391 4636 5 5446 17 2009 1 35 52 PM Img__1 106 3661 6681 5474 319 9865 3 9760 17 2009 1 35 52 PM Img__1 139 5050 11875 5584 434 9877 7 0669 17 2009 1 35 52 PM Img__1 159 2555 14631 5642 497 4977 8 7069 17 2009 1 35 52 PM Img__1 125 0543 9356 2864 384 0695 5 5677 17 2009 1 35 52 PM Img__1 146 3044 12296 0259 435 5593 7 3171 17 2009 1 35 52
34. D Calibration steps Place R1C1 spot in the view field by moving the lt Y table Set marker at the R1C1 center for automatic grid inspection Bone Figure 6 46 Deposit calibration window CellEctor gt Calibrate Deposition or Deposition in the system panel the user has to set two markers at two locations at the R1C1 spot e the center of the spot e the place where the capillary has to deposit the cell s The first location is used by the software to center the image on the spot during the automatic grid inspection The second location is used for the deposition of cells It may deviate from the spot s geometrical center in order to compensate for the asymmetry of the droplet formed during the pumping of liquid out of the capillary Calibration of a single cap is made simultaneously with the calibration of the capillary po sition within the cap Please refer to chapter 6 9 4 for the detailed description of the calibration procedure The user is guided through the calibration with a self explanatory wizard 81 6 MMI CELLECTOR 6 9 8 Service slide calibration If one works with one of the MMI standard deposition slides AmpliGrid or single cap of Known geometry there is no need to calibrate the service slide For non standard deposition slides a dedicated button Service Slide shows up in the system panel and opens a panel for a manual calibration procedure of the service slide CellE ctor CellCut
35. In the dialog showed in Fig 6 20 the upper part Acquisition defines the behavior of the single acquisition process the lower part While scanning for cells scan that of the fully automated cell acquisition Acquisition mode is presently limited to single cells This means that the user cannot set more than one marker at a time to select a cell and to acquire it However several cells may be collected in the capillary by repeating the acquisition step before the deposition of the cells takes place In such a case one has to adjust the volume that is pumped out during the deposition process to at least multiple of the volume pumped in during a single acquisition step in order to ensure that all the collected cells will leave the capillary upon deposition Two other options allow further modification of the acquisition process Allow for acquisition without markers if this option is checked the system does require setting a marker to acquire a cell If the option is unchecked a message is issued when the user clicks Acquire button without setting a marker prior to it 50 Configuration Acquisition Mode Single cell v Allow for acquisition without markers v Leave capillary down after acquisition While scanning for cells scan 1 screens horizontally 1 screens vertically Search each screenshot 1 time s minimize fluorescence excitation Cancel OK Figure 6 20 Cell acquisition configuration dialog
36. SIS USING MMI CELLEXPLORER Color Power J 50 C Multimode Figure 8 1 Color panel Move the mouse cursor onto the displayed image and left click on the color of the object you would like to count The color will be displayed on the left side of the first of the eight color fields in the settings panel right click LL FL Figure 8 2 Detecting objects of uniform color With a left mouse click the color under the cursor will be entered into the left side of the first color field The computer would search for all objects with color ranging from green to white The gradient in this color field displays the search color A right mouse click will choose the end color of the color gradient to search for In our case of a unicolored object select the same object with a left and the right mouse click The color field will now be filled with green only Only green objects will be searched for A left and right mouse click onto a unicolored object searches unicolored objects with the same color Fig 8 3 If the objects are small it can be tricky to hit the right color By using the zoom options of the mmi CellExplorer section 8 2 2 the selection will be very easy and precise 104 8 1 2 8 1 3 MMI CellEctor Figure 8 3 Detecting objects of uniform color Resulting objects Encircling Objects with different uniform colors In order to search for objects wi
37. a negative value since the capillary narrow end part will be deflected by the slide surface However be aware that the capillary may break if the negative value exceeds 10 um Please observe the safety precautions described in the first chap ter of this manual Capillary size tilt and orientation Some hints concerning the size have already been mentioned above The tilt and the orientation can be arbitrary except for the case when the tip bevel should be parallel to the slide surface and the tilt should be equal to the bevel angle Collecting cells to a matrix of spots grid Since there is a variety of commercially available grids we will describe col lecting of cells to AmpliGrid as an example Most of the steps apply however to all other grids as well If a new grid is placed under the microscope the following operations have to be performed in order to make the grid ready for cells Procedure 1 Notify the software about the new grid Open the deposit configura tion dialog select the type of the grid set its parameters if necessary custom grid and check the option New grid inserted if it has not au tomaticallybeen checked This deletes all the data stored for the previous grid 2 Set the number of cells you want to deposit at each spot Please note that this number is used to determine whether or not the spot is full i e whether the next cell can be added to the current spot Close the deposition configuration
38. a used should also be reflected in the setup name On single camera systems you should normally select lt default camera gt Objective editor The objective selection box contains all objectives defined for the active setup To change the database press the edit button to open the objective editor Fig 5 4 To use this editor follow the steps for the Slide Editor described above If you create a new setup all objectives will automatically be copied from the active slide Objective Editor 14 4 ANN SA UN A Objective name Nosepiece pos Lamp voltage m NominalMagnification a gt 4x UPlanFLN ph 1 4000 4 10 UPlanFLN ph1 2 5000 10 3 20 LUCPlanFLN ph 3 7000 20 i 40x LUCPlanFLN ph2 4 10000 40 60x LUCPlanFLN ph2 5 12000 60 100x PlanFL 6 12000 100 4x UPlanFLN phL New as copy of selected Aca Figure 5 4 Objective editor To rename the defined objective click on the objective name and type in a different name The Nosepiece and Lamp Voltage settings are only used with motorized mi croscopes If specified Nominal magnification will be used to compute various objective 12 MMI CellEctor dependent properties In addition to the parameters displayed in the Objective Editor window the following information is stored separately for each objective e Camera settings see section 5 3 2 e Objective related stage calibration see section 6 9 1 e Lens offset see section 6 9 3 If one of these se
39. ae Hct Ps Y v Enable plane tilt focussing v Define plane point 1 EN K v Define plane point 2 ba v Define plane point 3 pm Reset plane tilt Z E Figure 7 2 Plane tilt user interface Procedure 1 Move to a location near the edge of your sample and focus the micro scope 2 Select Define plane point 1 from the plane tilt menu Fig 7 2 or press Ctrl 1 3 Move to a second suitable location and focus 4 Select Define plane point 2 or press Ctrl 2 98 7 4 3 7 4 4 MMI CellEctor 5 Move to a third suitable location and focus 6 Select Define plane point 3 or press Ctrl 3 7 Repeat this procedure for any other slide on the tray Note that the positions of the plane points are displayed in the overview scan After the plane is defined the sample plane focussing aid can be switched on and off through the plane tilt button or menu Enable plane tilt focussing Any of the three points can be redefined individually afterwards to refine the plane definition The orientation of the sample plane is stored independently for each slide When changing to a slide for which no sample plane has been defined plane tilt focussing will be disabled You may use Reset plane tilt to remove all points and start defining a plane from scratch Neither the single cap nor the service slide require the determina tion of the plane tilt Automatic Z drive control Each time you move the stage with the arrow keys or mouse the b
40. alibrations To estimate the correct focus position requires e Correctly measured location of the membrane slide in three dimensions Sample plane definition e Correctly measured z drive offsets for different objectives 7 4 2 Sample plane definition The sample plane focus mechanism is a simple focussing aid based on the assumption that the sample is planar but slightly tilted against the horizontal plane of the stage As a consequence the focus always lies on this plane which can be defined by three points on the slide see Fig 7 1 When moving to a different location on the slide the focus is automatically adjusted according to the sample plane definition 97 7 AUTOMATED MICROSCOPE CONTROL Figure 7 1 Definition of a tilted sample plane Given three arbitrary base points 1 2 and 3 the software computes the tilt angles a and with respect to the horizontal plane While plane tilt focussing is a useful focussing aid on glass slides and for small areas of interest the approach is limited By prin ciple this method cannot compensate for rugged and non planar surfaces For such samples it is recommended to switch plane tilt focussing off see below To properly define a plane in three dimensions three points must be set These points should not be on a line To get most precise results use a high magnification objective for this procedure 20x or higher Overview Slide Scanning Navigation w
41. ary in two steps e Step 1 Define the vertical reference position of the capillary in the con tact with the planar slide e Step 2 Define the XY position of the capillary tip on the screen Defining the vertical reference position of the capillary at the contact with the slide is done in the 3D positioning dialog accessible through the link Switch to system or the menu Cellector gt Capillary and than click on the button Contact Point One can choose between the simple see Fig 6 27a and the advanced positioning method see Fig 6 27b In the simple method a step by step wizard guides you through the calibration and there is no need to use joystick This method is recommended for most users The advanced method is recommended for those who prefer to work with joystick and or numbers As both methods result in the same accuracy of the positioning it does not matter which one you actually use The wizards for the simple method differ slightly depending on whether the calibration has to be done on a flat slide or in a cap Neither of the methods will work properly if the plane tilt of the slide is unknown or incorrect The simple method on a flat slide runs through 5 steps described below and has to be done on both the sample as well the deposit slide in a deliberate order Just carefully follow the instructions marked blue and warnings red in the wizard and perform the required actions For documentation purposes the detailed
42. by double clicking into the over view area with the left mouse button Define the area of the scan with the Select 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 Begin the scan by pressing the Start Scan button You can always interrupt the scan with the Stop scan button Also pressing the Esc key will interrupt the scan The scanned image can be made available to other applications Use the Save button or the corresponding menu item to save the overview image to disk Alternatively select Copy image to clipboard from the popup menu next to the Save button to copy the overview image into the Windows clipboard and paste it in other applications To erase the scanned image click on the Clear overview image button With the magnifier button you can open the large slide overview window Fig 5 12 Overview scan O E Slide Scanning Navigation RSH Pa E 1 2 3 E Figure 5 12 Large slide overview window The window provides the same tools as the small slide overview panel Only the magnifier button behaves differently In this big slide overview window the magnifier button opens the standard Windows photo viewer Multi slide handling The mmi Multislide holder grants the user the opportunity to use more than one slide in the xy stage To move from one slide to another you can directly
43. c Slide to slide motion pm 1000 To prevent collision between the objective and the sample slide the focus drive is restricted by the upper limit specified below Objective sample collision um 10000 Reset K ca Figure 7 3 Microscope safety parameters Open the dialog using the menu item Microscope Safety parameters Objective collision prevention There are two situations in which mmi CellTools automatically lowers the ob jective turret to prevent potential damage to the objectives The first situation is when switching to another objective This function is car ried out automatically and does not require configuration The second situation is when moving the stage to a different slide If the two slides are not perfectly aligned the surface of the objective may scratch against the slide s lower surface To reduce the risk of damage the objective turret is 100 7 5 2 MMI CellEctor lowered by a certain amount which can be specified in the safety parameters dialog Slide to slide motion If the slides used in your setup exhibit very high tolerances you may wish to increase this value When you are using the small step arrow keys or the mouse the z drive escape function is switched off Objective sample collision To prevent contact between the objectives and the specimen and to protect the microscope itself the range of the focus drive is automatically limited to a certain maximum value which ca
