CELL-IQ IMAGEN USER MANUAL

Transcription

1 CELL-IQ IMAGEN USER MANUAL FOR CELL-IQ, FOR YOUR RESEARCH NEEDS 2010 Chip-Man Technologies Ltd. All rights reserved. Protected by copyright law and international treaties.

2 Contents 0 Introduction 2 Cell-IQ applications 6 Normal single layer assay 6 Horizontal migration assay 7 Proliferation assay 7 Vertical migration assay 8 Cell colony assay 9 Embryos and oocytes assay 9 Fluorescence assay 11 Preparing for a cell test 14 Program workspace 18 Cell test focus settings 24 Setting up a cell test 27 Cell test settings 28 Gas flow settings 31 Phase contrast settings 32 Fluorescence settings 33 Imaging positions 37 Multiple positions and grids 41 Position specific settings 43 Saving and opening cycle 45 Monitoring cycle 47 Changing medium 50 Error recovery 52 Contents

3 Introduction Introduction I 1

4 PUBLISHER: CHIP-MAN TECHNOLOGIES LTD. Contact details Address: Biokatu 12, Tampere, Finland Phone: Fax: URL: Chip-Man Technologies Ltd. All rights reserved. Protected by copyright law and international treaties. TERMS All-in-focus image All pixels in image are in focus. Pixels are collected from different images in the Z-stack. Autofocus Method that finds the single best focus position. If depth of focus of optics is smaller than target thickness, all pixels in image are not in focus in the focused image. Binning size Binning size defines how many pixels of the sensor is used to collect light to one pixel in the image. The saved images are always the same size as with 1x1 binning, when other binning is used the images are scaled to that size by stretching them. By using larger binning the image resolution drops but better sensitivity is achieved and prolonged exposure times are avoided. Cell-IQ well plate Lid Well plate lid with gas connectors that enables gas and humidity control and phase contrast imaging for the cells in the well plate. Channel Imaging method. Maximum of four channels for each position can be used simultaneously. These are the phase contrast and three different fluorescence channels. Cycle A set of defined XYZ-positions in the wells of one or two well plates. Connector Piece that connects two gas line components airtightly together. Focus measure Measures single pixel focusing accuracy. This represents normally the strength of high frequency component in the image position. Horizontal migration Movement of adherent cells along the well plate bottom. Introduction 2

5 Image grid NxN set of non-overlapping consecutive images. Imaging interval The period of time between two consecutive images taken from the same xy-position during imaging cycle. Incubator Temperature, gas and humidity controlled space. IRI Information rich image. Cell-IQ implementation of the all-in-focus principle. Medium Nutrition rich liquid for cell cultivation. Position Location in xyz space. Vertical migration Cells drop from horizontal migration filter towards the well bottom. Well plate Synonym for microtiter plate. They are intended for long term cell cultivation and they typically enable phase contrast and fluorescence imaging. x-direction Horizontal direction in well plate row direction. y-direction Horizontal direction in well plate column direction. z-direction Vertical direction. Z-stack Several images from the same XY-position are combined by focusing to different depths with even Z spacing. Introduction 3

6 ALL-IN-FOCUS IMAGE Depth of focus in Nikon 10x objective is about 3 µm. Therefore it is not sufficient to take a single focused image and assume that entire imaging area is well focused. Cell-IQ uses focus stacking method to capture a stack of images and combines focused pixels into a single result image, information rich image (=IRI). The imaging principle is generally called All-in-focus image. Normally the Z-stack step size is 0,8 µm and Z-stack thickness is 16 µm. There can be small XY-positioning error between different Z-stack images. This error is corrected before IRI is built. The source of the error can be someone just touching the Cell-IQ or a nearby freezer vibrating the system. Introduction 4

7 Cell-IQ applications Cell-IQ applications II 5

8 Imaging settings of specific cell test applications Proliferation assay Horizontal migration assay Normal single layer assay Adherent single layer assays Vertical migration assay Cell colony assay Embryos and oocytes assay Fluorescence assay NORMAL SINGLE LAYER ASSAY Default settings Z-stack: 16µm Auto-adjust focus active Use area in and near the well center Focus manually the first position in first well Autofocus will find the right focus for other positions and wells Cycle time If 48 well plate and 3 positions in each well: 24 min If 48 well plate and 1 positions in each well: 8 min Cell-IQ applications 6

