Acquiring Multi-Channel Images with Cameras
In the following chapters you will learn how to set-up and run multi-channel experiments quick and easy.
MicroToolBox (MTB)
Make sure that you work with a fully motorized microscope system. In advance all microscope components (e.g. objectives, filters, etc.) must be configured correctly in the MicroToolBox (MTB) software.
There are two variants for setting up multi-channel experiments. The first variant uses Smart Setup, while the second variant uses the Channels tool. Both variants have similarities and differences, which are presented in the following overview:
Commonalities
- Fluorescent dyes and transmitted light techniques can be selected from a database.
- Hardware settings for motorized microscopes, which take the properties of the selected dye and the available microscope hardware into account, can be created automatically.
- Bases for experiments can be created using both variants and experienced users can optimize settings further.
Differences
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Smart Setup |
Channels tool |
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A maximum of 4 camera channels, 8 confocal channels and transmitted light channels are available |
No restriction on the number and type of channels |
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Offers up to 3 proposals of variants of the experiment (depending on the selected combination of dyes and available hardware) |
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Offers more optimization of experiment settings by using the Motif buttons |
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Graphic overview of the expected signal strength for the selected dyes |
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Graphic overview of the expected spectral crosstalk with the selected dye combinations |
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Display of the excitation and emission spectra of the selected dyes |
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Channels can be configured for dyes that are not supported (or not supported sufficiently well) by the available hardware |
Variant 1: Smart Setup
If you see the error message Smart Setup calculation failed, it was not possible for Smart Setup to calculate any proposal. This may be because the filters and light sources available on the system do not allow an image of the dye to be acquired with a good signal strength or with little crosstalk. The channel for this dye or the contrast method cannot therefore be created. In this case, try selecting another, similar dye.
Should the error message be displayed for all dyes that you select, this may be due to one of the following causes:
- no light source has been configured or the light source is switched off.
- no camera has been configured on the system, the camera is not connected or (on some models) has been switched off.
- On the Acquisition tab, click Smart Setup.
- The Smart Setup dialog opens.
- Select the WF button on top of the dialog.
- To add a channel, click + Dyes & Contrast Methods.
- The Add Dye or Contrast Method dialog opens.
- Double-click desired dye or contrast method.
- The dye is then adopted directly into the experiment. You have added a channel to your experiment. To add further channels, repeat the previous step.
- To return to Smart Setup, close the dialog.
- You will now see a graphic overview in the Proposals section. This displays the spectra of the dyes, the expected signal strengths per dye and the spectral crosstalk schematically.
- Depending on which dye you have selected and the microscope hardware available, up to three different proposals (Best Signal, Fastest, Best Compromise) are displayed. These differ in terms of signal strength, crosstalk and speed. Select the proposal that best meets the needs of your experiment.
- To select a proposal (if there is more than one) for all active configured channels, activate the proposal.
- To optimize experiment settings additionally, click on a Motif button. The Automatic button is set as default.
- With the Motif buttons you can optimize acquisition parameters and camera settings automatically, either for a high quality image (Quality) or a faster acquisition but reduced image quality (Speed). Find a more detailed description of Motif buttons in Smart Setup dialog.
- To optimize experiment settings, adopt the suggestion, and leave Smart Setup, click OK.
- The added channels are adopted automatically into the Channels tool.
- Click Set Exposure at the top of the Acquisition tab.
- The exposure time is now measured for all three channels one after the other. This is adopted into the settings for the channels. Following the measurement of the exposure time, the multi-channel image is acquired automatically and displayed in the Center Screen Area.
- To save the experiment together with all the settings, go to the in the Experiment Manager and click
. - Options are displayed.
- Select the Save entry in the dropdown list.
- You have set up the multichannel experiment using Smart Setup, executed it and then saved the configuration. This means that you can repeat the experiment as often as you like using the same settings.
Variant 2: Channels Tool
This workflow does not work with LSM systems.
- Open the Channels tool in the Acquisition Parameter group.
- From the drop-down list, select the channel.
- The Add Dye or Contrast Method dialog opens.
- Select the desired dye or contrast method. You can search for a dye by entering its name (or starting letter) in the Search input field.
- Double-click the entry.
- The channel is added to your experiment. To add more channels, repeat the last 2 steps.
- Close the dialog.
- The added channels are displayed in the Channels tool.
- Click Set Exposure at the top of the Acquisition tab.
- The exposure time is now measured for all active configured channels one after the other. It will be also adopted into the settings for the channels. To set the exposure time channel specific, use the Set Exposure button in the channel settings of the channels tool.
- To save the experiment together with all the settings, go to the Experiment Manager and click .
- Options are displayed.
- Select the Save entry in the dropdown list.
- You have set up the multichannel experiment using the Channels tool, executed it and then saved the configuration. This means that you can repeat the experiment as often as you like using the same settings.