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v6.0

by Joana Raquel Delgado Martins

Introduction

In this guide of the Center for Microscopy and Image Analysis you will learn how to set-up and perform a simple screen on the GE InCell Analyzer 2500 HS widefield fluorescence microscope.

  1. The InCell microscope and the control computer are always running. Therefore, there is no need to power-on and to power off this microscope or the computer. Log-in with your ZMB credentials. Start the "IN Cell Analyzer" software via the desktop icon.
    • The InCell microscope and the control computer are always running. Therefore, there is no need to power-on and to power off this microscope or the computer.

    • Log-in with your ZMB credentials.

    • Start the "IN Cell Analyzer" software via the desktop icon.

  2. The standard sample format for the InCell microscope is a 96 well plate. However there are also holders and adapters for slides, slide sized live cell chambers as well as petri dishes. In the cabinet close to the entrance door you can find the accessories including different sample holders.
    • The standard sample format for the InCell microscope is a 96 well plate. However there are also holders and adapters for slides, slide sized live cell chambers as well as petri dishes.

    • In the cabinet close to the entrance door you can find the accessories including different sample holders.

    • Either use a well plate or choose a suitable sample holder.

  3. In the software click on "Eject" to open the microscope door. Insert your plate or sample holder. Make sure that the orientation is correct (A1 should be on the top left corner).
    • In the software click on "Eject" to open the microscope door.

    • Insert your plate or sample holder.

    • Make sure that the orientation is correct (A1 should be on the top left corner).

    • Remove the lid of your sample if you are imaging in Transmission/DIC/Phase contrast mode.

    • In the software click on "Load".

  4. Select a plate/slide template. Select an objective.
    • Select a plate/slide template.

    • Select an objective.

    • The InCell microscope is optimized for imaging regular arranged specimens such as well-plates. When producing/imaging slides we recommend to precisely mount them using the available ZMB templates. For such arranged slides we also have suitable slide definitions on the microscope.

    • Adjusting or generating plate/slide templates and using the water immersion objective is explained in separate guides.

  5. Choose a suitable polychroic: BGOFR_1: Blue (390/18), Green (475/28), Orange (524/27), Far Red (632/22) BGOFR_2: Blue (390/18), Green (475/28), Red (575/25), Far Red (632/22)
    • Choose a suitable polychroic:

    • BGOFR_1: Blue (390/18), Green (475/28), Orange (524/27), Far Red (632/22)

    • BGOFR_2: Blue (390/18), Green (475/28), Red (575/25), Far Red (632/22)

    • Add the wanted channels by using the "+" icon.

    • Hoovering with the mouse over a channel opens a context information box with additional information.

  6. To move the stage, click in the center of a well or cover slip. Activate the "Laser Autofocus". The needed power level depends on the objective as well as the sample. Check the laser auto focus trace by clicking "Verify":
    • To move the stage, click in the center of a well or cover slip.

    • Activate the "Laser Autofocus". The needed power level depends on the objective as well as the sample.

    • Check the laser auto focus trace by clicking "Verify":

    • If the signal is saturated, reduce the auto focus laser power.

    • If the "Measured Parameters" deviate more than 10% of the "Protocal/Plate Parameters" adjust the plate parameters.

    • Click "Apply Measured Parameters" for doing that.

    • Click "Apply" in the "Plate Editor" for adjusting the plate parameters to the measured values.

    • If you execute "Verify" without beeing within a well/slide, it can happen that the objective presses the sample up. Since the plate is fixed within the instrument, you end up with a tilted plate and focusing is not possible anymore. To resolve such issues eject and reload your plate.

  7. Under most conditions the laser auto focus system of the InCell microscope is very fast and reliable. Therefore, an additional, much slower software auto focus step is usually not required.
    • Under most conditions the laser auto focus system of the InCell microscope is very fast and reliable. Therefore, an additional, much slower software auto focus step is usually not required.