9 HORIZONTAL MIGRATION ASSAY Default settings Z-stack 16µm Auto-adjust focus is active Use near cell center area in well Focus manually the first position in first well Autofocus will find the right focus for other positions and wells Cycle time If 48 well plate and one 3x3 grid in each well: 72 min PROLIFERATION ASSAY Default settings Z-stack: 16µm Auto-adjust focus is active Use near cell center area in well Focus the first position in first well Autofocus will find the right focus for other positions and wells Cycle time If 24 well plate and three positions in each well: 12 min Cell-IQ applications 7

10 VERTICAL MIGRATION ASSAY Focusing Focus can be set manually if there's something visible in the well bottom to focus on, such as cells or garbage. Otherwise the focus is set on the outer well bottom surface and then shifted upwards by the thickness of well bottom plastic. Focus level can be copied to the other positions in the well. Special about this application Z-stack: 45µm. It should be so big that cells in well bottom stay focused No auto-adjusting the focus because it would adjust towards filter mat Use a 5x5 image grid in the wells Focus all wells manually. If the quality of the plate is good, z-values can be copied within a well. Auto-focus doesn't work in this application because it would focus to the filter mat. Adjust focus manually every 2 days Cycle time 3 hours with 24 well plate Cell-IQ applications 8

11 CELL COLONY ASSAY Special about this application: Z-stack: µm, depending on the colony thickness Auto-adjust focus active in <20µm thick colonies, in thicker colonies no Automatic focusing works in <20µm thick colonies, in thicker colonies no EMBRYOS AND OOCYTES ASSAY Special settings All-in-focus Z-range is set to 10µm. Larger stack would result into smudgy images due to too much information. To scan the entire embryo, preferable distance between the 12 Z-levels should be 10µm, as the embryo can be 120µm thick. The Z-stack size and the distance between Z-levels must be the same (10µm), otherwise some information may be lost as the stacks would not cover the entire Z-range of the embryo. It is important to disable the Auto-adjust focus during cycle option as the 12 set Z-levels would probably drift into a bundle. Make sure the checkbox is empty! Grids cannot be used, 1-3 single positions in each well is acceptable. Using grids with 12 overlapping positions would create a unmanageable result folder structure. Cell-IQ applications 9

12 ADDING THE OVERLAPPING POSITIONS MANUALLY 1. Focus into the well bottom. At this focus level, no details of the embryo are visible. 2. Click Add Pos. button. 3. Check the Z-value in the lower left corner in Imagen. 4. Click the right arrow (UP) button of the focusing bar slider until the Z-value has increased approx. by 10µm. 5. Click Add Pos. again. 6. Find the next Z-level 10µm up. 7. Repeat until all 12 positions are added and save the cycle. Cell-IQ applications 10

13 ADVANCED IVF SETTINGS To apply advanced IVF settings, go to Embryo and oocyte tracking in the IVF tools menu. Settings for IVF applications Embryo tracking ensures the embryo is always in the image, even if it moves due growth or G-forces. IVF gassing changes the automatic gassing setup to full time gassing. The short wait after movement allows the embryo to settle down after deceleration. Advanced settings for IVF applications Adjust the speed and acceleration/deceleration values to assure embryo stays in view. Lower the default acceleration and deceleration value if the embryo is very mobile. FLUORESCENCE ASSAY Cell-IQ applications 11

14 Settings for fluorescence channels Z-stack: From 1 image stack to 150µm thick stack. Big stack is not necessary for automatic focus adjustment, but if sample is thick, big stack guarantees that focused images are captured from all parts of the sample. The sample s exposure time can be minimized to the fluorescence excitation light with a small Z-stack size and with big camera pixel binning value, which lowers the resolution. A focusing level offset can be set for each fluorescence channel, if the fluorescencing level is different from the phase contrast which is used in focusing. Cell-IQ applications 12