44. calibration the microscope does not lose focus when the objectives are changed Paraxial lens offset Due to mechanical and optical tolerances two objectives never have exactly the same optical axis The difference between the position of the optical axes of two objectives is called paraxial lens offset This offset manifests itself by a small shift of pixels in the video panel when objectives are changed The mmi CellTools software can automatically compensate this shift if the paraxial lens offset has been determined for each objective in use To perform the calibration of the paraxial lens offset select CellEctor Calibration Objective gt Lens Offset paraxial from the main menu or press the button Objective in the CellEctor system panel and then click the link Offset paraxial In the lens offset calibration pro cedure the XY displacement of a sizeable object is measured while changing the objectives from the highest to the lowest magnification The panel shown below guides you through the following calibration steps Procedure 1 Change to the objective with the highest magnification and or click Start button to confirm it 2 Two tool buttons show up 59 6 MMI CELLECTOR CellEctor CellCut CellE xplorer Exit objective calibration Current objective 4x Magnification Offset paraxial Task measure the XY displacement of an object while changing the objectives Next step Change to the objective with
45. ch between camera and binocular 96 Define z drive offset for current slide 100 123 Keyboard shortcuts Navigation cursor keys numeric pad Insert Home Page Num f m Up Lock Del End Down EJE E 1 3 Enter press minor steps press major steps hold slow movement hold fast movement 124 MMI CellEctor C Technical data C 1 Required minimum workspace The table top for the microscope laser optical equipment computer monitor and keyboard requires a minimum workspace of 1 20 m x 0 90 m The computer should be positioned under or near the table The camera computer connection cable is 2 m long to ensure reliable data transfer C 2 System components C 2 1 Microscope Inverted or upright research microscope with fluorescence port Supported microscope models e Olympus IX 71 non motorized e Olympus IX 81 e Nikon Ti S U E e Nikon Ni U E e Nikon TE 2000 S U E C 2 2 Stage Scanning stage with stepper motors Scanning area 120 x 100 mm Repositioning accuracy lt 1 yum Step resolution 0 078 um 125 APPENDIX C TECHNICAL DATA C 2 3 Digital camera mmi CellCamera MXF285c e IEEE1394b FireWire color CCD camera e 2 3 interline transfer frame readout CCD Temperature controlled active Peltier cooling e signal to noise ratio gt 62 dB On board integrated color processor for high qual
46. changes the direction from in to out or vice versa Over pumping volume volume that is pumped in prior to the main vol ume to be pumped in and then pumped out after the main volume has been pumped in This value has no effect on the pumping out process 49 6 MMI CELLECTOR 6 8 4 Relaxation time waiting time spent for pressure to equilibrate after the pumping process is finished e Speed pump speed in percent of the maximum speed Acceleration pump acceleration in percent of the maximum accelera tion 40 number showing the current capillary size for which the pump param eters are displayed Click on it opens the list with available capillaries as shown in the figure above All the pump parameters are specific for a given capillary Up to five parameter sets can be stored for capillaries with different sizes You can retrieve a stored parameter set for a given capillary by selecting the corresponding capillary size Whenever the pump parameters are modified the changes are valid for the running session only In order to save the current set for the future sessions check the box Save this parameter set before you leave the above dialog Acquisition configuration One can adjust some parameters of the acquisition process in the configura tion dialog that can be opened either from the main menu CellEctor gt Configure Acquisition or by clicking any acquisition relevant button with the right mouse button
47. ction 6 3 2 6 6 3 Calibrating the capillary Contact point Determine the vertical reference position of the capillary see section 6 9 4 On a flat slide it is the position at which the capillary tip touches the slide surface XY position on the screen Once the contact point of the new capillary is known move the capillary down and calibrate its XY position on the screen see section 6 9 6 6 6 4 Finding the optimum conditions for collecting cells Solutions containing cells as well cells themselves exhibit a broad range of physicochemical and biological properties Thus the optimum conditions have to be found for the particular cells of interest While experimenting with new cells and buffer solutions one can vary the following parameters Size of the capillary tip In principle it should be larger than the size of the cells to be collected If the concentration of cells is not too large even relatively large capillaries tip diameter of 40 um can efficiently collect small cells approx 5 um Orientation of the capillary tip bevel Rotating the capillary around its axis may affect the efficiency of catching and depositing the cells Spacing The distance between the capillary tip and the slide surface spacing affects both the volume of the liquid necessary to be pumped in order to pick a cell as well the volume to be pumped out at the deposit to get the cell out of the capillary It should be adjusted for the sample and th
48. d ay tle ee le 126 C225 2D M nip lator tact ie Goce Sate ree ae dl hog aan Ae ha 127 CONTENTS C 2 6 mmi CellPump MMI CellEctor 1 Security advice 1 1 General safety e Do NOT disassemble the system The installation of the system is done by MMI service personnel or a designated MMI representative Repairs removal or exchange of components except for the operations described in this manual must be carried out solely by the MMI service personnel or persons expressly authorized by MMI If you have any problems with the instrument contact MMI e The responsibility for the correct functioning of the mmi CellEctor passes onto the owner or user if the system is maintained or serviced improp erly by persons not belonging to the MMI service team or if used for a purpose other than that intended MMI is not liable for damage occurring from non observance of these notes e The power supply is installed by MMI MMI ensures that the system is provided with the appropriate voltage Do not change the power cords Avoid wet or dusty conditions near the system Do not attempt to use the system if liquids get inside it Contact MMI in such a case Unplug all the electrical supply before cleaning the system Do not use cleaning fluids or sprays use solely a smooth and dry cloth e If the stage is not calibrated its movements can be sudden and fast Ensure that the work area around the stage is free of clutter and material Read the
49. dialog 91 6 MMI CELLECTOR 6 11 6 6 11 7 3 Determine the tilt of the grid plane 4 Optionally determine the contact point of the capillary on the new grid This is necessary only if the thickness of the new grid significantly differs from the previous one and or a high accuracy of the spacing between the capillary and the grid is required 5 Calibrate the position of the spot located in the first row first column R1C1 6 Perform the first deposition at the first spot of choice leaving the capillary down after pumping out If the buffer solution with a cell could not leave the capillary after having pumped out the preset volume increase the latter or decrease the capillary spacing to the deposit whatever appro priate Continue with filling all other spots with cells The software registers the num ber of deposition actions in each spot and assumes that one cell is deposited each time Once every spot in the grid is filled with the preset number of cells the entire grid is full no further deposition is possible and the user is prompted to replace the grid One can also fill a selected part or every second spot on the grid only To do the former simply define the current and the end row column in the deposit configuration dialog To do the latter de fine a custom grid with spacing between the columns that is double the spacing and the number of columns rows that is a half of the number of columns rows on the ac
50. diately after a marker is set Thus there is no opportunity to delete a marker that has erroneously been set on a wrong cell 6 10 5 Scan for cells workflow With the Scan for cells workflow the user does not need to mark the cells since it is done automatically be the software This workflow is usually applied under fluorescence conditions since the colored cells of interest can easily be distinguished from the black background A single click on the Scan for cells button in the action panel launches the following recurrent workflow Procedure 1 N O O A W 86 A snapshot of the video screen containing the cells is made at the start ing position of the stage and is then transferred to the image processing software mmi CellExplorer mmi CellExplorer finds the cells marks them and sends the set of the markers back to mmi CellEctor mmi CellEctor selects a single cell to be aspired acquisition deposition rinsing if required moving back to the sample 6 10 6 MMI CellEctor 8 Depending on the settings made by the user either another snapshot is made at the same stage location or the stage is moved to the next location and the loop restarts In order to scan through the sample the user can define the number of screens horizontally and vertically in the acquisition configuration dialog see chap ter 6 8 4 Since usually there are several cells of interest within one snapshot the recognition and se
51. e capillary towards the optical axis of the objective and to lower it towards the slide The first step is to change to the objective with the smallest magnification usually x4 Then press Next 63 6 MMI CELLECTOR 2 The capillary reached the leftmost X position note the red bar at the X arrow in Fig 6 29a and was lowered so that its shadow should already be seen in the screen see Fig 6 29b If it is not you have to adjust its horizontal position first Using the X and Y arrows place the capillary tip Switch to advanced YA x 4a z y y Speed max Y medium min 1 pm 3D Positioning Wizard SAMPLE SLIDE Step 2 of 5 Do NOT refocus the microscope b Capillary moved over the c Capillary lowered to the d Capillary positioned ex center marker but is still optical plane actly on the center marker above the optical plane and to the optical plane Figure 6 29 Step2 of capillary vertical positioning wizard in the center of the target marker as shown in Fig 6 29c Then move the capillary down or up using the Z arrows so that its tip is in focus like in Fig 6 29d Then press Next 64 MMI CellEctor 3 The system has now set the focus from the capillary tip back to the slide Change to the objective you anticipate to work with during collection of cells Then press Next Capillary 3D positioning m t t y y Speed max Y medium min 1 um 3D Posi
52. e deposit slide separately 41 6 MMI CELLECTOR Pumping parameters These parameters have the major influence on the efficiency of collecting the cells In most cases it is enough to adjust the main volume to be pumped in or out In some extreme cases one has to use the other pump parameters like pre and over pumping relaxation time or speed and acceleration of the pump see section 6 8 3 6 6 5 Performing cells collection 6 7 6 7 1 Cells can be collected using either the manual automatic or workflow mode In the manual mode moving the stage moving the capillary up or down and pumping are action separately launched by the user This mode is suitable learning the mmi CellEctor and for finding the optimum conditions The rou tine work is preferably done in the workflow mode that combines these single actions in series of actions see section 6 6 mmi CellEctor user interface As described in section 5 1 the special features of the mmi CellCut the mmi CellExplorer and mmi CellManipulator are installed as separate plug ins soft ware modules The mmi CellManipulator plug in appears as a separate tool panel on the right side of the program window To switch from one plug in to the other you only have to click on the appro priate tool panel see Fig 6 13 Once the mmi CellEctor tab is selected in the component control panel of the mmi CellTools main window mmi CellEc tor becomes the active component It adds its own menu
53. e microscope s controller hub must be connected to the PC either COM port or USB To make mmi CellTools connect to the microscope activate the menu item Microscope Remote control This activates control of objectives filter block condenser cassette fluores cence shutter and lamp brightness The focus knobs on the microscope body will still be useable mmi CellTools will remember these settings and restore them at startup Make sure the microscope is connected and switched on before starting mmi Cell Tools 7 2 Observation methods The setup editor is used to define the observation methods see section 5 2 2 In the setup editor you define for each setup the position of the 95 7 AUTOMATED MICROSCOPE CONTROL 7 3 7 4 Table 7 1 Optical path settings for Olympus and Nikon microscopes Olympus Nikon Left side port 1 5 Binocular 2 1 e Filter block Condenser e Optical path Camera port Binocular When you change the setup the motorized microscope will set these three items to the positions defined in the new setup automatically The optical path settings are specific to microscope vendors see Table 7 1 Filter wheel position numbers and condenser turret position numbers are clearly indicated on the microscopes A fast switch from any camera port to binocular and backwards is performed in the menu Microscope Binocular or by pressing F7 Objective control In the objective editor see sectio