    • Check the "Software Autofocus" checkmark to engage the software autofocus.

    • Specify a z axis search range.

    • Define the channel as well as the frequency for performing a software auto focus.

    • The InCell microscope uses the integrated image intensity as the optimization parameter for the software auto focus system. This means the software autofocus will focus on the brightest z plane.

  8. Usually there is a z offset between the found laser auto focus position and your brightest fluorescence signal. Click on "Auto Offset" to automatically measure the offset for the selected channels. When done, the software shows you the resulting offsets. They should be in a similar range for all channel. If a channel shows a quite different value then you can:
    • Usually there is a z offset between the found laser auto focus position and your brightest fluorescence signal.

    • Click on "Auto Offset" to automatically measure the offset for the selected channels.

    • When done, the software shows you the resulting offsets. They should be in a similar range for all channel. If a channel shows a quite different value then you can:

    • Move to another position and try again.

    • Check the signal in this channel, adjust the exposure (see next step) and try again.

    • Manually adjust the offset to the same value as in another channel.

    • The measured offsets will be set in the "AF Offset" window. They also can be manually adjusted there.

  9. On the InCell microscope the signal intensity can only be adjusted by the camera exposure time. Select the first channel and press "AF". A laser auto focus will be performed, the offset applied and an image generated.
    • On the InCell microscope the signal intensity can only be adjusted by the camera exposure time.

    • Select the first channel and press "AF". A laser auto focus will be performed, the offset applied and an image generated.

    • Check the image statistics. This microscope has a 16 Bit scmos camera so the dynamic range can be very high.

    • Depending on your statistics, adjust the exposure time.

    • Click on the camera symbol icon to make an image with the new exposure setting. When using the camera symbol no auto focus is performed.

    • Check again your image statistics.

    • Repeat this step for all channels.

  10. In the left menu select the "Fields" settings.
    • In the left menu select the "Fields" settings.

    • Here you can select the number of fields acquired in each well.

    • Next you can adjust the field placement, spacing as well as the acquisition strategy for the fields in a well.

  11. In the plate view you can select/unselect wells for acquisition.
    • In the plate view you can select/unselect wells for acquisition.

    • Shift and holding the left mouse button can be used to unselect wells.

    • Control and holding the left mouse button can be used to select wells.

    • This strategy also works for selecting/unselecting individual fields within wells.

  12. Before running a screen,  you have to save your modified acquisition protocol. Before running a screen,  you have to save your modified acquisition protocol.
    • Before running a screen, you have to save your modified acquisition protocol.

  13. You can give an additional experiment/plate ID which is used for naming the folders. Specify a storage location for your image data. We recommend saving on DataMover (X:). You can later on move your data to the ZMB data network drive via the DataMover (see also the corresponding guide). Click on "Scan" to start your scan.
    • You can give an additional experiment/plate ID which is used for naming the folders.

    • Specify a storage location for your image data. We recommend saving on DataMover (X:). You can later on move your data to the ZMB data network drive via the DataMover (see also the corresponding guide).

    • Click on "Scan" to start your scan.

  14. After a scan the review mode is automatically opened.
    • After a scan the review mode is automatically opened.

    • For some imaging modalities such as when using deconvolution, it is necessary to reopen the dataset after acquisition and automatic processing.

    • Here you can change the displayed parameters/preview images for the plate overview.

    • Select a field for its data being displayed.

    • Explore the channels by adjusting the image intensities.

    • We recommend to use the review mode to have a quick look at the acquired data as well as for the sometimes helpful visualization of plate parameters (e.g. z focus position).

  15. "Eject" and remove your sample. Click again on "Load" to close the microscope door. Close the InCell software.
    • "Eject" and remove your sample. Click again on "Load" to close the microscope door.

    • Close the InCell software.

    • "Sign out" from the computer.

    • Do not shut down the computer and do not turn off the microscope.

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