15 Preparing for a cell test Preparing for a cell test III 13

16 PREPARING FOR A CELL TEST PREPARING CELLS 1. Warm up the Cell-IQ well plate lid to 37 C before you connect it to the well plate. For example, place it into an incubator for at least 30 minutes. This prevents moisture from condensing in the lid. 2. Cells must be in the well plate a minimum of 4h (24h recommended) before imaging cycle is started. It takes time for the cells to stick to the well plate bottom. The attachment time depends on the cell type. Normally there is serum in the medium during this time, it makes cells attach to well plate much faster. 3. In toxicology experiments the medium is changed into serum-free medium before compounds are added. 4. Spaces between wells are filled half way with purified water to increase gas humidity and hence decrease drying of medium in well. 5. Seal the Cell-IQ lid to the well plate with paper tape 6. Connect the output connector and the filter 7. Place the well plate into Cell-IQ incubator 8. Connect the input connectors CELL NUMBER AND MEDIUM VOLUME Cell number in one well in Nunc 48 Well plate: Neural cells SHSY-5Y 48 h test: cells in a well HK2 (human kidney) cells 48h test: cells in a well MCF7 cells 7 day test: 3000 cells in a well Typical cell incubation medium is used. Use 300 (to 500) µl medium in one well in Nunc 48-well plate. GAS FLOW SETTINGS Gas flow rate 6 ml/min Gas control settings: Gas On 15 min, gas Off 30 min SEALING THE WELL PLATE Preparing for a cell test 14

17 CONNECTING THE OUTPUT CONNECTOR AND THE FILTER PLATE LOCATIONS AND POSITIONING Note! Inserting the well plate inside the holder can cause a relocation of the second plate if two well plates are used. Always check the position of the plate that has already been inserted in the holder after inserting a new plate. Preparing for a cell test 15

18 CONNECTING THE INPUT CONNECTORS COMPONENT SPECIFICATION Preparing for a cell test 16

19 Program workspace Program workspace IV 17

20 PROGRAM WORKSPACE 1. Wizard and start imaging -buttons: Define the imaging cycles and channel settings with the wizard and start imaging. All the settings in the wizard can also be found in the imaging, edit and incubator menus. 2. Cycle information e.g. interval and images taken: The cycle interval is set before starting a cell test in the Wizard. Interval corresponds to how often same well plate position is imaged. Inserted values are for h (hours), min (minutes) and sec (seconds). The images taken is the number of images captured so far in the cell test. Also saving directories, gas flow for each plate and temperature of the incubator are displayed. 3. Live image display: Before starting the cycle you can select the displayed channels by clicking on the small channel displays on the bottom. During the cycle, the channel currently being imaged is always displayed in the main display. Right click by mouse to toggle the cursor information box that includes the cursor coordinates and the RGB values in that point. The pixel [R,G,B] values are the red, green and blue channel values in that pixel coordinate. Right click by mouse to toggle the black or white scalebar. The scalebar adjusts when the image is zoomed. Scalebar is not saved while imaging cycle is running, because it would ruin the analysing. Right click by mouse to edit visualization of the image: Show/Hide/Reset visualization controls, the same as in Small displays for each channel, Visualization settings in this topic. Program workspace 18

21 You can move the live image area by clicking the main image display (the small channel displays don't have this feature), the image will center itself to the point clicked. You can also use the drag and drop feature on the image display to move around in the image. Zoom in and out in the image by scrolling with mouse wheel. 4. Temperature and gas flow logs: The current temperature in the Cell-IQ incubator and the gas flow rates are continuously displayed (2). However, a log file of the temperatures and gas flow rates during the cell test can be saved at defined intervals. To start the log and define saving settings click Environment charts button. 5. Current well plate, well and position: The well plate, the well and the position details of the currently viewed image in the large display. 6. Focusing bar slider: Image can be focused manually using the focusing bar just below the live image. The slider moves by holding down left mouse button. Image is refreshed when slider is not moving. For more focus settings, right click by mouse over the focus bar. You can change the focusing scale, reset the focus bar and perform autofocus. 7. Combined image (only Cell-IQ with fluorescence module) ADD COMPONENTS: Add channels to the combined image by right clicking over each channel you want to add. Choose Add to combined image from the pop up menu. Similarly, remove a component by selecting Remove from combined image in the pop up menu: SNAP COMBINED IMAGE: You can take a snap shot and save the image displayed in the combined image display Program workspace 19

