Image-Pro Plus 7.0 Product Note

Image-Pro Plus 7.0 Product Note Automated Microscope Configuration Introduction Cameras, software, shutters, stages, objectives, filters, turrets there are a lot of components in an automated microscopy system, and a lot of information required to use it properly. This Product Note is intended to walk you through the installation and configuration of an automated microscope system, including Image-Pro Plus 7.0 or later, lens and dye information, Scope-Pro and Stage-Pro automation controllers, and the AFA (Advanced Fluorescence Acquisition) module for synchronizing multiple channel and position acquisition. Following the procedures described should help get your system up and running. As an overview, here are the steps described in this document: 1. Install Image-Pro software. 2. Camera Installation... 3. Microscope Setup... 4. Image-Pro Settings Configuration..... 5. Scope-Pro Configuration.. 6. Stage Configuration/Alignment for Tiling...... 7. AFA Experiment Configuration....... p.1 p.2 p.5 p.6 p.8 p.16 p.22 STEP 1 Install Image-Pro Software: A) Refer to the Image-Pro Getting Started Insert for quick and easy install instructions. -or- B) Refer to the Image-Pro Startup Guide included with Image-Pro Plus for detailed installation instructions. NOTE: Do Not Restart Yet Once you have installed Image-Pro, it is recommended that you complete step 2, before restarting your computer. 1. Automated Microscope Configuration Updated 7/15/2009 1

STEP 2 Camera Installation: A) Install driver(s) B) Install hardware C) Run Image-Pro D) Test for a live picture and SAVE A) Install driver(s): i. Current supported drivers: To use a camera or frame-grabber with Image-Pro, you will need to install the appropriate capture drivers from the Media Cybernetics website (http://support.mediacy.com ). You must install the 32- bit version of the capture drivers to work with Image-pro Plus on 32-bit systems, or the 64-bit version on 64-bit systems. Keep in mind that the driver may run a board or library, and some support a dozen or so cameras. If you do not see your camera, look for the hardware used to interface your camera to your computer and then run the installer for your hardware. Legacy and 3 rd Party drivers: If you do not see your camera in the list, go to the www.solutions-zone.com website and check for drivers and updates. Select Compatible with all, Solution Type of Frame Grabber Driver or Digital Camera Driver to see the complete listings for those devices, or search on the name of the camera or frame grabber board. These drivers include third party drivers, older drivers that are no longer fully supported but still work, and updates to both. There are over 50 different cameras and frame grabbers in the complete listings. If you see an appropriate update for your legacy or 3 rd party driver camera, download it and install it according to the instructions for that driver. B) Install hardware: i. Follow Read-Me Files: Make certain that the hardware is installed. The exact steps for this will vary from camera to camera this can be as simple as plugging in a Fire Wire (1394) cable, or as complicated as installing a PCI board, drivers, and multiple cables. Many of the camera installers include Readme files that describe the installation process and it is recommended that you read these. You may want to consider printing them so that you have a hard copy when working on your computer. Reboot Machine: It is strongly recommended that you reboot your machine during this process, first to recognize the drivers, and then to recognize the hardware. You will generally be prompted to reboot after driver installation, but you may not be after the hardware install. Either way, a reboot after installing the hardware and drivers ensures that your system software recognizes it. 1. Automated Microscope Configuration Updated 7/15/2009 2

C) Run Image-Pro: i. Verify Driver Installation: With the camera powered on, Start Image-Pro and open the Capture dialog using the menu command Acquire Video/Digital Capture. Basic Dialog The Basic window lists the currently selected camera in the title bar. If you can t read the name, click on the More >> button to see the expanded dialog, and make certain that your camera driver is the current selection. This is generally set as the default when installing your driver; if a different driver is selected, such as Analog Simulation, select your camera or driver from the Current Driver combo box at the bottom of the expanded dialog. If the hardware is not connected, or your camera is not currently on, you may see an error message indicating that. If you do, check that everything is plugged in and turned on. 1. Automated Microscope Configuration Updated 7/15/2009 3

Advanced Capture Dialog D) Test for a live picture and SAVE i. Configure and Auto Set: There is a Configure button at the bottom of the expanded dialog that includes advanced settings for your camera driver, but starting with Image-Pro 5.0 and the current set of drivers, there is a simpler way to get a picture. Use the Less << button to go back to the Basic dialog, and press the Auto Set button at the top. This will set the driver to default settings such as 1x1 binning, initialize the correct exposure mode for that hardware, run a white balance for color cameras, and perform an auto exposure to get a useable preview running. For older cameras, or for frame grabber cameras that acquire by frame without a settable exposure time, start with the default settings as installed, and proceed from there. 1. Automated Microscope Configuration Updated 7/15/2009 4