54. either automatic or workflow mode with the au tomated microscope it is compulsory to determine the tilt of the plane both on the sample as well as on the target slide except for a single cap Manual pump use The pump is delivered ready for use i e it is filled with oil and with the piston set in the backward position rime a il wl Fame GUT 100 il wl Figure 6 48 Set the pump volumes in the action panel and initiate pumping by presseing Pump IN or Pump OUT Pumping a predefined volume The buttons Pump IN and Pump OUT in the Action Panel allow pumping a pre defined volume of liquid see Fig 6 48 The pump speed and acceleration can be configured as described in chapter 83 6 MMI CELLECTOR 6 8 3 Once launched the pumping cannot be interrupted Thus mmi CellEc tor estimates the pumping time for a given volume and issues a warning if the time exceeds 2 minutes CellEctor CellCut CellE xplorer Exit pump panel Move pump piston by Pumping out Stop Figure 6 49 nitiate continous pumping by presseing Pumping in or Pumping out Endless pumping Continuous pumping with the highest speed and acceler ation can be done using the pump panel accessible by pressing Switch to system Pump see Fig 6 49 The pumping can be stopped by pressing the Stop button 6 10 2 Moving the capillary up or down Manual Move capillary down L LY Refocus on capillary Figure 6 50 Moving the capillary up o
55. es apply 90 The smaller the spacing on the sample the lower the amount of the liquid that has to be pumped in to get a cell into the capillary One has however to take into account the size of the cells as compared to the diameter of the capillary tip and the height of the cells The above rule does not apply for pumping the cells out on the deposit If the deposition takes place on a dry surface the surface tension of the liquid has to be taken into account which at small spacing may prevent the liquid from flowing out of the capillary The optimum spacing on the deposit depends on the amount of liquid to be pumped out and vice versa Both parameters should be chosen so that the liquid touches the deposit surface and flows out of the capillary before a droplet is formed and falls down 6 11 4 6 11 5 MMI CellEctor e If the deposition takes place into a single cap spacing should in princi ple be set to zero Nonzero values may still be used to correct for the liquid boundary moving upon evaporation in order to keep the capillary tip within the liquid Capillary bevel vertical to the slide surface and cell scratching If the capillary bevel is oriented vertically to the slide surface the flow forces parallel to the slide surface can be combined with a scratching action of the capillary Scratching is achieved by sliding the capillary tip along the slide surface The capillary will move in this way if the spacing is set to
56. est Z drive position will be calculated and the Z drive motor moved to that position With flat samples you immediately receive a sharp image The more uneven the sample is the more you need to correct the Z position manually If you draw a shape or if you set a pin position the software saves the z position Each time you go back to the shape or the pin position the saved Z position will be recalled You never need to refocus during cutting Z drive offset for slide In the case of a slide which is non planar it is necessary to define a Z drive offset for the current area This distance can be measured and corrected automatically Procedure 1 Focus microscope 99 4 y i Je fi 1 l 7 AUTOMATED MICROSCOPE CONTROL 7 5 7 5 1 2 Select menu item Microscope gt Drive Offset gt Slide or press F8 After successful calibration the scope does not loose the focus when you change the position of the stage slightly Microscope safety parameters mmi Cell Tools provides functions to help prevent physical damage to the mi croscope objectives These functions are always in place and do not require manual intervention However under certain circumstances they can be con figured through the safety parameters dialog Dbjective collision prevention feat nt objective damage the obje arent is m ale jut of the way I ertain situatior The below speci e respective safety distance Objective change pm automati
57. ew grid cap inserted is automatically checked The basic parameters of a grid rows columns spacing can be changed only for a custom grid On the other hand the number and the location of the target spots can be selected arbitrarily on both AmpliGrid as well as on the custom grid by changing the current row the end in row the current column and the end in column The number of cells per spot is used by the software to determine whether or not a given spot is already filled and the stage has to be moved to the next spot No further deposition is possible if all the spots of a grid are filled with the pre defined number of cells The same applies to a single cap The remaining three options facilitate the visual control of the deposition of cells during the automatic acquisition and deposition In the standard depo sition process the capillary is moved up as soon as the pumping out step is completed including the pump relaxation time This corresponds to N 0 set in the spin box After having pumped out stay down for N seconds Change this number if you want the capillary be left down for an additional amount of time maximum 20 seconds before it is moved up and the XY stage moved back to the sample This number becomes irrelevant if the option Wait for spot inspection is checked In this case the XY stage is not moved back to the sample until the user clicks Back to sample from the context menu on the video image Workflows editor In
58. explanation is given below 61 6 MMI CELLECTOR Capi ary 3D positioning Capillary 3D positioning Switch to advanced Speed _ max medium min 1pm 3D Positioning Wizard AMPLIGRID Step 1 of 5 Change to the objective with the smallest magnification x4 Then click lt Nexb and wait for the capillary be moved Warning the capillary may break if the plane tilt of the slide is unknown or incorrect Switch to simple Current position pm x 25000 j Y 10657 zlo a Joystick Fixed positions pm x Y zZ Set IDLE position 25000 10657 0 Set CONTACT position 14367 10657 12640 Initialize motors Calibrate motors a Simple method The wizard only shows up if plane tilt is active 62 b Advanced method Fixed positions only show up if plane tilt is active Figure 6 27 Capillary vertical contact point calibration windows MMI CellEctor Capillary vertical contact position Ampligrid or custom grid simple method Procedure 1 Once the 3D positioning dialog is opened a large target marker is shown in the center of the screen facilitating the positioning At this moment the Figure 6 28 Center of the screen indicated in the video panel capillary stays in the idle position which is at the maximum distance from the slide This is indicated by a red bar above the Z arrow no further raising of the capillary is possible The system is now ready to move th
59. g Two spin boxes that allow adjusting the distance be tween the capillary tip and the surface of the sample or deposit slide The values in these two boxes are in effect before the capillary starts its movement down For changing the spacing when the capillary is already down use the fine positioning arrows shown below These two arrows show up as soon as the capillary arrives to the down position Pressing one of them moves the capillary up or down in fine steps giving the opportunity to adjust the capillary spacing The spacing value in the corresponding spin box is automatically adjusted and is then used in the future movements down Once the fine adjustment arrows show up the relevant spin box is disabled Rough spacing mode warning shows up when the Z drive control of the automated microscope has been turned off or you are working with the manual microscope It indicates that the numbers in both spin boxes are approximate Like in the system panel clicking some buttons capillary pump acquisition deposition rinse with the right mouse key launches the relevant configuration dialogs If some system components are not calibrated the affected buttons in the Action panel are temporarily disabled grayed The link Switch to system opens the system panel Distance measurements Pressing the Measure button the Action panel changes the mouse cursor to a hand with an arrow that allows measuring the distances in the video panel F
60. he camera operates at a lower resolution but yields brighter black and white im ages at a very fast frame rate Color shifts mainly caused by changes in lamp brightness can be corrected using the white balance function To set the white balance first locate an empty transparent part on the sample slide then click the Set white balance button If the image is too bright or too dark the white balance fails The white balance function is also available in the drop down menu next to the camera button in the main menu under Video Adjust white balance or using the keyboard shortcut Ctrl W 15 5 MMI CELLTOOLS MAIN APPLICATION 5 3 3 5 3 4 Do not use the Set white balance option in fluorescence imag ing All camera settings including white balance are stored separately for each objective This eliminates the need to adjust the camera after each objective change Optionally the software uses a single white balance setting for all objectives This is recommended for non automated microscopes Select Video Save white balance per objective to enable or disable this function Freeze video live video In fluorescence applications it makes sense to freeze the video when you have acquired a good image After freezing the video you can close the fluores cence shutter and go ahead with drawing and cutting your dissectates without further photo bleaching of the fluorescence dye To freeze the video use the men
61. he hardware components of the system pump capillary all the operations are launched at PC by the user 30 MMI CellEctor The mmi CellEctor is an adjustable open platform for any assay format It closes the gap between frequent and rare cell sorting It is dedicated for the use in the following disciplines cancer research amp oncology immunology virology stem cell sciences as well as many other applications in biomedical research cellular diagnostics microbiology amp virology environmental ecology forensics MMI is committed to establish specified and detailed application related protocols in a close co operation with the users All the methods applied to measure molecular changes require 100 pure cell populations to ensure unbiased results For the analysis of DNA copy number variations or single nucleotide polymorphisms are being measured Changes of RNA expression are analyzed by microarrays or massive paral lel sequencing approaches Protein patterns are analyzed with spectroscopic or biochemical protocols and epigenetic changes are detected by changes in methylation patterns FACS sorting and bead based protocols are the major state of the art methods to isolate single cells Also a wide variety of physical and immunological methods are employed The mmi CellEctor helps to over come the hurdles and drawbacks of the above mentioned methods The mmi CellEctor technology has been used for the enrichment of tu
62. illary 1 is mounted in a transparent capillary holder 2 that connects to the pump The capillary holder is fixed in the manipulator holder 4 by a knob 3 An additional knob 5 allows changing the rotational position of the capillary holder Furthermore there is a screw at the rear side of the manipulator holder 4 that allows adjusting the capillary tilt The standard tilt is 45 deg Figure 6 8 The entire 3D manipulator can be tilted by 90 deg down to a position at which the replacement of the capillary is facilitated For this purpose unscrew the knob 6 and gently rotate the manipulator to the required position 35 6 MMI CELLECTOR Procedure 1 Place the 3D manipulator in the position shown in the figure 6 8 2 Slide the capillary cover tube along the transparent capillary holder down to the lowest position 3 While rotating the capillary cover tube gently press it down until it steps into the screwing position 4 In this position slightly loosen the o rings that tighten the capillary by rotating the capillary cover tube 5 Pull the old capillary out of the holder 6 Pump oil out until it reaches the outer o ring that tightens the capillary Take care than there are no air bubbles in the oil 7 Insert the new capillary While inserting ensure that the capillary passes both o rings 8 Tighten the capillary by rotating the capillary cover tube At this stage it is not recommended to fill the capilla
63. image editing It mainly helps with manipulating color images with a color depth of up to 32 bits The three sliders modify the hue saturation and inten sity value New colors are defined with the cursor in the color field located at the lower part of the register The primary colors are defined with the rainbow tool displayed in the larger part of the window The HSL register In the HSL register you can further adjust the colors apply ing the same principles as described for the HSV register The RGB register In the RGB register you can control the individual primary color values All true colors can be modified by adjusting the three sliders for red green and blue The Equalization register In the Equalization register halftones can be con trolled exactly The distribution and frequency of the halftones 0 255 for 8 bit images 0 65535 for 16 bit images is displayed in the histogram By adjust ing the sliders halftone ranges can be shielded from the image Just move the slider with the left mouse button held down You can also edit color images 113 8 IMAGE ANALYSIS USING MMI CELLEXPLORER 8 2 3 4 Just click into the small windows below the word histogram The respective primary color will be displayed Now the individual distribution of the colors red green and blue RGB can be filtered Bump Map filter The Bump Map Filter produces a surface structure like those of freeze cuts It simulates a metallic surface where