22 by choosing Snap combined image from the drop menu CHANNEL NAME BOX: The lower right corner of the small image display is a list of the channels that have been added to the combined image. The name box appears every time a channel is added to combined image or by selecting Show channel name box This list can be hided by selecting Hide channel name box. VISUALIZATION SETTINGS: The intensity of the combined image can be adjusted and the intensity value is saved into the cycle.dat file. Right-click the combined image to see adjustments and settings to the combined image. Select Show, hide or reset visualization controls. Adjust brightness, contrast and gamma parameters Small displays for each channel (only Cell-IQ with fluorescence module) VIEWING CHANNEL IMAGES: The name of the channel is above each display. To view the channel live image click on the round no camera symbol. If the name of the channel is shown light grey, the channel ratio was set zero in the wizard/channel settings. VISUALIZATION SETTINGS: Once the channel image is displayed, right click over the display to adjust visualization. Choose Show visualization controls from the pop-up menu to adjust brightness, contrast and gamma: Program workspace 20

23 IMAGE COLOR: Image color can be changed for each channel image. Set color by selecting Show image color menu in the pop-up menu. Select color from the predefined options or type in custom RGB values. The selected color is displayed in the big square. Click OK to close or Cancel to restore the previous pseudo color. The color and intensity values are saved into files Imagen_MFU.ini and cycle.dat. Program workspace 21

24 12. Well plate control panel: The well plate control panel is composed of the well plate image with well names in the top, the well position view of the selected well with position cursor in the middle, and the bottom part with arrow keys, function buttons and position information. In this important interface you can e.g. Select well plate Select well Define imaging cycles for each plate Add, copy, move or delete positions Define and preview grids Change position coordinates 13. (x, y, z) offset coordinate:the x, y -coordinate values is the offset in millimeters from the system origo. 14. Image dimensions: The dimensions of the image are displayed in the bottom information bar of the window. Image dimensions are in both pixels and micrometers (µm). 15. Full screen mode for main image: Hide the plate control frame on the right to view the image in the big screen larger. Click Show controls to restore the plate contol frame. NOTE! Cell-IQ Imagen single label fluorescence has no images in fluorescence channel 2 and channel 3 windows naturally. Cell-IQ Imagen with only phase contrast channel the lower part of the GUI is invisible as there is no fluorescence option. Program workspace 22

25 Cell test focus settings Cell test focus settings V 23

26 CELL TEST FOCUS SETTINGS ALL-IN-FOCUS Z-RANGE Cell-IQ takes stack of images and collects focused pixels to the single result image (All-in-focus image). The default z-stack thickness in phase contrast imaging is 16 µm which is the best setting for typical single layer cell test, but it can be adjusted if necessary. The z-stack step size is 0,8 µm in phase contrast imaging. In Cell-IQ with fluorescence module the z-stack thickness is 10µm due to the smaller 0,4µm step size used in fluorescence imaging. Larger z-stack would increase the image data collected for each stack excessively. FOCUSING SCALE If the focus is not found by moving the focusing slider from one end to another, you can select deeper focusing area. AUTOFOCUS WHEN IS AUTOFOCUS RUN? Cell test focus settings 24

27 AUTO-ADJUSTING FOCUS DURING CYCLE Cell test focus settings 25

28 Setting up a cell test Setting up a cell test VI 26

29 SETTING UP A CELL TEST USING WIZARD Settings wizard will take you through the simple parameter settings of the cell test easily and fast. With the wizard you can: 1. Define well plate type for each plate separately 2. Define the saving locations for the results 3. Define the imaging cycle interval 4. Edit phase contrast settings 5. Select fluorescence channels and define settings for each. Start wizard by clicking the Wizard button. First define the basic settings for the cell test. START AND STOP IMAGING After defining the cycles click Start imaging button to start the automatic imaging cycle. All-in-focus images are saved to result folders selected earlier in the settings wizard, and they can be viewed with Cell-IQ Analyser program during image capture or anytime afterwards. Stop the imaging cycle by clicking Stop imaging. This may take a little while. Setting up a cell test 27