i Live Preview: Bring up the live preview with the Preview button, and check that the preview changes if you block the light going to the camera. Using your microscope system and shutters or by simply waving a hand in front of the detached camera should change light input enough to show a result. Any change in the intensity of the preview indicates that you have a live image. Snap a picture or two as a test. When Preview and Snap both work, the camera is up and running. Once you have the camera on your microscope you can refine the exposure times and the white balance as appropriate. SAVE Video Preference Files (*.VPF): At this time you should save one or more video preference files (*.VPF) using the Save and Save As buttons at the bottom right of the Advanced Capture dialog. These describe the camera settings you are using, such as gain, offset, and frame size. These will be used later on by AFA. For example, you can set the camera to use the full field of view, with grayscale output, and name it Grayscale.vpf. Also, it is recommended that you save at least one main VPF file that specifies the settings you plan on using when acquiring in AFA. You can also save additional VPF files for particular channels if different channels require different capture setups (for instance, a dim dye that requires a higher gain setting). Each one of these modes can be edited later on through the AFA control panel. STEP 3 Microscope Setup A) Set up microscope B) Capture simple test images C) Tips for successful digital imaging using microscopy A) Set up microscope: i. MICROSCOPE ASSEMBLY AND CONFIGURATION IS BEST DONE WITH THE ASSISTANCE OF YOUR DEALER: If it is not already assembled and aligned, install your microscope as directed by the dealer, and ensure that it is working properly before continuing. This means checking that automated components move, that the proper optics are installed, and that you have your filters mounted correctly. B) Capture simple test images: i. Start with simple samples: Once your microscope is set up, mount the camera on the system. As a first run, you might try imaging a simple transmitted light sample to ensure that you re still getting a live feed from the camera. Do not start with your most difficult sample in order to avoid complication. Once you have established that the camera works, try imaging more complex samples that you know the appearance of, in order to compare the camera with previous observations. C) Tips for successful digital imaging using microscopy: i. Koehler illumination: Remember that consistency in your observations is a key to reproducible results. Configuring the light sources for even Koehler illumination helps make intensity measurements and segmentation consistent across your field of view. 1. Automated Microscope Configuration Updated 7/15/2009 5

i Reference backgrounds: Tracking your light conditions with background and flat field images allows you to correct samples for light variations these reference images should be kept and associated with the experiments taken under those lighting conditions. Acquiring and saving references along with your experimental data should become part of your procedure, so that you maintain consistency regardless of lamp and microscope adjustments. Using the correct modes: Different staining, lighting, and imaging conditions can make the difference between simple and a difficult image processing and measurement procedures. If the images are hard to segment or measure, consider whether different sample preparation, lighting, or even microscopy modes might simplify what you are trying to determine. STEP 4 Image-Pro Settings Configuration A) Set up Lens List B) Set up Dye List C) Set up Spatial Calibrations D) Set up Capture Settings NOTE: Start Image-Pro If Image-Pro is not yet running, open it from the desktop shortcut or from Start Programs Media Cybernetics Image-Pro Plus 7.0. A) Set up Lens List: i. Characteristics of Lens Files: Lens files keep track of the optical details such as magnification, refractive index of immersion fluid, and numerical aperture (NA) properties that will be attached to images when they are captured, used by SharpStack, SharpStack Plus, and SharpStack Total for deconvolution, and maintained to help document the images and their origins. These lens details are also used by Scope-Pro later in the setup for assigning optical characteristics to objective positions in the microscope nosepiece. This will create a simple automated solution when the setup is complete. Save Lens Files (*.ipl) for each objective: Using Edit Lens List, create and save a Lens File (*.ipl) for each objective or lens you are going to use. a. Select the New button. b. Type in the name of the lens you are adding to the List. The name should contain the magnification and objective type, such as 40x UPlanFl which allows the AFA configuration wizard to easily check lens files against positions. It is suggested to enter the Magnification first. If your microscope has specific names for the various lenses, it is useful to match the names of your lens files to the names supplied by your microscope. The default lens is 10X Dummy Lens Please Replace!.ipl and should be replaced by a list specific to the objectives. 1. Automated Microscope Configuration Updated 7/15/2009 6

c. Adjust the Magnification, RI, and NA for each Objective. d. Select Save after the parameters have been entered. e. If new objectives are ever added to the system simply return to this same dialog to add the new len(es). Lens List Editor NOTE: Name Consistently It is suggested that you name all Lens Files and Calibration Files the same as the associated objective lenses in order for the AFA configuration wizard to automatically check lens files against positions. NOTE: Default Folder All Lens Files (*.ipl) will save to the C:\IPWIN70\Documents and Settings\ folder by default and can be changed to a different location by choosing the Save As instead of Save. B) Set up Dye Lists: NOTE: Set up only for NEW Dye s There is no need to save new dye files if the current list contains all the dyes you will be using. i. Characteristics of Dye Files: Dye files associate a dye name with excitation and emission wavelengths, and select a viewing color for that dye. If AFA has a dye file for each acquisition channel, it can use the information to build appropriately shaded full color composites, and the wavelength information is passed along to SharpStack for deconvolution. Save Dye Files (*.ipd) for each filter or mode: Review the existing dyes with Edit Dye List. You should have a dye for each filter set or mode used in your microscope. Note that there are Dye Files for Brightfield, DIC, and Phase microscopy containing excitation and emission wavelengths of 550nm added for your convenience. Image-Pro comes with a large number of pre-defined dye files; if you are using dyes outside this list, just add them with the New button. It is suggested that you review the relevant dye files to ensure that the numbers used match your filter sets, and that the display color selected either matches what you see in the microscope or is an otherwise reasonable assignment. You can also remove dyes that you will never be using, if you wish to shorten the list but be conservative about this or move them to a different folder, so that you don t have to re-enter them later. 1. Automated Microscope Configuration Updated 7/15/2009 7