64. ime At this moment the capillary Z position and the position of the Z drive of the microscope are stored for future calculations The controls of this advanced capillary 3D positioning dialog Fig 6 42 perform the following operations X Y Z show the current position of the capillary If you change any of these values using the spin controls the changes are in effect after the Go to button has been clicked e Go to moves the capillary to the point defined by the current X Y Z values e Refresh updates the X Y Z values Move down moves the capillary to the point defined as the contact point minus spacing This button is invisible if the contact position is unknown Move up moves the capillary to the point defined as the idle position usually the uppermost one e Joystick activates deactivates the joystick Activation of the joystick dis ables the seven controls mentioned above The position indicators X Y Z are however continually refreshed while the joystick is in use 76 pili ey POSTE Switch to simple Current position um x 25000 j Y 10657 f ZO a Joystick Deam 1 eee Fixed positions pm x Y zZ Set IDLE position 25000 10657 0 Set CONTACT position 14367 10657 12640 Initialize motors Calibrate motors MMI CellEctor Figure 6 42 Capillary vertical contact point calibration windows for the advanced method Fixed positions only show up if plane tilt is active
65. ing of cells If this option is checked the motorized fluorescence shutter is opened just before a screen shot is made and closed immediately afterwards 6 8 5 Deposition configuration Configuration Deposition Deposit type AmpliGrid 4 12 4500 4500 New grid inserted Current row Current column End in row End in column au e cell s per spot After having pumped out stay down for additional 1 sec 4 Wait for spot inspection with capillary down Figure 6 21 Cell deposit configuration dialog One can adjust several parameters of the deposition process in the configura tion dialog that can be opened either from the main menu CellEctor gt Configure Deposition gt Deposit or by clicking any deposition relevant button with the right mouse button see 52 6 8 6 MMI CellEctor Fig 6 21 The choice of the deposit type has effect on many other settings of the system Presently one can choose among three types of the deposit e AmpliGrid commercially available grid Custom grid grid defined by the user Single cap transparent cap placed in a holder If the user changes the deposit type the configuration parameters irrelevant to the selected type are disabled For instance the number of rows and columns as well the spacing are not applicable to the single cap Changing the type also deletes all the settings and data associated with the current deposit target and the check box N
66. involve either the entire grid check the box Scan entire grid or only a part of it For the latter define the starting and the ending row and column For visual inspection define the number of seconds the XY stage will stay at each spot 55 6 MMI CELLECTOR MK Deposit scan area E Scan entire grid Row Column Statin 1 1 de ar Endin 1 5 de oF sec Stay at each spot for 1 wr __ Apply image processing Cancel OK Figure 6 24 Configure inspection of the deposit slide For automatic inspection of the spots check Apply image processing In this case the XY stage waits until the recognition of cells is completed by mmi CellExplorer A report will be created automatically also see chapter 6 10 6 Prior to starting the deposit inspection with the image processing the correct image processing parameters have to be loaded to mmi CellExplorer 56 MMI CellEctor 6 9 Calibration routines 6 9 1 In order to efficiently perform the acquisition and deposition of cells mmi Cel lEctor requires the microscope objectives be set up properly The objective setup includes the following Procedure 1 XY calibration of the objectives magnification 2 calibration of the parfocal lens offset automated microscope only 3 calibration of the paraxial lens offset Objectives magnification When the mouse is dragged within the video panel in the movement mode the stage move
67. ion or manipulation A carefully selected and application adapted set of functionalities guarantees a high throughput at a comfortable level of automation Nevertheless a high degree of flexibility is supported The mmi CellExplorer finds counts and sorts objects primarily based on the object colors Secondly the automatic distinction of various object shapes is a particularly innovative feature Round shapes undifferentiated cells for instance can be automatically distinguished from oval forms differenti ated cells Objects adjacent to each other are automatically separated and counted by the software Finally the object shapes found can automatically be optimized for laser cutting For advanced users the mmi CellExplorer comprises complex image pre processing that can be performed before the object finding For this pur pose the software has many different filters like image sharpness color inten sity brightness color space transformations background separation by fast Fourier transformations gamma corrections Getting started Switch from mmi CellCut or mmi CellEctor to the mmi CellExplorer by choos ing the mmi CellExplorer tab The live image is loaded to the mmi CellExplorer Encircling objects of one uniform color By clicking on the plus left to the color label the small color panel Fig 8 1 opens This panel shows eight fields representing eight different object types you can search for 103 8 IMAGE ANALY
68. is off the movie is played back at the same rate at which it was recorded When using time lapse the recording rate is automatically adjusted The dialog displays the recording interval i e the time elapsed between two frames This interval significantly influences the size of the resulting video file The Movie folder input field allows you to specify the destination folder where movie files are saved For maximum performance this should not be a net work folder Check the Open movie after recording option to have movies automatically open and playback in Media Player after recording Motorized stage control The movement of the motorized stage is controlled by the mmi CellTools soft ware Mouse movement Choose the move mode by clicking the hand tool button In the move mode the cursor in the video panel always appears as a hand By clicking and dragging the left mouse button the stage directly follows the mouse movement If stage movement does not exactly follow mouse movement you may need to carry out an objective calibration see section 6 9 1 You can quickly switch to move mode and back again by pressing Space Keyboard movement The main arrow keys and numeric pad arrow keys can be used to move the stage in discrete steps or continuously at constant velocity Fig 5 8 A single press of a cursor key moves the stage by a defined distance By default this is 10 of the screen for the cursor keys and 90 of the scree
69. ity color calculation e 1392 x 1040 pixels with up to 20fps e Ultra high sensitivity mmi CellCamera DXA285cF e IEEE1394a FireWire interline transfer color CCD camera e 2 3 interline transfer frame readout CCD e Super HAD technology signal to noise ratio gt 56 dB e 1392 x 1040 pixels with up to 15 fps e Ultra high sensitivity mmi CellCamera DXA285F e IEEE1394a FireWire interline transfer CCD camera e 2 3 interline transfer frame readout CCD e Super HAD technology e 1392 x 1040 pixels with up to 15 fps e Ultra high sensitivity Andor iXonEM EMCCD Camera 897 e EMCCD Technology Even single photon signals are amplified above the noise floor Full QE of CCD chip is harnessed no intensifier e RealGain Absolute EMCCD gain selectable directly from a linear and quantitative scale e TE Cooling to 100 C Critical for elimination of dark current detection limit C 2 4 Computer Suitable computer workstation models are preselected and tested by MMI Only use computer hardware supplied through MMI Before performing hard 126 MMI CellEctor ware modifications contact service mmi CellTools supports Microsoft Windows XP Windows 7 and Windows 8 in 32 bit and 64 bit configuration C 2 5 3D Manipulator Travel range 25mm Resolution 0 025 um C 2 6 mmi CellPump 127
70. ius e 10 is a large radius By clicking the Check button you can verify the effect Image Effects For manipulation of digital images the mmi CellExplorer con tains additional image editing algorithms Pressing the Image Effects button opens a dialog Fig 8 13 You can see two small versions of your image The left image shows a copy of the original while the right one directly displays the effects of the changes made by adjusting the controls A UI pa o Contrast HSV HSL RGB Equalization Bump map FFT Gamma correction Shits Contrast D g Brightness 0 g Figure 8 13 mage effects dialog How to use the combination of several image effects Evaluate the best set of image effect parameters 112 MMI CellEctor Procedure 1 Open the Image Effect panel 2 Change a parameter and press Copy Results to Original 3 Change as many effect parameters and press Copy Results to Original until you found the best set of parameters 4 Close Set the best set of image effect parameters for the use in combination with the green arrow key Procedure 1 Open the Image Effect Panel 2 Set the best set of parameters without using the Copy Results to Orig inal buttons and close the Image Effect Panel with the OK button Contrast and Brightness Optimize the image contrast and brightness by adjust ing the sliders The HSV register The HSV register contains further important functions for
71. ive Capillary Service Slide Inspect Deposit Capillary spacing um to sample 30 deposit 0 Figure 6 15 The mmi CellEctor plug in panel in calibration mode the calibration procedure of this component Clicking the button with the right mouse button launches the configuration dialog of this component if the cor responding item exists in the CellEctor main menu Clicking the link Switch to action opens the action panel The button Inspect Deposit shows up only for grids as deposition targets and the button Service Slide only for non standard deposi tion targets Action panel Most of the routine work with the mmi CellEctor is done using the Action panel It consist of the four groups of controls e Manual operation Automatic operation 44 MMI CellEctor e Workflow e Capillary spacing Here the controls will only briefly be explained e Move capillary down up Moves the capillary down to the point and in the way defined in the capillary calibration and configuration procedures Once the capillary is down press the button again to move it up to the idle position e Refocus on capillary slide This button toggles the focus between the capillary tip and the slide surface NOTE The button shows up only if the Z drive control of the automated microscope is on Marker button Changes the mouse cursor to a cross with which you can mark a cell to be acquired e Measure button Changes the mouse cursor to a hand
72. lection of cells can be repeated several times at the same stage location For the fully automatic scanning to work correctly it is compulsory to load the correct set of parameters to mmi CellExplorer software before you start the scan Please refer to the mmi CellExplorer manual for the explanation how to do it Automatic inspection and reporting of deposit grids Inspect deposit Figure 6 53 Inspect deposit button By pressing Inspect deposit mmi CellEctor is capable to automatically scan through the spots of a grid giving the user opportunity to verify the presence of cells in the spots The verification may be either just visual or using auto matic cells recognition by mmi CellExplorer The inspection process can be configured as described in chapter 6 8 7 Reports If the reporting is actived in the confiration dialog see chapter 6 8 7 a snapshot of each inspected spot is stored in the JPG format for documen tation purposes The image files are named RxCy jpg where R C represent row and column and x y represent their number respectively The images are stored it the folder My DocumentsimmiCellToolslMAutoDocA CL_InspectData_yyyy mm dd_hh mm ss RxCy jpg Furthermore a report file can be created in the ASCII tab separated format that contains the following information Spot ID number RxCy Number of cells automatically recognized in each spot during the inspec tion of the grid Total amount of liquid
73. manual of your microscope for microscope specific precau tions If you do not have the manual contact your microscope provider or MMI 1 2 mmi CellEctor safety e mmi CellEctor uses very thin glass capillaries that may break into splin ters and injure eyes or other parts of the human body Thus it is com pulsory for the user to wear protective gloves and clothes while working with the mmi CellEctor 1 SECURITY ADVICE e Never point capillaries at persons Capillaries can suddenly be loosened from their grips under high pressure and become missiles e The mmi CellEctor is to be used solely for collecting cells in accordance with the appropriate legislative requirements e Please ensure that all work is carried out in accordance with this manual 2 MMI CellEctor Installation The MMI system may only be installed by an MMI service engineer or our des ignated representative in the laboratory of the customer After the installation training will be provided in the use and operation of the system The customer should not change the installation of the equipment Should you want to move an installed unit please contact MMI for assistance With any malfunction of the device please contact our service department service molecular machines com After installation or modification of the MMI system an authorized specialist must perform a thorough check to ensure that the system is in perfect condi tion If your system comprise