30 CELL TEST SETTINGS BASIC SETTINGS 1. Open the Wizard and select one or two well plates to be used in the test. 2. A list of predefined types is available. The default plate is Nunc 48 well plate. Details and dimensions of the selected plate type is shown in Plate Details sheet. The Edit.. button opens the details sheet for each plate. You can also define a new plate type that is not found in the drop menu. 3. Define the saving directory for image folders from each well plate that is used. Images of selected wellplate positions are saved in automatically created sub folders. The program also generates image names using image capture times, so not write any filename in the File name field, just click Save after browsing correct saving location in the Save in field. 4. Interval corresponds to how often same wellplate position is imaged. Insert values for h (hours), min (minutes) and sec (seconds) according to the desired time between two images. Note: If interval is set smaller than the time it takes to go through all selected positions, images are taken without waiting between cycles, and the interval may slightly vary between consecutive cycles. Otherwise there is a pause between cycles to fill in the cycle interval time. E.g. If you have a 48 well plate and one imaging position per well, one imaging cycle takes a maximum of 8 minutes with default settings. If the cycle interval were set under 8 minutes, the imaging would proceed continuously with no pause between cycles. 5. An estimation of the number of images per cycle with current settings. Note that it does not include position specific settings that may vary. Setting up a cell test 28

31 ADVANCED SETTINGS OBJECTIVE TYPES To change the objective click Objective types button in advanced settings. Drive down the objective in order to change it. Choose an objective from predefined options in the drop menu: Setting up a cell test 29

32 Note: The phase slider has to positioned according to the objective. The right position of the phase slider for 4x, 10x, 20x and 40x phase contrast objectives is shown in the image below of the detached slider. The right-most aperture is for bright field imaging. Setting up a cell test 30

33 GAS FLOW SETTINGS The default cycle is gas flow on for 15 minutes and off for 30 minutes. Edit the gas flow cycle if other than default is necessary. The default settings ensure the correct gas control. They are restored when Imagen is restarted. A constant gas flow can be manually turned on for both plates separately. A red spot blinking on the settings window and in the program window indicates that constant gas flow is on. FLOWMETERS There are flow meter displays for gas calibration that show the flow value behind the left side panel of the Cell-IQ device (looking from the front). Ask further guidance about gas calibration from the local distributor. Setting up a cell test 31

34 PHASE CONTRAST SETTINGS BASIC PC SETTINGS (WIZARD) ADVANCED PC SETTINGS Setting up a cell test 32

35 FLUORESCENCE SETTINGS BASIC FL SETTINGS (WIZARD) 1. Cycle ratio for each fluorescence channel With the cycle ratio option you can choose how often each method is used in relation to the phase contrast that is always done each cycle.the entire cycle is always completed with one imaging method before switching into another. If the cycle contains a high amount of positions it can take hours to complete. The channel ratio is therefore very application specific setting. The channel ratios need to be adjusted after loading a cycle file. Example of the channel ratios: value 0: the channel is not used for imaging. value 1: the channel is imaged each cycle. value 2: the channel is imaged every second cycle. 2. Z-stack size for each channel The z-stack is the thickness of the imaging stack in vertical direction in mm. The z-stack size may vary a little if the objective is changed. With the 10x fluorescence objective the step size is 0,8µ m. 3. Binning size for each channel The saved images are always the same size as with 1x1 binning, when other binning is used the images are scaled to that size by stretching them. By using larger binning the image resolution drops but better sensitivity is achieved and prolonged exposure times are avoided. 4. Exposure time in ms for each channel The exposure time defines the time period the light is collected on the sensor. For example, with 1x1 binning the normal value for GFP is about ms. 5. Apply modified exposure times and binning sizes 6. Save and copy settings to all positions in this plate This applies the changes to all new positions added and the existing ones. Otherwise the changes made in the wizard don't apply to any existing positions but only those to be added. To change the settings of the existing positions use the Edit pos. button in the right side panel in Imagen. Setting up a cell test 33