Dye List Editor NOTE: Save Default Folder All New Dye Files (*.ipd) will save to the C:\IPWIN70\Documents and Settings\ folder by default, but a good tip is to create a new folder inside the default folder for dyes pertaining to the current experiment and copy *.ipd files into it. Lastly, you can change the default location of the Dye List by choosing the next to the dye name and directing to your new folder. C) Set up Spatial Calibrations: i. Refer to the Spatial Calibration Product Note: Once the camera is properly connected and the microscope is installed and configured, you can calibrate your objectives, and determine just how large a pixel is on your sample. This is covered in another Product Note, Spatial Calibration. This shows how to use the Spatial Calibration Wizard to create reference calibrations for your objectives, optics, and cameras, so that you can directly measure the physical size of features in your images. The Spatial Calibration Product Note can be found on the Media Cybernetics website, www.mediacy.com or you open a PDF version installed with Image- Pro 7.0 in the Documentation folder, typically C:\IpWin70\Documentation Reference calibrations like this work in any optical system with a fixed imaging distance like your microscope. For images taken at varying distances, you can use the spatial calibration wizard to establish spatial relationships using an internal reference, such as a known measure or ruler, or feature of known size, in the image. Use the Spatial Calibration Wizard to Calibrate the active image, then take a picture and test the calibration with measurements. Once calibrated, snap an image and check measurements. Spatial Calibration from stage movement: If a micrometer or calibrated specimen is not available, then it is possible to calibrate from Stage-Pro using Lens/Mag Calibrate Lens XY, and selecting Calibrate by stage movement. Stage-Pro will ask you to move a recognizable object from one corner of the field of view to the other, clicking on it at each position. The relative positions of the object and the number of stage steps between will determine the pixel size with that objective. IMPORTANT: If you would like to generate spatial calibrations from stage movement, this should NOT be performed now, but during STEP 6c Part NOTE: Multiple Cameras If you have multiple cameras that will be viewing objects through the same optics, you will need to calibrate each one separately. Again, it is more convenient (and much easier to follow) if you name your reference calibrations after the lens magnification, for example 40x Digital Camera. 1. Automated Microscope Configuration Updated 7/15/2009 8

NOTE: Calibrated Status Bar that the pixel readout in the status bar at the bottom of the Image-Pro main window can be set to show calibrated pixel positions using Edit Preferences, going to the Status Bar tab, and selecting Show Calibrated Cursor Position. When a calibration is applied, all measurements, such as from Count/Size or Manual Measurements are reported in calibrated values. STEP 5 Scope-Pro Configuration: A) Connect and test hardware B) Select system components C) Component configuration D) Optional graphical interface E) Save multiple configurations A) Connect and test hardware: a. Connect: If you have not already, physically connect the hardware for Scope-Pro to control. This module allows you to control automated microscope hardware, such as stages, focus motors, filter wheels, shutters, and so on. The full list can be seen on www.solutions-zone.com by searching for a Solution Type of Other Hardware Support, and on http://support.mediacy.com. This includes the latest versions of component controllers. b. Test: Ensure that the automated components work. If you are using an automated microscope, run the various motors and shutters from the manufacturer s control panel. If you are using a separate controller for components such as a filter wheel, stage, or shutter, you may want to wire it and run it before attaching the components to the microscope. Also connecting your stage to the stage controller and moving it with the front panel or the joystick, using the control panel on your automated microscope to move objectives, and so on. The stand-alone setup is the best place to determine if everything is actually connected and plugged in. That way you can ensure, for example, that you have the filter wheels connected in the right order. NOTE: Man ufacturer Controller Settings Read the installation instructions for your automated components! While Scope-Pro will usually default to the manufacturer s factory settings, it pays to double-check the settings for COMM ports - speed, parity, stop bits. For USB port interfaces, see if the component will draw its power from the USB line; certain USB ports may not supply enough to run a component, limiting which ports can be used. B) Select system components: i. Open Scope-Pro Configuration : With Image-Pro running, select Acquire Configure Scope-Pro to close Image-Pro and open the Scope-Pro configuration wizard. You will have an option to restart Image-Pro after the configuration is complete. If you do not have Image-Pro open, you can run the configuration wizard directly from the Scope-Pro Configuration desktop shortcut, or select Start All Programs Media Cybernetics Scope-Pro 7.0 Configuration. Be aware that if you invoke the configuration utility directly, you will be required to restart Image-Pro before the configuration changes will take effect. For illustration, we will walk through installing an external controller, with a stage, focus motor, filter 1. Automated Microscope Configuration Updated 7/15/2009 9

wheel and shutter, as well as a manual objective turret. Select system components: When asked what components to install, mark the items you have on your system. Note that these will be listed by manufacturer first, then by component. For this example the stage, focus, shutter and filter wheel are selected, as well as manual objectives. Component selection If you are uncertain of the presence or absence of a particular component, for example, components in a highly automated microscope, it is acceptable to select them at this time. During the final configuration you can mark as disabled those that Scope-Pro does not find, and later run the configuration utility to turn those components off. However, it s much easier if you have an accurate list of installed components to start with. iv. Choose focus control option: Click Next and choose to Control the Focus from Scope-Pro. Since you are using this guide to setup for AFA it will not matter where you choose to control focus, as AFA will control the Z focus during acquisition. If you were configuring Stage-Pro and Scope-Pro for standalone use, then you would want to consider whether you will be most often acquiring with tiling, X/Y scans or well plates, in which case you would want to configure with Stage-pro. If you would not be performing many of these X/Y stage acquisitions, you would want to configure with Scope-Pro to make it more convenient to access the microscopy hardware automation on the Scope page. v. Check the Launch imaging application checkbox: Verify that the checkbox is checked (which is on by default) in order to launch Image-Pro after finishing the Scope-Pro Configuration. 1. Automated Microscope Configuration Updated 7/15/2009 10