74. mmi CellEctor a workflow is a combination of acquisition deposition and the capillary treatment on the service slide that is launched by a single mouse click as a continuous series of actions The workflow can be customized in the following workflow editor see Fig 6 22 In the example shown in the above figure the workflow consists of four con secutive steps 53 6 MMI CELLECTOR mmiCellEctor Workflow Editor 1 0_RCO1 ay Workflow Editor Step Name Well No In vol nl Out Yol nl Stay Time s x Times E Acquire 2 Addwater 1 30 3 Deposit 4 Clean Hints Left click selects a cell Double click or F2 edits a cell Right click pops up a menu Capillary Service Slide Figure 6 22 The workflow editor Procedure 1 acquisition 2 diluting the reagent in the capillary by adding some water placed in well 1 of the service slide 3 deposition 4 cleaning the capillary in well 5 of the service slide The parameters of a single step can be modified by editing the values in a single row of this table The entire rows can be deleted inserted or appended using the commands from the pop up menu see Fig 6 23 The parameters in the rows Acquire and Deposit cannot be changed and these rows cannot occur more than once in this table that is why the menu items Insert Acquire and Insert Deposit are disabled in Fig 6 23 54 6 8 7 MMI CellEctor Insert Append Delete Row Em
75. mor cells and im mune cells from disaggregated lymphnode material blood and bone marrow of cancer patients For the outlined workflow of an application example the following purification steps were established Lymphnode metastasis is dis Y A Y hy p 3 lt gt lt gt a Preparation of cell sus b Lyse erythrocytes from pension disaggregate lym blood or bone marrow sam phnodes and filter cells ples Figure 6 2 Preparation of the patient material aggregated by the use of scalpels and filtered through a 70 micron filter to get a cell suspension Blood and bone marrow is treated by erythrocyte lysis to remove present erythrocytes Tveito et al 2007 In a first enrichment step Reference Tveito S Maelandsmo M G Hoifodt K H Rasmussen H Fodstad O Specific isolation of disseminated cancer cells a new method permitting sensitive detec 31 6 MMI CELLECTOR AS z a specific immunomagnetic b magnetic separation of beads and incubate under ro immunomagnetically rosetted tation cells microscopic evaluation and mmi CellEctor mediated cell picking Figure 6 3 Pre enrichment of target cells by immunmagnetic selection via targeting a cell specific surface marker and mmi CellEctor mediated cell picking Figure 6 4 Lymphnode metastatic cells targeted with an anti EP CAM antibody paramagnetic beads coated with an appropriate surface antibody were used for the
76. n 5 2 3 you can save nosepiece position number and the lamp voltage used When changing the objective the corresponding nosepiece position and lamp voltage will be established automatically by the microscope Nosepiece posi tion numbers are clearly indicated on the microscopes The lamp voltage setting may be different for different objectives Therefore it is recommended to enable the per objective camera white balance setting see section 5 3 2 Z drive control CellTools features built in focusing aids and functions that rely on motorized z drive control These are 96 MMI CellEctor e Sample plane definition e Focus memory for pin positions mmi Cell Tools offers three ways to manually focus the z drive 1 Using the vertical slider on the right hand side for coarse focussing 2 Using the two arrow buttons at each end of the slider for fine focusing and 3 Using the mouse wheel In order to use the mouse wheel for focusing click the mouse wheel focus button While the button is activated the mouse wheel is linked to the micro scope z drive Of course it is possible to refocus using the microscope s built in focusing controls When using the manual focus wheel on the microscope some microscope models may not feedback the changed focus values into the software In order to notify CellTools that the focus has changed click on the numerical focus display below the slider to update its value 7 4 1 Required c
77. n be specified in the safety parameters dialog To use the current focus setting as objective sample collision limit press the Read current button To reset the limit to its default value press Reset The recommended procedure for setting the objective sample collision limit is Procedure 1 Select the objective with the shortest working distance normally the ob jective with the highest magnification 2 Focus the microscope 3 Open the safety parameters dialog Microscope Safety parameters 4 Press the Read current button 5 Add the working distance in um to the displayed value and go to step 8 or if the working distance is unknown proceed with step 6 6 Carefully move the objective as close as possible to the specimen with out touching it 7 Write down the value displayed under Objective sample collision 8 Press the Read current button again 9 Now enter an Objective sample collision limit half way between the two positions 10 Click OK 101 7 AUTOMATED MICROSCOPE CONTROL 7 6 Fluorescence shutter control If installed the microscope s epifluorescence shutter can be opened and closed via software Select Microscope Shutter or press F6 102 8 1 8 1 1 MMI CellEctor Image analysis using mmi CellExplorer mmi CellExplorer is an optional image analysis software module for mmi Cell Tools lt was developed to find count and measure microscopic objects for subsequent extract
78. n for numeric pad keys 19 5 MMI CELLTOOLS MAIN APPLICATION cursor keys numeric pad Insert Home Page Num x Up Lock Page 7 9 E P E 1 3 Del End Enter press minor steps press major steps hold slow movement hold fast movement Figure 5 8 Stage movement using the keyboard NumLock must be turned on for numeric cursor keys to work as expected For moving larger distances hold down the respective key The velocities for the two sets of movement keys can be set independently see section 5 4 3 By default the cursor keys move slowly and the numeric pad keys move fast In applications where you need movement by well defined distances only the continuous movement can be suppressed by enabling Caps Lock Stage movement settings To change stage movement settings press the Stage movement settings button click the menu bar item Stage Stage movement settings or press Ctrl P to get the window in Fig 5 9 Acceleration This value determines the stage motors acceleration for both keyboard and mouse movement Reducing this value may facilitate the handling of liquid suspension sam ples For all other samples we recommend using 100 Units For the arrow keys on the keyboard you can set the stage settings in two different units e Screens or percentage of screen e Micrometers um When using micrometer units n
79. n the tool bar Click the Record button to start recording a new movie CellTools will auto matically create a new AVI file in the movie folder and record video until you press the Stop button If Auto open is enabled see below the movie will open in Media Player immediately after recording Otherwise you can access recorded movies through the drop down menu items Playback open in media player and Open movie folder By default the movie folder is located in My Documents mmiCellTools Movies Movies are compressed with a video codec currently either Win dows Media Video 9 or XVID To play back those movies your computer will require installation of the same codec Codecs are found on the mmi CellTools installation media Also note that any shapes drawn will not be recorded 17 5 MMI CELLTOOLS MAIN APPLICATION 5 3 5 1 Movie settings Detailed aspects of movie recording can be controlled by opening the Movie settings dialog Fig 5 7 i Movie settings Encoding Compressed Windows Media Video v Time lapse ix Playback frame rate max 10 0 Hz Record frame every min 0 1005 Movie file location 3H Documents mmiCellTools Movies a 2 open movie after recording Y ok MX Cancel Figure 5 7 Movie settings The Encoding specifies the format codec to store the video frames Ta ble 5 1 gives an overview of available encodings and their application Table 5 1 Movie encoding options
80. nical limits Ensure that the mechanical limits which are always set on slide 1 entirely cover the area of interest on the other slides otherwise the stage may not reach some destinations on the latter ones see section 5 5 1 6 6 2 Setting up the slides the capillary and the pump 40 Type of slides Decide what kind of slides will be used for the sample and the deposit For the deposit one can choose between AmpliGrid custom grid or single cap see section 6 8 5 Slide assignment Place the sample and the deposit slides in the se lected slide positions on the stage and assign the selected positions to the sample and deposit slides in the software see section 6 8 1 Deposit configuration and calibration For the grid deposits tell the sys tem the geometry of the grid see section 6 8 5 custom grids only and where the first spot of the grid is located on the microscope stage see section 6 9 7 MMI CellEctor Capillary tilt Insert a new capillary into the capillary holder adjust its tilt and set the tilt value in the software see section 6 3 2 Slide plane tilt Select the working objective and determine the plane tilt of both slides see section 7 4 2 Setting of the plane tilt does not apply to a single cap Pump Ensure that the pump is filled with oil the pump piston is away from its limit positions see section 6 4 there are no air bubbles and no leaks between the pump and the capillary see se
81. nl for both in and out e Pre pumping volume 30 nl e Over pumping volume 20 nl During the acquisition 1 of the cell 30 nl pre pumping are pumped in at first We assume here that pumping out took place just prior to it thus the pump changes the pumping direction and 30 nl are necessary for the com pensation of the overpressure remaining after pumping out In the second step 20 nl are pumped in over pumping that will be then pumped out in the fourth step Thus over pumping does not add net volume to the main volume It helps however to pump in cells that adhere to the slide surface In the third step the main volume of 10 nl is pumped in Thus totally 40 nl is pumped in during the acquisition process During the subsequent deposition 2 of the cell 30 nl are pumped out at first pumping direction again changed and then 10 nl of the main volume totally 40 nl The next acquisition action C proceeds exactly the same way as the first one A However the user launches an additional pumping in action D in order to get the cell into the capillary Since the pump did not change its direction the over pumping volume that is pumped out at the end of the pumping in does not count no pre pumping volume is added to the main volume this time There are no universal rules for choosing the values of the pump settings however several practical hints can be given 89 6 MMI CELLECTOR The smaller is the diameter of the capillary
82. nnected to the system switch between the cameras by selecting a setup dedicated to that camera To define the camera used by a setup open the setup editor and click on the camera field showing lt default camera gt as default A drop down list shows up in which you simply select the camera you like to connect to that setup Do not use the setup editor to switch between cameras The setup editor is only used once to create at least one setup for each camera Then change cameras by selecting the corresponding setup Camera settings For best imaging results the camera can be controlled through a settings win dow Fig 5 5 To open it click the camera button in the toolbar or select Video gt Camera settings from the menu Ctrl R Image exposure is controlled through the top elements in the dialog Unless Automatic exposure is selected the exposure time can be controlled through the Exposure time slider or the adjacent input field In Automatic exposure mode the slider is inaccessible If the auto exposed images are too bright or too dark use the Exposure correction slider to compensate Finally the amplifier Gain can be adjusted to yield brighter but noisier images Higher gain settings will however reduce the required exposure time For high quality images e g for publications it is preferable to use a longer exposure 14 MMI CellEctor T rh mmi CellCamera MXF285c Settings Exposure settings Ex
83. objects show high con trasts You can control the illumination from punctual to flat by adjusting the sliders left right Left and top down Top You can also dye the surface In order to do so just move the mouse pointer within the color window In case you would like include the original colors of your object into the metallic surface increase the percentage value below the Source image quantity Fast Fourier Transformation The fast fourier transformation searches for edges in the image This transformation can mainly be used to reduce background noise Gamma correction The Gamma correction is a special brightness and contrast Special function Especially very low light images can be improved by using the gamma correction Sharpen With the sharpen function you sharpen the contour of the objects Color tool panel Color Power 3 50 C Multimode Figure 8 14 Color panel Color fields for different object types Open the color panel to choose up to eight color gradients as settings for counting eight different object types This en ables you to find count and measure object types with different colors simul taneously the counting results are shown separately for each color The color fields are characterized in Fig 8 15 114 MMI CellEctor a Color field inactive for b Color field active for edit c Color field active for ob editing and inactive for ob ing only