36 ADVANCED FL SETTINGS To apply advanced fluorescence settings, go to Set channel parameters in the Imaging menu. You can set all the imaging parameters separately for each channel: 1. Channel name: the channels can be named freely, e.g. GFP, TRTIC. The channel name is displayed over the little channel images on the program interface. 2. Filter location: The filter cube that is used. Possible values are [1, 2, 3] from left to right. There is a fixed phase contrast in location 1 and a GFP filter. The locations 2 and 3 can be something else or an empty cube. 3. Light source: The light source that is used. Possible values are [1, 2, 3]. Usually these are 1=red, 2=green and 3=blue. A blue light is commonly used with GFP, that is source number 3. The light sources can be changed by the user. 4. Offset: Z-axis offset to Phase contrast focusing level in micrometers. This number can be positive or negative. Setting up a cell test 34

37 FL LIGHT SOURCE SETTINGS To apply advanced fluorescence settings, go to Set advanced parameters in the Imaging menu and click Fluorescence Settings button. Setting up a cell test 35

38 FILTER CUBE CHANGING To change filter cubes, go to Position Calibration in the Imaging menu and click Change Cubes. 1. Open the Cell-IQ system front main door to locate the cube changer 2. Remove the cover Setting up a cell test 36

39 3. Carefully lift the middle or the right cube directly up. Avoid leaving fingerprints on the filters. Left cube is fixed 4. Insert new cube, close the cover, close the front door and click Change Cubes from the Imagen software. IMAGING POSITIONS CREATE THE IMAGING CYCLE WITH THESE SIMPLE STEPS 1. Activate a well with cells by clicking it on the plate map. 2. Focus the image manually using the focusing bar slider. 3. Add the position by clicking Add pos. 4. Copy the position to all other wells by clicking Copy. 5. Repeat for plate Click Start imaging. MOVING TO POSITIONS Click on a spot inside the light gray circle to choose XY-offset from well center in the well plate control panel. You can also move around with mouse using the live image display, see directions. The position is shown with a yellow rectangle. Setting up a cell test 37

40 MANUAL FOCUSING Live imaging is automatically launched when program is started. After selecting a well the center position of the selected well is displayed. The image can be blurry before focusing. Focus the image manually using the focusing bar. Hold down left mouse button and move the slider until the image is in focus. The image view is refreshed when you are not moving the slider. You can select deeper focusing area using different focusing scale. Right click with mouse over the focusing bar to view the pop up menu. The options are 1, 2 or 4 times the scale of 700µm. With deeper area (2 or 4 times the scale), single step in focusing bar corresponds to bigger focusing step size. The focusing bar slider is centered when the scale is changed. You can also find the focus using Autofocus. Check the focus manually from the live image view before saving a position. Make sure the image is in focus by eye even after running autofocus. On the well plate map, cyan color in a position indicates that the program assumes that it is in focus and will not perform autofocus on that position during first cycle! TO ENSURE THE RIGHT FOCUS 1. Select first well on the well plate that has cells in it and click the well on the plate image. You can see the cells of the active well in the live image view. 2. Move the focusing bar slider until the image is in focus. 3. Save the position by clicking Add pos. button. Setting up a cell test 38

41 ADDING POSITIONS COPYING POSITIONS Click Copy to copy the added position to all other wells on the active well plate. This does not copy the focus (Z) values, only the XY coordinates, but Imagen will automatically find focus for the copied positions during first imaging cycle. The position can't be copied to wells in the other well plate. The wells that the position is copied to, turn light blue on the well plate map. Setting up a cell test 39

