This module enables you to acquire multi-channel images. The Smart Setup functionality delivers proposals for combinations of dyes or contrast methods based on the available hardware.
This module enables you to acquire multi-channel images. The Smart Setup functionality delivers proposals for combinations of dyes or contrast methods based on the available hardware.
In Smart Setup you configure channels. Select the fluorescent dyes and/or contrast methods that you want to include from a large dye database. Smart Setup takes the configuration of your microscope hardware and the properties of the selected dyes into account. Based on this information, it makes one or more suggestions for acquisition.
Note that Smart Setup tries to configure the components of your system for the acquisition of multi-channel images.
To open Smart Setup, select Free Mode > 2D Multi-Channel Acquisition workbench > Channels tool > Open Smart Setup. You can also open it in Job Mode.
Operator workflows are designed by Supervisors. In the following you see the operator workflow Counting Cells Live, which allows to count fluorescently-labeled nuclei that can serve as a proxy for the cell number.
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Tasks |
Description |
To Do |
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Form |
The input form ZEISS Cells is provided. The form is displayed in the report. |
The default form is displayed. To change the form, in the Form Selection tool, select the form, see Form Selection Tool. Fill in relevant data according to your sample. |
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2D Multi-Channel Acquisition |
Acquire images. |
To acquire multi-channel images, open Smart Setup and add dyes and contrast methods according to your sample. The first channel should be the fluorescently-labeled nuclei. Smart Setup provides proposals setting the light source, camera and filter setting, see Configuring Multi-Channel Acquisition with Smart Setup. Adjust the acquisition parameters, e.g. measure the exposure time, and then take a snap. |
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Image Segmentation |
Generate one mask for all nuclei per cell. |
Generate masks for nuclei, which are then counted, see Automatic Segmentation. To separate neighboring nuclei use a separation tool, e.g. Watersheds. If a large part of the image is selected initially, use background subtraction. |
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Region Filter |
Results are refined based on area and circularity of the objects. |
To refine the selection to limit the analysis to one object or nucleus per cell, use region filters for area and circularity, see Region Filter. |
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Measurement Data |
Check the results based on the previously conducted image analysis. The results are displayed in a table. |
To select an object, click a value in the table, and to check the size of a certain object, click the object in the Center Screen Area. For more information, see Measurement Data Tool. |
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Report |
Creates a report document, see Reports. You can print the report. The report contains the following information:
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For your documentation, you create a report. |
This workbench enables you to acquire 2D multi-channel images.
With this tool you configure the color mode for the selected camera. The tool is only available for color cameras.
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Parameter |
Description |
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Color Mode |
Switches to the desired color mode. |
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– |
RGB |
Based on the RGB (Red-Green-Blue) color model. Transmits the image data of a color camera unchanged. This corresponds to the standard operating mode of a color camera. |
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– |
B/W |
Treats the image data of the color channels as grayscale. The data of related color channels are averaged. The saturation of the camera appears reduced as a result. This process does not change the spectral properties of a color camera. The image information of the color sensor still undergoes color interpolation. An infrared filter also restricts the spectral sensitivity of the color camera compared to the spectral sensitivity of a genuine black and white camera. Exposure time measurements with the auto exposure button use the B/W mode for calculations and therefore individual pixels of the color camera might be oversaturated, even if the histogram displaying the black and white calculation does not indicate overexposure. One time intensity measurements switch to the RGB mode for exposure time determination. |
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1 |
+ Dye & Contrast Methods Opens the Add Dye or Contrasting Method dialog, see Configuring Multi-Channel Acquisition with Smart Setup. |
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2 |
Table with selected channels You can add up to 8 fluorescence channels and contrast methods. The added dyes or the contrast methods are shown in the list. If necessary, change the color by clicking the arrow and select a user defined color. |
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3 |
Buttons to optimize Acquisition Parameters Click on a button to optimize image acquisition regarding particular requirements and to influence parameters like the camera, detector, and lighting settings, see Buttons to Optimize Acquisition Parameters. |
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4 |
Proposals for Specific Filter Settings Displays various Smart Setup proposals for further experiment settings. See Graphical Display of Proposals and Smart Setup Proposals. |
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5 |
Show Excitation checkbox Activated: Shows the excitation spectrum of the selected dyes in the graphical display. |
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6 |
Show Emission checkbox Activated: Shows the emission spectrum of the selected dyes in the graphical display. |
The bars in the graphs only show relative values. The actual strength of the emission signal and the crosstalk in the image can deviate substantially from this estimate, as Smart Setup has no knowledge of the strength with which the sample has been dyed with the individual dye components.
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1 |
Emission Signal |
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2 |
Speed |
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3 |
Crosstalk |
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4 |
Tracks display Only visible if the Show Excitation and/or Show Emission checkboxes are activated. The various tracks are labeled with T1, T2 etc. The white lines show the excitation and emission spectra of the dyes schematically. The spectra are filled in color in the places that will be acquired by the acquisition configuration suggested by Smart Setup. Transmitted light channels are displayed as a white field. |
This workbench enables you to load and execute experiments based on the contained settings.
Result document not split
If you have designed an experiment using the EDF (motorized focus) workbench with the Z-stack option, you receive a single result document containing two phases: EDF and Z-stack.
Stitching is not executed
The Tiles Stitching tool is not executed in the experiment. To stitch the experiment result, in the Image Processing workbench, use the Stitching tool, see Stitching Tool.
This workbench allows the combination of different acquisition dimension, for example, multi-channel acquisition with tiles acquisition.
By default, the first channel in the channels list of the Channels tool is used as the reference channel for focus actions or stitching during acquisition. If two or more channels are created, you can assign the reference to one of these channels. It is possible to assign the reference channel to a channel that is not active for acquisition. For example, a brightfield channel is the reference, but is not to be imaged instead of being used only for SWAF (Software Autofocus) runs. In this case, activate the Activate Stitching During Acquisition for New Experiments option, see Tiles & Positions Section.