84. one color field can ject search but inactive for ject searching be edited at once but inac editing tive for object searching Figure 8 15 Color fields state indicators Standard color selection With a left mouse click on the displayed image the color under the cursor will be entered into the left side of the first color field E ACIL Figure 8 16 Standard color selection A right mouse click on the displayed image will choose the end color of the color gradient to search for If the objects are small it can be tricky to hit the right color By using the zoom options of the mmi CellExplorer as described in section 8 2 2 the selection will be very easy and precise Advanced color selection With a double left mouse click in the right or left part of the color field a standard color menu is opened to select a new colour for the right or left part of the color field Select the Custom file card in the Color menue to customize the favoured color The Power slider Fig 8 14 adjusts the intensity of the marking You should try to get homogeneous dark objects and a light background 100 is the lowest intensity Ois the strongest intensity Important Please try to obtain objects as dark as possible Multimode If multimode Fig 8 14 is selected the parameters in the lower setting panels defining the object shape like object area object roundness 115 8 IMAGE ANALYSIS USING MMI CELLEXPLORER
85. ons are nec essary capillary deposit plane tilt Thus it is recommended to change the slide assignment before you start calibrations while preparing the system for use Capillary movement configuration The capillary tip moves from the idle position towards the destination point along the three vectors shown in Fig 6 17 A horizontal movement 47 6 MMI CELLECTOR Idle position SY Tilt p Spacing Slide T Figure 6 17 Movement pathway of the capillary from idle to down position A horizontal movement B vertical movement C axial movement immerse distance e B vertical movement e C axial movement immerse distance The user can adjust the movement characteristics in the following dialog Fig 6 18 that can be opened from the main menu CellEctor gt Configure Capillary Movement Capillary Movement E Tit 45 deg Immerse Slow down distance distance Sample 2 mm 200 ym 1 4 Deposit 1 mm 10 ym o7 0 7 Cancel OK Figure 6 18 Capillary movement configuration dialog The tilt of the capillary should be set equal to the value read from the capillary holder Immerse distance corresponds to vector C in the above figure Slow down distance is the last portion of the immerse distance indicated as a dashed line on vector C in the above figure where the capillary moves with a highly reduced speed in order to minimize pushing away the cells
86. or this just drag the cursor from the start to the end point of the interest and the distance between these two points will be indicated on the screen Every subsequent measurement deletes the previous one Once you are done with the measurements press the measure button again which is now shown as an arrow to change to the cursor default shape 46 MMI CellEctor 6 8 Configuration routines 6 8 1 6 8 2 Before you start to use mmi CellEctor for the first time some preliminary work is necessary to make the system ready for use Most of this work has to be done only once and is usually done by the MMI service technician during the installation of the system Some activities are recurrent like capillary 3D positioning of a freshly inserted capillary Choosing the position of sample target and service slide The multi slide holder of the stage can hold up to three slides of the standard size One can change the slide assignment in the menu item CellEctor gt Configure Slides a Slide assignment o 5 fees Slide position sample 1 2 03 deposit 1 A Figure 6 16 The slide configuration window to select the position of the sample target and service slide The picture above shows the default slide assignment The sample and the deposit slides can be placed at arbitrary positions The third unoccupied posi tion is reserved for the service slide Once you change the position of the slides re calibrati
87. ormed by qualified MMI personnel or our desig nated representative s The MMI system contains no user serviceable parts For questions about your instrument technical consumables warranties please contact Europe and Asia amp Molecular Machines amp Industries AG MMI Flughofstrasse 37 8152 Glattbrugg Switzerland 41 0 44 8 09 1010 XN 41 0 44 8 09 10 11 North America ex MMI Molecular Machines amp Industries Inc P O Box 348 Haslett MI 48840 USA 1 603 629 9536 FAX 1 321 978 0304 www molecular machines com service Omolecular machines com 121 APPENDIX A SERVICE 122 MMI CellEctor B List of Keyboard Shortcuts General Caps Lock Ctrl P Esc F1 Space Camera control Ctrl R Ctrl S Ctrl W Suppress continous stage movement 20 Stage movement settings 20 Interrupt current process 23 83 Display user manual 26 Switch between moving and drawing mode 18 Open camera settings 14 Save an image o o o ooo ooo 16 Set white balance o o o o 15 Microscope control Ctrl 1 Ctrl 2 Ctrl 3 F6 F7 F8 Define first sample plane point 98 Define second sample plane point 98 Define third sample plane point 99 Open close fluorescence shutter 102 Swit
88. ositioning with respect to the slide surface If the remote control is off or you work with a manual microscope only a rough positioning of the capillary is possible 4 2 System preparation Follow these steps Ensure that the following microscope settings are correct camera orientation objectives calibration illumination settings for each objective camera settings for each objective On new systems this has already been done for you by the service technician e Prepare the mmi CellCellector for work as described in sections 6 8 and 6 9 4 GETTING STARTED 4 3 System turn off e Shut down CellTools by using the Project Exit command or press the close button in the top right corner of the program window e Switch the microscope off Shut down the PC by using the standard Windows command e Let the illumination housing of the microscope cool down to the room temperature and protect the system with the microscope cover MMI CellEctor 5 mmi CellTools Main application 5 1 Main window and plug ins Fig 5 1 shows the extended tool area and explains the main components of the mmi CellTools user interface mmi Cell Tools user database common tools active application microscope panel application panel slide status messages overview Figure 5 1 Structure of the mmi CellTools user interface The major portion of the window is occupied by the live video panel which displays the cur
89. ote that you may have to adapt distances with every objective change 20 MMI CellEctor Acceleration Keyboard control Units Screens Cursor keys Numeric pad Distance 10 00 90 00 Velocity 10 00 mms 15 00 mms max stage velocity 20 00 mm s X Cancel Figure 5 9 Stage movement settings Distance and velocity These values can be chosen independently for cursor and numeric pad keys By default cursor keys are used for minor steps and numeric pad keys for major steps Values that are outside the allowable range will be shown in red 5 5 Slide navigation and scanning 5 5 1 Mechanical limit calibration In order to use the navigation and scanning features mmi CellTools needs to know the the position and dimensions of the sample This is the purpose of the mechanical limit calibration Under normal circumstances the calibration remains valid unless the stage is moved manually or using a software other than mmi CellTools If the software detects an invalid calibration slide scanning will be disabled and the overview scan will show a red warning sign Procedure 1 Use a small magnification objective e g 4x and move the stage to slide 1 rearmost slide 2 Move the stage such that the top left corner is just visible at the edge of the field of view 3 Select Define origin from the slide geometry popup menu bottom right Bk corner of the overview panel 4 Move the stage such that the bottom
90. panel Now press the right arrow key in the number pad Best using standard step sizes for big step movement 90 of screen If you move the object horizontally to the right upper corner and the object is still on the same level the camera orientation is correct If the result shows a shift between the two corners ie the object does not move perfectly hor izontally loosen the hex screws on the camera port and rotate the camera carefully Repeat this move and observe operation again until 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 carefully 29 5 MMI CELLTOOLS MAIN APPLICATION 5 8 5 9 5 9 1 5 9 2 Multi user report Users can get information about time spent using mmi Cel Tools for each Win dows user account User name 0000 00 52 7 0000 58 27 0000 19 46 Figure 5 16 Multi user report Select the menu item Project Usage report or start it from Start All Programs mmi CellTools mmi MultiUser Report A time format is hhhh mm ss Help Help topics The user manual can be opened directly inside the mmi CellTools software By pressing F1 on the keyboard the PDF file of the User Manual shows up You also launch the user manual by selecting the respective item in the Help menu MMI online If your PC is connected to the internet you can launch the MMI web page by the men
91. pective tool panel on the original image Use the button to check the settings you have defined so far 110 MMI CellEctor 8 2 3 1 Process tool panel The process panel Fig 8 10 contains the following controls from left to right gt Process ku gt Stopped Q Obj Figure 8 10 Process panel Undo Undo the last change when editing settings Reset Reset the current photo to its initial state Calculate Performs a complete analysis on the current image using all pa rameters defined in the control panel After this calculation the objects can be transferred to mmi CellCut or mmi CellEctor Objects Displays the number of counted objects 8 2 3 2 Settings profile panel Save your settings by entering a name in the settings profile toolbar and click ing Save Settings Profile Figure 8 11 Settings profile panel 8 2 3 3 Advanced imaging panel The advanced imaging functionalities allow preprocessing and optimizing the image before an object search is performed Advanced Imaging Smooth radius 0 0 Figure 8 12 Advanced imaging panel 111 8 IMAGE ANALYSIS USING MMI CELLEXPLORER Smoothing You can smooth your image with the smoothing function in order to round over edges and achieve a better counting and splitting result By adjusting the Smooth radius slider you can define the level of smoothing The radius used for the calculation of the smoothing can be set with the slider Ois a small rad
92. posure time Gan U 0 0 dB v Automatic exposure g Exposure 7 4 correction 0 0 dB Imaging Gamma 0 75 Mode Best image x Color Y Set white balance Compact view OK Cancel Figure 5 5 Camera settings for mmi CellCamera models time and low gain Note that the allowed gain range is greater when automatic exposure is off The controls in the bottom part of the dialog allow control over color and con trast Use the Gamma slider to control image contrast The default Gamma value is 0 75 and it may be adjusted over a range of 0 01 to 2 0 Low gamma values are recommended especially to brighten low light fluorescence images High gamma values reduce noise and improve the black level of the image The camera Mode provides color settings optimized for various applications For bright field the color quality can be optimized with the Best image mode The camera transfers a high quality YUV4 4 4 data stream with full pixel reso lution By selecting Fastest rate the image rate frames per second fps will be maximized In this mode up to 20 fps depending on your camera model with full pixel resolution are displayed For fluorescence applications the con trast can be optimized with the Low light option The camera transfers a high quality RGB data stream with full pixel resolution This setting is used mainly in combination with the Gamma and Gain setting If you select Binning t
93. protocols that use nanoliter volumes for the separation process and provides the basis for molecular downstream analysis in only 1pl 29 6 MMI CELLECTOR Figure 6 1 Sample area of the microscope with a ampligrid and microcapillary There is no mechanical stress to non adherent cells the acquisition occurs solely by flow of the liquid surrounding a cell Under optimum conditions even partly adherent cells can be acquired in this way There is no contact between the cells and the capillary The capillary diameter 10 15 20 30 or 40 um may significantly exceed that of the cell For example cells of 6 umdiameter can efficiently be acquired with a capillary of 40 um diameter The suspension of cells can be placed either on a flat standard slide or in a dedicated container e g an IBIDI y Slide The deposition of cells may occur on various targets deposits Grid deposits are matrices of either spots like AmpliGrid or small containers like IBIDI well slides Single spot deposits may consist of a transparent cap a PCR tube or a microfluidic device Whatever the kind of the target is its size must not exceed the standard slide size that can be inserted in the multi slide holder of the stage The acquisition and deposition of cells can proceed in a variety of modes from manual mode through full automation with cells recognition by the mmi CellExplorer However even in the manual mode there is no need to manually operate t
94. pty Row Figure 6 23 The workflow editor Once the workflow has been edited and the workflow editor closed it becomes the active workflow in mmi CellEctor Depending on which button is pressed in the action panel either the entire workflow or only its part is launched For example pressing the button Rinse launches the actions on the service slide only Add water and Clean in the above example Pressing Acquire and deposit button with the check box With rinse checked launches the entire workflow otherwise only the acquisition and deposition steps are performed The workflow editor can be opened by either selecting the corresponding menu CellEctor gt Configure Workflow or by pressing the right mouse button over the relevant buttons Rinse Acquire and deposit A running workflow can be interrupted any time by pressing the ESC key on the keyboard In such a case the workflow terminates after the currently running basic step is completed capillary move ment pumping moving the stage If a workflow is on idle i e waiting for the next step a predefined amount of time Stay time in the workflow editor pressing ESC terminates the workflow imme diately Deposit inspection The deposit inspection calibration can be launched by the menu item Cellector gt Configure Deposition Deposit scan or pressing the inspect deposit button with the right mouse button opens the configuration dialog shown below The scanning can
