Multi-Channel Acquisition

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.

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.

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.

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.

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.

Report

Creates a report document, see Reports. You can print the report.

The report contains the following information:

• Form
• Counts and counts/mm² for each image and their mean
• Display of all images with the detected nuclei

For your documentation, you create a report.

Open Smart Setup

Opens the Smart Setup dialog, see Smart Setup.

Measure Exposure

Starts an exposure time measurement for all currently active channels.

List of channels

Displays a list of channels you have selected from the Add Dye or Contrasting Method dialog, see Configuring Multi-Channel Acquisition with Smart Setup.

If you change the selection of a channel, the live image is switched accordingly. If you modify light source or channel-specific camera settings, e.g. exposure time, the modification is immediately updated in the live acquisition.

Moves the selected channels up or down.

Deletes the selected channels.

Ref.

Sets the selected channel as reference channel for focus actions or stitching during acquisition.

Lightsource

Displays a list with light source settings depending on the used light source. Adjusting the parameters of the light sources is possible without saving it to the hardware settings.

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HXP 120/200 V

Adjusts the intensity in discrete steps with the arrow keys or the slider.

• Intensity slider
• Sets the intensity of the sample illumination. 1.0: Illumination off; 100.0: Maximum illumination.
• Adjusts the values in % with the arrow keys.

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TL Lamp

For Brightfield channel. LEDs refer to %.

• Manual
• Use your individual settings.
• 3200K
• Adjusts the intensity to a color temperature of 3200K comparable to working with a halogen bulb.
• Standby
• Sets the intensity to 0% or 0V.
• Intensity slider
• Sets the intensity of the sample illumination. 0.0: Illumination off; 12.3: Maximum illumination.
• Adjust the values in % or V with the arrow keys.

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Colibri 7/5/3

Up to 7 LEDs are displayed depending on the hardware. The number of available LEDs depends on the used Colibri version.

The following Colbri 7 versions with the corresponding LEDs are available:

• RGB-UV
  630, 555, 475, 385
• RYB-UV
  630, 590, 475, 385
• R[G/Y]B-UV
  630, 590, 555, 475, 385
• R[G/Y]CBV-UV
  630, 590, 555, 511, 475, 430, 385
• FR-R[G/Y]BV-UV
  735, 630, 590, 555, 475, 430, 385

The Colbri 5 version RGB-UV is with the corresponding 385, 475, 555, 630 LEDs available.

It is possible to activate all LEDs simultaneously which means that either 555 or 590 nm can be turned on, if both are available.

• Intensity slider
• Sets the intensity of the sample illumination.
• Adjust the values in % with the arrow keys.
• Excitation Filter of LED 567 button
• The 567 nm LED is combined with an excitation filter, which determines whether 555 nm (yellow) or 590 nm (green) excitation is used in.

The following Colibri 3 versions with the corresponding LEDs are available:

• UV-BGR
  385, 470, 555, 625
• UV-BG
  385, 470, 555
• BGR
  470, 555

Deactivate all LEDs/Reactivate LEDs

Only visible with Colibri 7/5 lightsource.

De- or reactivates all active LEDs to the status when the deactivate button was last used.

Exposure

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Auto Exposure

The exposure time is calculated automatically every time an image is acquired. For color cameras of the Axiocam series in b/w mode, the exposure time is determined in the b/w mode. This might lead to overexposure of individual pixels. The histogram displays the b/w mode and therefore does not reflect overexposure of individual pixels, which can be detected in the RGB mode with the same exposure time.

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Set Exposure

Starts a one time measurement of the exposure time, which is then used for all subsequent images. Deactivates the Auto Exposure checkbox. For color cameras of the Axiocam series in b/w mode, the exposure time is determined in the RGB mode to avoid unintended overexposure of pixels. Depending on the light source, this might lead to less usage of the histogram, i.e. dimmer images, than specified by the intensity value. To compensate for under- or overexposure, the intensity value can be adjusted.

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Time

Adjusts the exposure time for the camera. Use the slider or spin box.

Select the unit of time from the drop down list:

• ms
• s
• μs

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Intensity

Defines the range of the histogram that is used for exposure determination and therefore compensates for underexposure or overexposure.

• 5% - 100%: Darkens the image, compensates for overexposure.
• 100% - 200%: Brightens the image, compensates for underexposure.

Use the slider or spin box to adjust the value.

Shading

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Correction

Uses the calculated shading correction for this channel.

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Define

Automatically calculates the shading correction, see Applying the Shading Correction.

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Global

Performs an objective-specific shading correction.

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Specific

Default method.

Performs channel-specific shading correction. The fluorescence filter block used is saved with the shading file. If the fluorescence channel is changed, a previously created reference image is also loaded.

EM Gain

Only visible for EMCCD camera models. Sets the EM gain value.

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.

Select from

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Archive

Selects the archive as the source and opens the Open Template dialog to choose the experiment file.

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File

Selects the file system as the source and opens the file browser to choose the experiment file.

Not available in Job Mode.

Experiment

Opens the file browser or the Open Template dialog to select the desired experiment file.