C) Component configuration: i. Run Scope-Pro Configuration: Once running, select Acquire Scope-Pro. NOTE: No Stage-Pro Do NOT choose Acquire Stage-Pro since AFA requires the Scope-Pro setup alone. You will be prompted to run the Configuration Assistant or Configure Manually. It is recommended that you follow the configuration assistant as it walks you through the setup and configuration of each individual component and only Configure Manually once you have become familiar with the setup. The configuration assistant will walk you through the system configuration step by step, starting with the controllers themselves. Once communication has been established with a controller (such as the Media Cybernetics system), the Configuration Assistant will step through the other items on that controller and allow you to configure them. a. If initialization FAILS: If the initial communication fails, check the cabling and communication settings listed beneath the component name. The communications settings for each controller are set to the factory-defaults for that controller, so you should rarely need to change these settings. It is far more likely that the COMM port is incorrect, or that the wrong cable is in use. NOTE: Connect Hardware Make certain that your hardware is turned on and connected to your computer with the appropriate cable before running the Configuration Assistant. In the case of RS-232 controlled systems there may be issues of null modems, gender changers, and exactly which port to connect to. Refer to your controller manufacturer s documentation for these cables, and be prepared to experiment to obtain a good connection. If you are uncertain as to whether the problem is the controller, cable, or Scope-Pro installation, you may need to test the controller and cable with HyperTerminal or a similar program. View the list of components: The list of components that were previously selected in the Scope-Pro configuration will now be located in the drop-down list at the top left of the Scope-Pro dialog. List of components in Scope-Pro i Choose configuration settings for each component: At this time you must assign COMM ports and speeds, USB ports, or whatever is appropriate for your controller. Once again, check the installation instructions for your components as this will help you to avoid selecting the wrong options. The defaults are generally correct, although the COMM port will 1. Automated Microscope Configuration Updated 7/15/2009 11

vary depending on the socket you have plugged the component into. If you have more than one component, such as with an automated microscope and a separate XY stage, you will need to set one to COMM 1 and the other to COMM 2, as appropriate. Controller and communications Examples: a. Objectives: Choose the number of positions on an objective turret. Name each position in the dropdown list of objective positions. To choose the names that were previously setup in the Lens List, choose the button and select the correct name from the populated list. b. Filter Wheels: Choose the number of positions on the filter wheel. Name the various filters in the wheel, or the name of a light source. It will be helpful later in the configuration process to name the filter wheel positions with the name of the dye that the installed filter will excite or detect. You can select each filter wheel position that you will be using in the Filter Wheel Positions drop-down list, and then use the button to select the corresponding dye: c. Shutters: Choose whether a shutter is on the transmitted or epifluorescence light path. After configuring and naming the positions on the component, choose the next component in the dropdown list until all components have been completed and confirmed. When you have configured 1. Automated Microscope Configuration Updated 7/15/2009 12

all the components, click OK at the top right and Scope-Pro will show additional tabs (Scope & Acquire/Focus) for setting component positions. NOTE: Filter Wheels If you have initialized the controller, as you select filter wheel positions in the drop-down list, the corresponding filter wheel will rotate to the selected position to assist with determining which filter is in each position. NOTE: Communication Failure If the initial communication fails, check the cabling and communication settings. If another component on a functioning controller fails, check the settings (filter wheel number, light path, etc), on it, and make certain that component is actually installed. If everything else fails, you can disable the component at this point and move on to other items. That particular component may not be installed on your microscope. Component configuration D) Optional graphic interface: i. Setup: On the Configure tab select an upright or inverted microscope in Select Type, as appropriate. Restart Scope-Pro and go to the Configure tab, and you will be presented with a number of places to put each component on the graphical microscope. Selecting a position number for a component will put it on the graphic, which can be useful when setting up a particular configuration. See the Scope- Pro manuals for details. Graphical interface 1. Automated Microscope Configuration Updated 7/15/2009 13

Test: At this time you have Scope-Pro up and running. Spend a few moments testing various filters and shutters from the Scope-Pro interface to ensure that all components are configured correctly. E) Using the Scope Tab i. Attach Calibration/Camera Now that all the components have been selected and configured, you will need to assign associate the objective position on the nosepiece with the Lens List entry and the Spatial Calibration file. At the top right of the dialog choose Fill across and all the lenses will associate with the positions and calibrations. This is where it comes in handy to begin naming all the files with the same naming convention such as 10X UPlanFL, 40X APO, etc. Attach Calibrations Button Attach Calibrations Dialog Whenever an image is taken using Scope-Pro, the associated calibration is assigned to that image. All measurements will then use the calibration, allowing you to measure immediately in (for example) microns, rather than pixels. If you are taking images without Scope-Pro, you can set any reference calibration to be the system calibration, which then causes Image-Pro to automatically assign that calibration to newly captured images. If you are using pixel binning (2x2 or 4x4 grouping of pixels) for more sensitivity, Scope-Pro will derive a calibration matching those pixel sizes from the reference calibration, and assign it to the captured images. Test component movement Use the controls in the Scope tab to control the movements of each component and verify that each one is operational. 1. Automated Microscope Configuration Updated 7/15/2009 14