95. pumped in from the sample and pumped out on the deposit e Collect cells using either the automatic or the workflow mode 39 6 MMI CELLECTOR The following chapters describe these steps in more detail In a routine work under the same experimental conditions same type and size of cells buffer solution capillary size etc steps 1 2 and 5 can usually be skipped 6 6 1 Checking the microscope Setting up the microscope for working with cells is described in detail in sec tion 6 8 and 6 9 Here some measures you can apply to verify the setup Camera orientation Select the objective you are going to work with then move an object to the upper left screen corner so that the upper edge of the object touches the upper edge of the screen Using the arrow key on the keyboard move the object horizontally along the screen edge The object should not change its distance to the screen edge while moving Magnification factor for each objective Click an object with the mouse and drag it into four corners of the screen The object should not change its position with respect to the mouse cursor see section 6 9 1 Parfocal lens offset for automated microscope only If itis correct there should be no focus loss while changing the objectives see section 6 9 2 Paraxial lens offset If it is correct there should be no displacement of markers with respect to the objects they mark while changing the objec tives see section 6 9 3 Mecha
96. r down by presseing Move capillary up or Move capillary down Moving the capillary up or down is done by pressing the button Move capil lary up or Move capillary down The final down position of the capillary is calculated from the capillary contact position with the slide surface minus the spacing value set by the user in the action or in the capillary calibration panel If the user changes the spacing value while the capillary is down it will be in effect yet in the subsequent down movement Button Move capillary up or Move capillary down are enabled only if both the upper idle and the lower positions of the capillary are known i e the capillary is calibrated 84 MMI CellEctor Whenever the capillary is down the movement of the XY stage is arrested except for the movement by dragging the mouse if the user checked the latter option in the acquisition configuration dialog The software automatically recognizes if the capillary is up or down as long as the capillary movement is done solely by using the but ton Move capillary up or Move capillary down The actual po sition of the capillary may mismatch the position concluded from this button if the user moved capillary with joystick In such a case repeatedly press the button Move capillary up or Move capillary down to remove the mismatch 6 10 3 Automatic modes Acquire deposit and rinse Automatic Figure 6 51 Panel representing the automatic functions of mmi CellEctor In
97. rent field of view of the microscope and serves as the main area for interaction between the user and the system mmi CellTools is a single piece of software that controls all micromanipulation devices of the mmi instrumentation family see chapter 3 The instrument specific controls are located in the application panel Switch between appli cations using the tabs at the top 5 MMI CELLTOOLS MAIN APPLICATION 5 2 Those user interface controls that are common to all applications are located in the tool bar at the top of the window The user database controls provide access to instrument parameters for specific samples for certain imaging sit uations and for each objective see section 5 2 The remaining common tools on the tool bar provide elements for camera and stage control On systems equipped with an automated microscope the microscope panel provides access to z drive focus control and other microscope specific fea tures see chapter 7 The slide overview panel provides controls for slide scanning and navigation see section 5 5 2 Finally the status messages bar at the bottom displays current stage coordinates and camera frame rate To accommodate for left handed users the application and microscope panels can be moved to the left hand side using the menu item Edit Left handed UI User specific database All settings saved in the mmi CellTools are unique to the current user logged in mmi CellTools fully suppo
98. right corner is just visible at the edge of the field of view 21 5 MMI CELLTOOLS MAIN APPLICATION 5 Select Define far corner You are now ready to use slide navigation and scanning The field of view indicator and the dimensions of the slide overview will adapt during calibration The suggested positions to use as limits for different types of slides are illus trated in Fig 5 10 Origin Far corner a Glass slide 1 Far corner b mmi Membrane slide Figure 5 10 Suggested mechanical limit positions on standard microscopic slides The mechanical limit calibraiton needs only be carried out on the first slide The other slides automatically use the same mechanical limits 5 5 2 Overview scan peal Navigation aega Ap aa a as Ber Figure 5 11 Slide overview G The overview scan is started by pressing the Start scan button This window shows you an overview over your sample the roadmap image If no area of interest is defined the whole slide will be scanned within the limits set in chapter 5 5 1 The current field of view is indicated with a red blinking rectangle or point You can move this red frame by clicking and dragging the left mouse button 22 5 9 3 MMI CellEctor The motorized xy stage moves automatically to the chosen detail With this navigation method you always see the position on the slide You can also move to the position of interest
99. rts Microsoft Windows user management During program startup the last settings saved by the active user are loaded The database represents a hierarchical structure Slide gt Setup1 gt Objective 1 gt Objective 2 gt Setup2 gt Objective 1 gt Objective 2 Basically the user can save all parameters for each experiment Slide he she runs separately If the user changes settings for an experiment settings from recent experiments are still saved and accessible The Setup represents all necessary parameters to define an illumination meth od bright field fluorescence DIC If you change a parameter in a setup the change will only be reflected in the current slide The Objective represents all objective related settings and calibrations If you change an objective calibration or objective related parameter the change only will be reflected in the current setup and slide 10 5 2 1 5 2 2 MMI CellEctor Slide editor The slide selection box contains all samples you defined in the past All doc umentation is saved under this name To change the database open the slide editor Fig 5 2 by pressing the edit button Slide Editor AN O UN EN SA A A Name a factory settings slide 1 slide 2 slide 2 New as copy of selected Y OK x Cancel Figure 5 2 Slide editor Use the button to define a new slide All parameters from the active slide will be copied Rename the slide by
100. ry with the oil since it is much easier to perform the 3D positioning when the capillary tip is empty Once the capillary 3D positioning has been done one can proceed with filling the capillary with oil and the buffer solution dedicated to collect cells The following procedure is recommended Procedure 1 Fill almost the entire capillary with oil The oil boundary must not enter the narrow part of the capillary i e leave some air in the conical part of the capillary see Fig 6 9 2 Stop pumping out and wait approx 30 seconds for the pressure in the capillary to equilibrate 3 Immerse the capillary into the buffer solution placed on a slide 4 Pump in the buffer solution up to the moment the air gap between it and the oil moves into the transparent part of the grip head see Fig 6 10 5 Stop pumping in and wait for the pressure in the capillary to equilibrate 6 Move the capillary up from the buffer solution The air gap between the oil and the buffer solution works well for capillaries with the tip diameter of at least 30 um The narrower the capillary tip is the longer time it takes to equilibrate the over or underpressure after each pump ing step which makes this method impracticable for smaller capillaries In the later case it is recommended to collect cells in small volumes with the capillary filled with oil and air only 36 MMI CellEctor Figure 6 9 Fill almost the entire capillary with oil The oil boundar
101. s a laser it must be ensured that the laser safety features are functioning correctly and that the covers to protect against laser radiation are fitted 2 INSTALLATION MMI CellEctor 3 The mmi CellTools instrumentation family The mmi CellTools 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 tweezers to manipulate cells or beads with an optical trap mmi CellEctor automated micro pipetting to mechanically manipulate cells or beads with a capillary and mechanical micromanipulator mmi CellExplorer pattern recognition software for PC based image analysis Any or all of these modules can be combined in one microscopic environment 3 THE MMI CELLTOOLS INSTRUMENTATION FAMILY MMI CellEctor 4 Getting started 4 1 System start up To start the mmi CellTools with mmi CellEctor follow the steps below e Start up the PC and allow the boot process to complete reaching the Windows desktop Turn on the microscope control box e Start mmi CellTool software and wait until the software has finished the start up and self test procedure For the system to work properly all the hardware components have to pass the hardware monitoring procedure without errors e Remote control of the microscope is compulsory for the mmi CellEctor to achieve the highest precision in the capillary vertical p
102. s by a distance depending on the magnification of the objective in use Thus a Calibration of the X and Y coordinates is necessary for each objective separately To perform the XY calibration of an objective select CellEctor Calibrate Objective Magnification from the main menu or press the button Objective in the CellEctor system panel Several things happen now e two transparent rectangles show up in the top left and the bottom right video panel corner mouse cursor changes to a target shape objective calibration panel shows up The objective calibration panel guides you through the following calibration steps Procedure 1 Press the spacebar to switch between drawing and moving mode and move a distinct object to the rectangle in the top left corner of the video panel The object does not need be centered within the rectangle 2 Toggle the cursor back to the target shape and click theobject with the left mouse key This marks the object with the target marker 57 6 MMI CELLECTOR 6 9 2 CellEctor CellCut CellExplorer Exit objective calibration Current objective 4x Magnification Offset paraxial Task measure the XY table distance necessary to move an object diagonally through the screen Step 1 of 5 Move a conspicuous object to the rectangle in the UPPER LEFT comer Figure 6 25 The mmi CellEctor plug in panel in objective calibration mode 3 Toggle the cursor back to the hand shape and move
103. st object area and diameter which are counted Max area and Max diam defines the largest object size and diameter which are counted Use Min c a and Max c a to filter by roundness All objects with roundness between the Min c a and Max c a value are counted Definition of the roundness in mmi CellExplorer c circumference a 4r totalarea 8 1 The roundness will separate different geometrical shapes such as circles squares rectangles and ellipses The results are independent from shape size and allow you to separate shapes by geometry Attention In binary images objects appear as a collection of pix els therefore objects are never entirely round or rectangular Table 8 1 Roundness of simple objects geometry roundness circle 1 0 square 21 5 rectangle zx 2 0 ellipse 1 2 x 4 0 118 MMI CellEctor a Original image E cee Unit Diameter Total rea Circumference Roundness pm 11849754 702083 3185 3351 6165 1 5708 pm 534 9537 54970 8773 1331 2057 4 0887 3 pm 632 2779 311343 6148 2088 5129 1 0085 4 um 1260 5487 622489 1109 3213 6582 2 0048 5 pm 630 9560 155816 8586 1611 3605 2 0067 6 7 8 um 1071 0476 2216311996 2606 0141 4 0651 um 600 6331 1814130935 1819 0820 1 5619 wm 6324457 311378 9932 2084 1057 1 0089 Spm 11928757 7147204644 3475 2633 1 5637 10 pm 1066 2970 219678 3148 2511 0524 4 0650 um 841 7502 5531723134 2779 7572 um 840 5515 276778 9687 2144 4515
104. stick is used occasionally for the manual adjustment of the capillary position in 3D space The horizontal XY position of the capillary can be changed by tilting the joystick to the left or right X coordinate or forth and back Y coordinate lts vertical Z position can be changed by turning the joystick clockwise for lowering Z value increases or anticlockwise for raising Z value decreases the capillary The speed with which the capillary moves is proportional to the deviation of the joystick from its equilibrium position In order to get the feeling how quick the movement is please observe the X Y and Z current values in the advanced 3D capillary positioning dialog while using the joystick Typical workflow in collecting cells While collecting cells one picks a cell on the sample slide and deposits it on the target slide The typical workflow for achieving this task with mmi CellEctor consists of the following steps Ensure that the objectives are calibrated and the Z drive control is on for automated microscope e Setup the mmi CellEctor major components the slides sample and deposit the capillary and the pump e Calibrate the vertical contact point of the capillary on the sample and the deposit slide e Calibrate the capillary tip position on the screen on the sample slide Working on the sample slide in the manual mode adjust the capillary spacing and the pumping parameters so that the cells can efficiently be