42 If there are already selected positions in other wells: by selecting Yes the previously selected positions are replaced with new positions. by selecting No the new positions added to the selected wells while preserving the existing positions. When copying positions from one well to another, the position specific settings remain, even if they are different from the well specific settings, e.g. exposure times. So new position specific settings overrule the existing well specific settings. This applies also when making changes in the wizard or Settings Form; the changes don't apply to those positions that were added prior to these changes, only to any new positions added. OPERATING ON A SELECTED GROUP OF WELLS Select or unselect wells by clicking a well and then Select button or hold the shift key down and click the wells. Each selected well will turn green. When selecting multiple wells, hold the shift key down and click the wells you want to select/unselect. When selecting all the wells on a well plate hold the ctrl key down and click any well on the well plate map. The Copy and Delete functions can be applied to the selected wells in all these three cases. E.g. Click the well C2, add a position as described above. Hold down shift key and click wells C3, C4, C5 and apply Copy function. The positions in well C3 are copied only to the other three cells. To clear the positions on a plate quickly, hold down control key, click any well and apply Delete. To clear only a selection of wells, hold down the shift key, click the wells to be cleared and apply Delete. CHANGING POSITION COORDINATES To change the coordinates of a added position, point the new position with mouse on the well image and click Set Pos. XY. The position is moved. The procedure is the same for image grids. Only entire grid can be moved or deleted, not single positions in grid. DELETING ADDED WELLS To remove all positions in a well, select the well and click Delete. REMOVING ONE POSITION IN A WELL Point the well in the well plate map. Added positions in that well are displayed with colored squares. Select the position you want to delete from the drop menu under Delete Pos. button. The yellow rectangle is placed on the position. Click Delete Pos. to remove the position. RESETING CYCLE Reset cycle by selecting Reset cycle 1 or Reset cycle 2 from the Edit menu. Choosing Reset cycle 1 will delete all positions from all wells in the well plate 1. Setting up a cell test 40

43 MULTIPLE POSITIONS AND GRIDS ADDING CUSTOM POSITIONS IN A WELL For each well and each position in that well, the three steps are repeated to add the positions to be imaged: Point position in the well: Click a position inside the light gray circle or search position by moving image in the live image display to choose XY-offset from well center. The position is shown with the yellow rectangle. The cursor can be returned to well center with the center button of the arrow keys.see Moving to positions. Check that first position to be added is in focus: Focus the image manually using the focusing bar slider. Hold down left mouse button. See Manual focusing. Save the position position: When the yellow rectangle is in the desired XY position and the image is focused, click Add pos. See Adding positions. Repeat these three steps for all positions in a well. Move on to the next well by clicking on it in the well plate map. Always select positions near well center where the image quality is best. There is 3.5 mm unusable area near well edges due the phase contrast microscopy method. GRIDS Positions to be imaged can be selected in grids, which dimensions are the number of images in vertical and horizontal direction, e.g. 3x3 grid is formed by nine images. Setting up a cell test 41

44 ADD GRID POSITIONS The same can be repeated for several positions in a well. You can select more positions with various grid sizes or combinations of single positions and image grids. All added positions in the source well, including grids, can be copied to other wells with Copy function. A grid position can be removed with Delete function. Note! When using Nunc 6 well plates focus one image manually in each grid you add. The well bottom z-levels can have more than 200µm differences within the plate. Setting up a cell test 42

45 POSITION SPECIFIC SETTINGS WELL SPECIFIC SETTINGS To set well specific settings, click Edit pos. button on the well plate control panel or right-click on a specific well on the well plate map. Select the Well tab: Setting up a cell test 43

46 PLATE SPECIFIC SETTINGS To set plate specific settings, click Edit pos. button on the well plate control panel or right-click on a specific well on the well plate map. Select the Plate tab: POSITION SPECIFIC SETTINGS To set position specific settings, click Edit pos. button on the well plate control panel or right-click on a specific well on the well plate map. Select the Position tab: Setting up a cell test 44

47 SAVING AND OPENING CYCLE SAVING CYCLE LOADING CYCLE : If the well plate was not removed, click No. The saved focus values (z-values) are loaded. If the well plate was removed, click Yes. The focus values are not loaded, because they have probably changed and the images need to be focused again. Point the first position in first well and focus the image before starting imaging. Setting up a cell test 45