F) Save multiple configurations: i. Setup modes of microscopy: Your microscope is now ready to be configured for your desired modes of microscopy. Use the Scope-Pro Scope tab to move filters, objectives, and shutters for a particular observation mode. Save each mode as a (*.scp) settings file: Once you have defined a mode, Save that configuration. These modes should define a Scope-Pro settings file for each mode or dye that you wish to use, so that the microscope configurations are available for selection when configuring the AFA capture. Examples: a. Bright-field imaging: Using the transmitted lamp with neutral density filters, condenser and an open epifluorescence filter position. b. DAPI staining: Switch to the arc lamp or any illumination device and move in the appropriate excitation and emission filters. NOTE: Save all Configurations in SAME Folder All configuration files of a particular type must be in the same location. For example, all microscope.scp files must share the same directory for an AFA experiment. That directory is used to fill the drop-down list of configuration files in each column. Saving Scope-Pro Settings NOTE: Save Specific Components You can define.scp settings files that ignore the positions of selected components. For instance, you can define settings ignoring objective positions, so that you can reuse the same set of microscope modes with any objective if desired. You will determine this when using the Save from Scope-Pro. Click the button next to Select Components to save in Settings files on the bottom left of the Save dialog box and choose your preferred components from the list that appears (illustrated on the next page). 1. Automated Microscope Configuration Updated 7/15/2009 15

Items to be Saved In order to take full advantage of the accelerated acquisition capabilities of Scope-Pro and AFA, it is very important to save per-channel Scope-Pro configurations in which you select only the components that are needed to change the emission and/or excitation filtering for each channel that you will be acquiring. If you will be changing observation modes, for instance from brightfield to EPI fluorescence, you should also include the shutter state of the closed shutter on the unused light path. However, for these per-channel configurations, you must also make sure NOT to check the current Shutter component, meaning the active shutter for the observation mode. AFA will be driving the current shutter for each observation mode on a per-experiment basis, as determined by the AFA settings for Shuttering. i i Launch the mode: Once you have saved the modes, you can just select a configuration file from the combo box on the Scope tab and Scope-Pro will move all automated components to those positions. Optional use of the minimal Scope-Pro dialog: An additional way of launching the saved modes (*.scp files) is using the Mark and Minimal Dialog. If you Mark up to six of your configuration files using the Mark button in the upper left, the reduced Scope-Pro dialog will show these as buttons for ease of use. To reach the minimal dialog, click on the minimize button on the upper right of the Scope-Pro main dialog, the one containing the underscore _. Minimal Scope-Pro dialog 1. Automated Microscope Configuration Updated 7/15/2009 16

STEP 6 Stage Configuration/Alignment for Tiling A) Run Stage-Pro B) Set Area of Travel C) Optional lens/calibration attachment D) Square the camera with the stage E) Perform a Tiling Test F) Adjust stage Step Size A) Run Stage-Pro: i. Stage-Pro: Choose Acquire Stage-Pro. Stage-Pro is the Image-Pro component that controls X-Y stages. B) Set Area of Travel: When starting up, you will be asked to set the area of travel. The following dialog will appear: Area of Travel i. Use joystick: You have several options in terms of setting a new area of travel. The first is to use the stage joystick to mark the valid limits of movement, which allows you to hold all travel within, for example, the area of the cover slip. 1. Automated Microscope Configuration Updated 7/15/2009 17

i Use physical limits: The second is to use the physical limits of the stage, although that is likely to be considerably larger than your sample. Use current position as center: The last is to specify a region of travel around your current location. This third option, Use current position as center of Area of Travel, is the simplest when setting up your system. a. Area of Travel option: If you have previously defined a scan area, you can specify Park at shutdown on the Stage tab, and use the current position as the origin of the area of travel. Do not do this if you expect to be moving the stage independently of Image-Pro. iv. Run Area of Travel: After the choosing the proper options, click OK to run the routine. Once Stage-Pro is up and running, try moving the stage using the Stage tab buttons for Left, Right, Up, Down, and Focus Up and Down. Alternatively, go to the minimal dialog using the minimize button _ at the upper right, and use the arrow keys. If Stage-Pro opens with only the Configure visible, check the communication settings, in the Scope-Pro configure dialog. C) Optional lens/calibration association: i. If only two tabs are visible: If the Lenses and Calibrations were not associated with the objective positions in Scope-Pro then it is possible that only the Configure and Lens/Mag tabs are visible. If this is the case, click on the Check Lenses button on the Lens/Mag tab to associate calibrations with the various lenses. At this time you can just associate any calibration with the lens to obtain stage movement, and follow the steps below to create a correct calibration for the lens. Stage-Pro Stage Controller with 2 tabs Optional calibration of lenses from Stage-Pro: If the lenses show a (Not Calibrated) to the right of the lens name then you can calibrate the lenses following STEP 4c Part i. or or use the Calibrate Lens XY button on the Lens/Mag 1. Automated Microscope Configuration Updated 7/15/2009 18