105. th different colors you need more than one color field Corresponding to the eight color fields you can define up to eight object types for your search In order to define the colors for those objects activate the respective color field Activate the field for color editing with a left click Only one color field can be edited at once The outer frame of the color field indicates the selection status black for active light gray for inactive Activate deactivate the field for object search with a right click The inner frame of the color field indicates the status See section 8 2 3 4 You can find count and calculate objects with different colors separately with the mmi CellExplorer software see Fig 8 4 Encircling Objects with color gradients Many objects show a color gradient The mmi CellExplorer can detect them as well as uniform colored objects Select the start color of your gradient with a left click and the end color with a right click Differing brightness or intensity of the same color can form the gradient too Note Only activated color fields will be used for object search 105 8 IMAGE ANALYSIS USING MMI CELLEXPLORER Figure 8 4 Two colors selected a Color field b Result Figure 8 5 Green was selected with the left mouse click red with the right mouse click The selection is displayed in the color field as a gradient All colors contained in the gradient will be searched for during a calculation
106. the automatic mode the work is divided in three steps acquisition depo sition and rinse In order to separate a cell of interest from the sample and transfer it to the deposit perform the following actions Procedure 1 Select a cell on the video screen by setting a marker 2 Click the Acquire button 3 Once the selected cell has been aspired click the Deposit button 4 If required click the button Rinse The capillary moves up the stage moves to the to service slide and the capillary is rinsed 5 Open the context menu by clicking on the video image with the right mouse button and select Back to sample The stage moves back to the point on the sample where the cell has been aspired If the cell could not be aspired in step 2 the user has the possibility to per form additional pumping step s by pressing button Pump in in the manual part of the action panel provided that the option Leave capillary down after acquisition has been checked in the acquisition configuration dialog 85 6 MMI CELLECTOR Workflow Acquire and deposit Full auto Scan for cells C with rinse Figure 6 52 Panel representing the workflow functions of mmi CellEctor 6 10 4 Acquire and deposit workflow To acquire a cell from the sample slide and deposit it automatically to the deposit slide mark a cell and click the button Acquire and deposit If the check box Full auto is checked acquisition and deposition of the cell is launched imme
107. tioning Wizard SAMPLE SLIDE Step 3 of 5 Change to the working objective Do NOT refocus the microscope a The control panel b The system has now set the focus from the capillary tip back to the slide Figure 6 30 Step3 of capillary vertical positioning wizard 65 6 MMI CELLECTOR 4 The system further lowered the capillary thus it is roughly in focus again see Fig 6 31b The XY position of the capillary tip on the screen can de Capillary 3D positioning YA t x 4 i z y y Speed max medium Y min 1 pm 3D Positioning Wizard SAMPLE SLIDE Step 4 of 5 Move the capillary tip to the preferred landing point on the screen and to the current focus plane a The control panel b The system further low ered the capillary thus it is roughly in focus again Figure 6 31 Step4 of capillary vertical positioning wizard liberately be chosen i e does not have to be in the screen center any more You can adjust it using the X and Y arrows The vertical position of the capillary must be adjusted so that its tip gets in a gentle contact with the slide Use the Z arrows and the minimum speed to change the vertical position Then press Next 66 MMI CellEctor 5 At the contact position both the slide and the capillary tip should be in focus as illustrated in Fig 6 32b Press Finish to set the contact Capillary 3D positioning YA t x 4 i
108. tons b using joystick and position numbers Figure 6 44 Capillary 3D positioning without active plane tilt support Also used for manual microscopes 79 6 MMI CELLECTOR 6 9 6 6 9 7 Capillary horizontal position on the screen This calibration step is performed on the sample slide only Exit capillary calibration Vertical Position Capillary spacing um to sample deposit 30 a 20 S Automatically refocus on capillary capillary slide slide Contact point Horizontal Position Move capillary down then mark its position with the left mouse key Figure 6 45 Capillary horizontal contact point calibration window Calibration of the position of the capillary tip on the screen is done in the panel shown in Fig 6 45 Click Move capillary down mark the tip on the screen with the mouse and click Done Deposit calibration Calibration of the deposit is done in two different ways depending on the type of the deposit Calibration of grids Once the basic parameters of a grid number of rows and columns spacing between them are known it is enough to determine the location of the first spot row 1 column 1 R1C1 on the XY stage for the software to be able to calculate the locations of the remaining spots In the calibration routine 80 MMI CellEctor a CellEctor CellCut CellExplorer Exit deposit calibration Current deposit AMPLIGRI
109. toolbar split is used for separating these overlapping objects Split Split Factor J 0 5 Min line 3 12 771 Max line J 403 8 O split Multisplit Singlesplit Figure 8 19 Split tool panel For the separation of overlapping objects activate the box Split Adjusts the intensity of the split factor 0 is a low split factor e 1 is a high split factor The Min line slider selects the shortest length of the split line If you select a very small value many small segments of larger objects are separated as individual objects If you enter a very large value overlapping objects are no longer separated The Max line slider selects the longest length of the split line The value is preset to maximum If large individual objects are separated into two parts you have to decrease the value The split feature allows the separation of overlapping objects The feature Singlesplit separates objects once while the feature Multisplit cross checks after separating whether another separation of the objects is necessary Sep arating with Multisplit is time consuming 117 8 IMAGE ANALYSIS USING MMI CELLEXPLORER 8 2 3 7 Find tool panel With the find tool panel you may adjust the size and roundness of the selected objects Find Min area 7 328 428 Min diam um 20 449 Max area J 1280918 Max diam 1277 1 Min c a 7 0 1000 i Max c a J 20 000 Figure 8 20 Find tool panel Min area and Min diam define the smalle
110. ttings is not correct please refer to the corresponding chap ter 5 2 4 Default database reconstruction Each user handles his her own database Changes in this user specific data base will not be visible for any other user A default database is always stored separately in a central file location This default database is specific for your instrument and will be set up and handled only during installation and service lf for some reason a user s database becomes unusable the user can recover the default database by the following procedure Procedure 1 Close mmi CellTools 2 Delete the following folder from the hard disc C Users username AppData Roaming MMImCUTDataBase or for Windows XP Systems C Documents and Settings username Application Data MMImCUTDataBase 3 Open mmi CellTools The default database will automatically be recovered and is directly visible in mmi CellTools You will lose all slide and setup data contained in the user database You will need to recalibrate the entire system 13 5 MMI CELLTOOLS MAIN APPLICATION 5 3 Camera operation 5 3 1 5 3 2 mmi CellTools supports a range of scientific digital cameras supplied by MMI or third parties e The mmi CellCamera range of digital cameras for general microscopy applications The Andor iXonE EMCCD camera for ultra sensitive detection Multiple cameras mmi CellTools also supports multiple cameras If more than one camera is co
111. tual grid Standard settings for beads The settings in the Table 6 1 apply to 10 umpolystyrene beads in aqueous solution Concentration of the beads should be chosen so that approximately 20 beads are seen at once in the field of view with a 20X objective Collecting cells to a single cap To collect cells to a single cap one works with a transparent cap placed in a cap holder on the XY stage Assuming that all the operations necessary to optimize the acquisition process on the sample have already been done one has to calibrate the position of the cap and that of the capillary in the cap Once the cap position has been determined one can proceed with the acqui sition and the deposition of cells using one of the automatic modes 92 Table 6 1 Standard settings for beads Capillary Bevel orientation Parallel to slide Tip diameter 40 um Spacing to sample slide 5 10 um Spacing to AmpliGrid 50 um Pump Pump in volume 10 nl Pump out volume 100 nl Pre pump volume Onl Over pump volume Onl Relaxation time 0 ms Speed 100 Acceleration 100 MMI CellEctor 93 6 MMI CELLECTOR 94 MMI CellEctor 7 Automated microscope control mmi CellTools supports the following automated microscope types e Olympus 1X81 Nikon Eclipse Ti e Nikon TE2000 E The automated microscope control functionality must be installed by an autho rized MMI technician 7 1 Activating microscope control To enable automated microscope control th
112. u item 26 MMI CellEctor Help gt MMI online Questions about the system can be sent to MMI service staff by the item Help Online Support 5 9 3 Version info Information about the currently installed software version can be found under Help gt Version info 27 5 MMI CELLTOOLS MAIN APPLICATION 28 6 6 1 MMI CellEctor mmi CellEctor Principles The molecular analysis of pure enriched cell populations or even of single cells is an important prerequisite of medical genomics and proteomics and may become essential for molecular patient profiling in the future Up to now this goal is often very difficult and tedious to accomplish Therefore MMI developed a new analysis and cell sorting device the mmi CellEctor The isolation of single and rare cells from tiny sample sources is a prerequisite for their genotypic and phaenotypic characterization The mmi CellEctor is the starting point for a new integrated work flow It facilitates the automated isolation of any type of single and rare cells in three steps e Cell recognition e Cell acquisition e Cell deposition The mmi CellEctor is based on e automated microcapillary automated pump motorized stage To encrease the automation level the mmi CellEctor can be upgraded with e motorized microscope e mmi CellExplorer the MMI cell recognition solution Acquisition and deposition are controlled by a high precision pump This allows establishing
113. u item Video Video freeze To go back to live video presentation select Video Video live Saving images To save an image simply press the Save image button use the menu item Video Save Image or press Ctrl S The file dialog allows you to specify the image filename and image type JPG BMP PNG and TIF The image will be saved with maximum pixel resolution By selecting the Include drawings check box you can save a screen shot from the current video image including all markers and drawings When saving images with drawings the system will produce a screenshot from the live video area resulting in reduced pixel res olution of the saved image 16 5 3 9 MMI CellEctor Press the Copy image to clipboard button to make the current image avail able to other applications The image will be copied to the Windows clipboard in order to paste it into e g office and image processing applications The same result can be seen with the menu item Edit Copy Image The Include drawings does not affect the Copy image to clip board function The clipboard always receives the original camera image Recording movies mmi CellTools allows you to record live camera images into video files AVI format It supports compressed and lossless video codecs and allows you to record time lapse movies Movie and time lapse settings Open movie folder Playback open in media player Figure 5 6 Movie recording functions i
114. y must not enter the narrow part of the capillary i e leave some air in the conical part of the capillary Figure 6 10 Pump in the buffer solution up to the moment the air gap between it and the oil moves into the transparent part of the grip head 37 6 MMI CELLECTOR 6 4 The mmi CellPump Figure 6 11 The mmi CellPump allows to pump in and out by manually turning the rotation knob Manual pumping The mmi CellPump can also be driven manually For this raise the rear cover of the pump and rotate the turning knob located there see Fig 6 11 Manual pumping is preferably used to fill the pump or the newly inserted capillary with the oil While pumping manually you must not move the pump piston outside the allowed range see below While using capillaries with small opening quick manual pump ing may increase the pressure in the pump so that the capil lary may burst Figure 6 12 The mmi Cel Pump allows to pump the shown range Pumping range The pump piston has a limited range of movement as indicated in Fig 6 12 Ifone of the limits has been reached and one tries to pump further 38 6 5 6 6 MMI CellEctor in the same direction using one of the buttons mentioned above the software issues a warning like Pump reached limit pumpin IN impossible In such a case you have to move the piston in the opposite direction to get out of the end switch Use 3D manipulator with joystick In mmi CellEctor the joy

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