48 Monitoring cycle Monitoring cycle VII 46

49 MONITORING CYCLE TEMPERATURE CONTROLS Monitoring cycle 47

50 TEMPERATURE AND GAS FLOW LOG The incubator temperature and the gas flow rates are continuously displayed in the left sidebar in Imagen and can be saved into a log file. Click Environment charts to view incubator monitoring logs. Click Start log to start monitoring each variable. You can also Save Log into a file. GAS FLOW RATE ALARM The gas flow rate has an alarm function which raises alarm if the gas flow rate drops below smallest value allowed. The gas flow alarms understand the on/off cycle. Alarms are not raised when the gas flow goes below the alarming limit during the OFF period, only if the gas flow rate drops during the gas flow ON period and during the gas flow is on by manual control. Monitoring cycle 48

51 Changing medium Changing medium VIII 49

52 CHANGING MEDIUM 1. Click Stop Imaging. 2. Select correct well plate (1 or 2) in the well plate map. 3. Stop gas flow for that plate. 4. Remove the well plate, change medium and place it back to the Cell-IQ. 5. Re-start the gas flow. 6. Empty Focus (Z) memory. 7. Select first position in the first well. 8. Focus the position manually and save the focus by clicking Set image Z. 9. Click Start Imaging. Note! If you have two well plates and you change medium to one of them, the focus adjustment must always be done for both well plates because they both move a bit even if you touch just one of them. Changing medium 50

53 Error recovery Error recovery IX 51

54 ERROR RECOVERY Error recovery 52

55 Index Delete 37 Delete Pos. button. Deleting added wells.bmp 27.jpg 27 deleting mode 37 drag and drop 18 Draw lines 41 A Adding positions ensure the right focus 41 Error recovery exposure time 37 Adherent single layer assays All-in-focus 24 All-in-focus Z-range 24 6 File size 28 Find XY home position 24 B binning 33 Binning size blurry 37 bright field focusing bar 28 24, 37 G gamma gas flow cycle 31 Gas flow settings 31 C Capture interval GFP 33 grid sizes 27 Cell colony assay 9 Changing medium 50 Changing position coordinates 37 Grids H 43 Connection specification constant gas flow 31 contrast 18 Hide visualization controls 18 Horizontal migration assay 7 I 14 Image names Image quality copy the added position 37 Copying positions 37 copying positions from one well to another Create the imaging cycle cyan 37 cycle ratio 33 Cycle ratio for well plates D interpolation method 32 Interval settings 27 L lancos Imagen default parameters input connector deeper focusing area 41 Grid view 41 Grid view functions channel name box 18 Choose grid size 41 clear the positions 37 copy function 37 Focusing scale 32 Channel name 52 fixed phase contrast 33 flow meter displays 31 fluorescence channel Basic settings 27 filter 14 filter cube 33 Filter location 33 Auto-adjusting focus Auto-exposure F Applications 6 arrow keys 37 attachment 14 comment 37 E add comment 43 Adding custom positions aperture lid 14 light gray circle 37 Light source 33 Line color 41 List of consumables 14 53

56 Loading cycle 45 Loading the well plate location 1 Sealing the well plate 14 selecting multiple wells serum location information 47 M Manual focusing 37 medium 14 MEDIUM VOLUME 14 Set Pos. XY 37 Setting up a cell test 27 Settings Wizard 27 Snap image 18 Starting wizard T Monitoring cycle 47 Moving to positions 37 tape N Normal single layer assay 14 Thick formation assay time stamp 43 6 Tubing O 9 14 U objective 28 Objective types Update 28 Operating on a selected group of wells output connector V Vertical migration assay 8 visualization controls 18 P phase slider 28 plate description 43 W Plate specific settings 43 well description Position calibration 52 Position specific settings 43 Preparing the well plate 14 Preview grid functions Proliferation assay Pseudo color 18 pseudo color menu Using wizard 43 Well specific settings 43 X XY calibration Y yellow rectangle Z R Recovering the calibration Zoom in and out 18 z-stack size Refresh connection 31 Removing one position in a well Z-stack step size Reset button 37 Reset cycle 37 Reset cycle 1 Reset cycle Reset focus axis position reset the brightness Reset window size 41 Reseting cycle 37 S save comment 43 Save grid image 41 save plate description Saving cycle 45 Saving format Saving interval