tab to either Calibrate by stage movement or Import Calibration. PRODUCT NOTE a. Calibrate by stage movement: The method of deriving spatial calibrations from the movement of the stage derives the pixel sizes from how many pixels are moved for a given number of steps. This assumes that the stage moves an accurate distance per step, when averaged over the number of steps required to cross an image. Stage-Pro will ask you to move a recognizable object from one corner of the field of view to the other, clicking on it at each position. The relative positions of the object and the number of stage steps between will determine the pixel size with that objective. Calibration of the stage by travel is an alternate method of spatial calibration, which depends upon the gearing of the stage and the movement per step. Use this ONLY if you do not have a stage micrometer available. This calibration will generally allow consistent tiling, but may not agree very well with absolute standards of measurement. The stage micrometer method is a much more accurate measure of absolute sizes, while the stage movement method is simpler to use when establishing how many steps across a frame is. The micrometer method is highly recommended for accuracy to established standards and can be read about in the Spatial Calibration Product Note referenced in STEP 4c Part i. & Whenever an image is taken using Scope-Pro the associated calibration is assigned to that image. All measurements will then use the calibration, allowing you to measure immediately in (for example) microns, rather than pixels. If you are taking images without Scope-Pro, you can set any reference calibration to be the system calibration, which then causes Image-Pro to automatically assign that calibration to newly captured images. If you are using pixel binning (2x2 or 4x4 grouping of pixels) for more sensitivity, Scope-Pro will derive a calibration matching those pixel sizes from the reference calibration, and assign it to the captured images. The vast majority of digital cameras have square pixels. When the calibration results from stage movement are returned, any calculated aspect ratio is most likely due to a pixel or two difference between where you clicked on the object in the corners. If the calculated spatial calibration aspect ratio varies much from 1.0 (the mm/pixel in X and Y are different), edit the calculated calibration in Measure Calibration Spatial Calibration. A good starting place is to set the X and Y values to their average. NOTE: Approach from Same Direction When using stage motion to calibrate the stage motion, always approach your reference object from the same direction, such as from the upper left or lower right. Otherwise backlash in the stage gearing may throw off the positions. This backlash can introduce an error of a few percentage points into your spatial calibration, which is enough to cause visible artifacts where tiles overlay each other. Such an error will also be reflected in inaccurate spatial measurements. b. Import Calibration: If you have already created calibrations by following STEP 4c, then simply select the calibrations that were previously defined from the dropdown list for each lens in the Stage- Pro lens list. 1. Automated Microscope Configuration Updated 7/15/2009 19

i Close and Reopen Stage-Pro: Once the calibrations have been associated, close Stage-Pro and reopen to view all tabs. Stage-Pro Stage Controller with all tabs NOTE: The Importance of Camera Squaring One of the major advantages of a motorized X/Y stage is the ability to tile adjacent images together in order to view a large area at high resolution. In order to do this you need to establish the size of the pixels and the movement of the stage. You also need to square the camera with the stage, so that adjacent tiles differ only in X or Y position. AFA also has options to tile non-aligned images if you are not able to precisely square the stage to the camera. D) Square the camera with the stage: i. Run stage/camera rotation procedure: Stage-Pro Configure tab Square runs a stage/camera rotation procedure. You will be asked to move an object near the center of the image, and take three images with the camera rotated at increments of 120 degrees. This establishes the center of rotation for your camera and mount, which may or may not be at the center of the image. Establish alignment: Once a center of rotation is established, Stage-Pro will display crosshairs on a preview at that point. Move an object to the crosshairs. Then, using just X or just Y motion, move that object to the edge of the image and rotate the camera until the object is on the line again. Properly done, this will line the camera parallel with the stage, allowing you to tile images edge to edge with less than a pixel of error. NOTE: Post-Process Tiling in Image-Pro In Image-Pro 5.0 and later versions, you can overcome incorrect calibrations and stage alignment by using the Process Tiling function to tile overlapping images. This requires an edge overlap of 20-100 pixels with some features crossing the borders, which are cross- 1. Automated Microscope Configuration Updated 7/15/2009 20

correlated to find the relative positions of the image tiles. However, this requires a large amount of post-processing computation. If you can line up your hardware sufficiently, you can generate tiled images directly with no further processing. E) Perform a Tiling Test: i. Tiling Test: The following steps allow you to test the calibration and stage squaring by generating a small tiled image. 1) On the Lens/Mag tab, set the Lens/Magnification combo box to the current objective, the one which you have been calibrating. Stage-Pro generates frame positions for tiling based on the current objective, and if this is incorrect your positions will be off by the difference in magnification scales. 2) Go to the Stage-Pro Scan Area tab, and set up a scan with two tiles in X and Y and 0 overlap. Save this scan with an appropriate name, such as 2x2 40x Oil. 3) Start a preview, and find an object or objects that cover more than one frame. 4) Go to the Stage tab, and Set XY Origin Current Pos.. This sets the beginning of the scan to the current location. Alternatively, check Set cur XY Pos as Origin on the Acquire tab. 5) Check Enable Anti-Backlash. 6) At the bottom of the Stage tab, there is an edit item entitled Frame. Use the arrow keys to the right to move between frames 0-3. This should slide the stage to adjacent and abutting positions on the sample. 7) If this looks good, go to the Acquire tab. Uncheck everything on the left side of the tab for the test. On the right, check Set cur. XY as origin, and Tile Images. 8) Acquire a 2x2 tiled image. If the calibration is correct, the images should meet with no overlap or gap. If the stage squaring is correct, there should be no sideways shift between the images in the final frame. 9) Repeat the stage squaring and calibration if necessary. i. Repeat, adjust calibrations, or Adjust stage Step Size: If the frame borders do not abut each other either repeat the movement calibration, or directly adjust the calibration. To do that, examine pixels in adjacent frames, and scale the calibration to correct it. For example: If a feature appears 5 pixels from the edge in one image, and 7 pixels from the edge in its adjacent frame, with a 1024x1024 image, the pixel calibration is off by 12 pixels. To adjust it, scale the calibration pixels/unit by [1 + (12 / 1024)] = 1.0117. If the frame borders still do not abut each other go to STEP 6F, Adjust stage Step Size. There you can adjust what Stage-Pro uses as the movement per step, to make it agree with the micrometer measurement. F) Adjust stage Step Size: i. Method for Micrometer Calibrations ONLY: If you derived the pixel size calibration from a stage micrometer image, you can accurately set the stage step size using that calibration. This is the reverse of deriving the calibration from stage movement if you calculate the calibration from stage movement, DO NOT change the stage step size. 1. Automated Microscope Configuration Updated 7/15/2009 21

In the Configure tab of Stage-Pro, use the Step Calibration button. This runs through a procedure similar to deriving the calibration from stage movement, but adjusts the step size so that Stage-Pro can move in full frame increments when tiling. If the adjusted step size is more than a few percent off and in particular if it is off by a factor of two or more, check that the original step size was set properly for your hardware. Once the step size has been set, repeat the Tiling Test phase in STEP 6E above. G) Optional Sample Pattern selection: Stage-Pro supports creating sample patterns that represent a regular row/column grid layout of an area, such as the wells in a well-plate. You can create new sample patterns with arbitrary number of rows and columns, or select the existing patterns for 96- and 384-well well-plates. If you will be working with wellplates, please refer to the Stage-Pro documentation for more information. STEP 7 AFA Experiment Configuration A) AFA Configuration B) Define dimensions of the Experiment C) Setup each dimension D) Test Experiment E) Acquire Sets A) AFA Configuration: i. Run AFA: Select Advanced Advanced Acquisition AFA Wizard: When opened, the AFA wizard will check to see if the necessary prerequisites are installed and configured. For example the wizard will verify the presence of the following. 1) Objective positions defined in Scope-Pro 2) Saved Lens Files (*.ipl) 3) Calibrations assigned to Objective positions 4) Saved capture settings files (*.vpf) 5) Custom or default dye lists (*.ipd) 6) Saved Scope-Pro settings files (*.scp) 7) Stage-Pro Scan Area files (*.scn) 8) Sample Pattern files (*.ptn) For each Objective, if the stage has not been calibrated, or if there are no Scope-Pro configuration files, or if the camera is not working, AFA will inform you. When you choose Next > the wizard will step you through configuring lens files (describing lens magnifications, NA s, and immersion fluids), objective calibrations (pixel scale as seen through that objective), and the other items required to fully describe your microscope system. 1. Automated Microscope Configuration Updated 7/15/2009 22

When the wizard setup is complete select Finish to move to the tabbed AFA control panel. AFA Configuration Wizard NOTE: Wizard Errors and Warnings The AFA Configuration Wizard may issue warnings and/or errors. Errors indicate that a required item, such as a dye file, cannot be found. Warnings indicate that an optional item is missing. Errors must be corrected before acquiring with AFA, while a warning may indicate that certain functionality will not be available. For example, if you do not have Lens files, you cannot append lens information such as NA (numeric aperture) to your images, and SharpStack will require you to enter it again prior to a deconvolution. B) Define dimensions of the Experiment: i. Choose the dimensions of the Experiment: AFA allows capturing multidimensional data multiple channels, focal planes, scan areas, stage positions, time points, and so on. From the Experiment tab, select the dimensions in your current experiment. Choose from any combination of the following: 1) Multiple channels 2) Z Stack/Focus 3) Scan Areas 4) Multiple Sites using a Sample Pattern or User Defined Sites 5) Multiple Time Points Additional tabs will become available across the top for each dimension selected. NOTE: Scan Area and Multiple Sites Scan Areas enable scanning contiguous frames and tiling, while Multiple Sites allows you to either image multiple wells in a well plate or multiple user-defined positions on the slide. If you have Scan Areas enabled, they can be combined with Multiple Sites such that an X/Y scan of the defined area is acquired at each site. 1. Automated Microscope Configuration Updated 7/15/2009 23

i iv. Capture Order: Choose a capture order of Focus first, then Channels or Channels first, then Focus. The preferred order of capture (channels or focus first) depends on the speed of your hardware and the purpose of your experiment. If your Z is considerably faster than your filter wheels, or you are looking for the highest precision/least drift for Z-stack deconvolution, then focus first is appropriate. If you are performing co-localization or ratioing studies, or have a fast monochromator for light changes, channels first may be a better choice. Destination: The destination will allow you the option to capture to system memory (RAM) which is typically faster in writing, but fills up faster than a hard drive, or to save each image to your hard drive after it has been captured, which could potentially be slower in writing to disk for older hard drives, but will not fill up as quickly. Enter Experiment information: This is also the time to enter a Title, Experimenter, and a Description of the Experiment. AFA Experiment tab C) Setup each dimension: i. Channel: a. Select the [Add ] button below the Channels column in order to add more channels to the Experiment. Right-click on the channel to delete it. b. Select the Dye or Mode being used in each channel by selecting it from the dropdown in the Channels column. This list populates from the Dye List that was setup earlier. When you select the dye that you will be using for a channel, the dye specifies wavelengths and display color for composites, as well as providing a unique name for that channel in AFA. The Microscope Setup column will also default to using a Scope-Pro setting file with the same Dye name. The channel names must be unique within an AFA set. If for some reason you are using identical settings for the same channel in one experiment, create another dye file with a new name, such as DAPI_1, so that the channel names are unique. 1. Automated Microscope Configuration Updated 7/15/2009 24

c. Check the check-box in the Color column to acquire this channel in the current Experiment (this is checked by default when new channels are added). d. The background of the Color column illustrates the color used to tint the resultant images. If the dye color is incorrect, you can double-click the Color column to edit the dye. e. Choose the saved Microscope Setup that corresponds to the selected channel by selecting from the dropdown list in the Microscope Setup column. These settings will be the hardware component settings saved in Scope-Pro during STEP 4F. f. Chose the saved Capture Setup settings files from the Capture Setup column. These were previously saved in STEP 2D, but they can be either added or edited and then resaved by choosing the [Add/Edit capture setup ] option in the dropdown. Using different *.VPF files can allow using different gains or offsets for each channel if desired. This can be empty, but it is strongly recommended that you specify a *.VPF file so that you don t capture using the current camera settings, which may vary considerably depending on what has been done last with your system. g. Enter the desired Exposure time in the Exposure Column. It is recommended that exposure be controlled through the AFA Channel tab and NOT the Capture dialog for best performance. h. Check the check-box in the Pvw column if you wish to open the preview window while acquiring the current channel. i. Check the check-box in the Focus column is you wish to apply Focus settings to the current channel. j. Double-click the Focus Offset column to manually add any differences in Z Focus for individual channels or run the Focus Offset Wizard by choosing the Set Focus Offset button. k. The Capture options include Shuttering around different dimensions of the Experiment, Always AutoExposing, and setting Background Correction Images. NOTE: Save all Configurations in SAME Folder All configuration files of a particular type must be in the same location. For example, all microscope.scp files must share the same directory for an AFA experiment. That directory is used to fill the drop-down list of configuration files in each column. AFA Channel Tab 1. Automated Microscope Configuration Updated 7/15/2009 25

Focus: a. Choose from your Retain options which include: Single Image at Focus, Best (In-Focus) Image from Stack, Composite (EDF) from Stack, and Full Stack. Note that Single Image at Focus is the default if you wish to retain a full Z stack, you must change this selection to Full Stack. b. Choose whether to Focus while acquiring and at which intervals or to Manual Focus, Hardware auto-focus (which requires specific hardware), or Software autofocus (which requires no hardware). Select the Focus Now button to begin the chosen method. c. Set the Range of the Z depth you wish to acquire by choosing the number of planes and spacing. This will result in a Total Distance calculated the box below. d. Select the Set Range button to set the center of the Total Z Distance, or the top and bottom of the total Z depth being acquired. Use this dialog to view a Live Preview and step up and down in Z by the given spacing: Set Range Dialog e. Choose the Composite Type when retaining Composite Images from the Stack. AFA Focus Tab i Stage: There are two different types of multiple-site experiments supported in AFA. a. In many cases you will want to capture images from several different user-defined sites on your slide or well plate. For this type of acquisition, select the User Defined Sites option on the Experiment page: 1. Automated Microscope Configuration Updated 7/15/2009 26

1. Use the Site Map to add locations in X and Y by moving the stage to a new location and selecting the Add button. Add as many sites as you need. 2. To choose a pre-defined Scan Area from Stage-Pro choose from the dropdown list and check, Tile Images if needed. To create new Scan Areas open Scan Area in Stage Pro by selecting the Scan Setup button. 3. The alignment options in the right-hand pane allow you to use AFA with cameras proper aligned with the stage, but also provide two options (angle and calculated offsets) for overcoming misalignment. The 'Calculate' button will acquire a set of tile images for 'Camera at angle' or 'Derived offsets', calculate the realignment, and perform that while capturing and creating tiled images. AFA Stage Tab User Defined Sites b. You may wish to acquire several images in an X/Y scan of a particular area or areas. For this type of acquisition, select the Sample Pattern option on the Experiment page: 1. You will need to have selected the sample pattern that you will be using on the Sample Pattern page of Stage-Pro, verified the pattern layout, and set the pattern origin. 2. If you have Scan Areas selected for your experiment, you will be able to select the scan area definition to use, and whether to tile the acquired images. AFA Stage Tab Sample Pattern 1. Automated Microscope Configuration Updated 7/15/2009 27

iv. Time: a. Select the [Add ] button below the State column in order to add more Time Phases to the Experiment. Different phases support sampling at different rates. For example, you could acquire a great many sets of images at long intervals, and then switch to faster sampling when something interesting happens. b. If you would like to enable the Minimum Interval for the selected phase check the checkbox in the Min. Time. Column. c. Double-click in the Count column to multiply the number of cycles for each phase. d. Double-click in the Interval column to edit the Interval of Time for the selected Phase. e. To Duplicate a Phase, simply selected the Phase you wish to duplicate and select the Duplicate button, or Delete button to delete the Phase. AFA Time Tab v. Preview: a. The Preview tab gives you the flexibility of navigating the dimensions of your Experiment from the Channel, Z position, Scan Position, and the Site Position. b. The View All button will play through all dimensions to give an overview of your captured Experiment. 1. Automated Microscope Configuration Updated 7/15/2009 28

vi. Macros: AFA Preview Tab a. Select the Add button to browse for a previously created macro and select the Call Point you desire for it to be implemented at in the Experiment. Select Done to add the macro. Examples of macros that can be executed at different points in the acquisition are included in the DemoAFA macro script. AFA Macros Tab 1. Automated Microscope Configuration Updated 7/15/2009 29