Utilities

3D Processing

This option is only available for images with 5 or more z-positions.

If activated, this option improves the resolution in axial and lateral direction. The data set needs to have at least 5 z-sections acquired with an optimal step size. 3D Processing is slower than 2D Processing. For 3D Processing, the whole z-stack (single channel and time point) needs to fit into the physical memory.

2D SR Processing

This function is available for 2D images only. It enhances the 2D resolution.

Note this only results in increased superresolution when images are acquired with optimal settings and sufficient signal.

Auto Filter

If activated, a suitable Super Resolution parameter for the Airyscan processing is automatically determined for the selected data set.

To manually adjust the Super Resolution parameter, deactivate the checkbox. Then determine suitable values by using the corresponding function in the Airyscan viewer in the Airyscan view. Note that the preview is only suitable for 2D Airyscan processing. A preview for 3D Airyscan processing is not available. For adjusting 3D processing parameters, you should first process your data set once using the Auto Filter and then check the value that was actually applied by the Airyscan processing function. This value is stored in the metadata of the processed image and can be accessed using the Info view.

Note: High strength might look attractive at some images, Z planes or color channels, but other filtering artefacts might occur which appear like small rings in the image. Also, the results will become very sharp, but grainy. So carefully check your image data in order to avoid such artefacts.

Adjust per Channel

Only visible, when the Auto Filter is deactivated.

Only available for images with two or more Airyscan channels.

If activated, you can manually set channel-specific Airyscan processing parameters.

Strength

Use this option for an increased (high) or decreased (low) strength of the automatically assigned filter value. This is especially useful for 3D processing, as the 2D preview of the processing filter value in the Airyscan viewer does not allow to conclude the result after a 3D data processing.

The increment of this parameter is ± 0.4 compared to the standard auto Airyscan processing. This setting is not available when manual processing strength is selected.

Display Mode

ApoTome images are acquired as raw data. The Display Mode sets how the image is calculated and displayed.

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Optical sectioning

The displayed output image is calculated/rendered by eliminating the excitation and emission light that originates in regions outside of the focal plane.

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Conventional fluorescence

The displayed output image is calculated/rendered like a conventional fluorescence image.

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Raw data

Displays the raw data as output image and disables all other parameters of the function.

Correction

Applies stripe artifact correction to the resulting image. It attempts to remove stripe artifacts which may be caused by bleaching of the sample during acquisition or by slight deviations in the grid phase position.

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No correction

No correction is applied to the image.

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Local Bleaching

Corrects the bleaching for each pixel individually (default setting). This is usually the best method.

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Global Bleaching

Corrects bleaching by means of global bleaching correction, which is applied equally to the entire image.

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Phase Errors

Corrects phase errors in the image without additional bleaching correction.

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Phase Errors and Global Bleaching

Corrects phase errors in the image with additional global bleaching.

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Phase Errors and Local Bleaching

Corrects phase errors in the image with additional local bleaching.

Fourier Filter

The Fourier filter attempts to remove residual stripes.

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Off

Uses no Fourier filter to remove stripes.

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Weak

Uses a weak Fourier filter to remove stripes.

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Medium

Uses a medium Fourier filter to remove stripes.

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Strong

Uses a strong Fourier filter to remove stripes.

Normalization

Here you can select how the gray/color values that exceed or fall short of the value range should be dealt with. If you use this method with Direct Processing, only the Clip method is available and preselected.

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Clip

Automatically sets the gray levels that exceed or fall short of the predefined gray value range to the lowest or highest gray value (black or white). The effect corresponds to underexposure or overexposure. In certain circumstances some information may therefore be lost.

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Automatic

Normalizes the gray values automatically to the available gray value range.

Columns

Define the measurement parameters for classification by entering the column numbers freely, e.g. 1,3,5, or 1-6 or 1,3-7,8.
Clicking on the button to open the Select columns dialog. Here the column names of the data can be activated or deactivated by clicking on the relevant checkbox.

Class Boundaries

Select here, how you want the class boundaries of the calculated histogram to be determined.

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>=,…,<

A numerical value falls into the histogram class if it is greater than or equal to the lower class boundary and less than the upper class boundary.

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>,…,=<

A numerical value falls into the histogram class if it is greater than the lower class boundary and less than or equal to the upper class boundary.

Automatic Classification

Activated: The class boundaries are calculated automatically from the data. The value range from the lowest to the highest data value is divided into as many classes of equal width as you have set in the Class Number input field.

Example:

Minimum value is 0
Maximum value is 10000
Range is 10000 units
Class Count is 4
Then the class boundaries are as follows:

Class 1: 0 .. 2500
Class 2: 2501 .. 5000
Class 3: 5001 .. 7500
Class 4: 7501 .. 10000

Logarithmic

Only active, if the Automatic Classification checkbox is activated.

Activated: The class boundaries are scaled logarithmic.

Example:

Minimum value is 0
Maximum value is 10000
Range is 10000 units
Number of classes is 4
Then the class boundaries are as follows:

Class 1: 0 .. 10
Class 2: 11 .. 100
Class 3: 101 .. 1000
Class 4: 1001 .. 10000

Class Count

Specifies the number of classes that shall be created.

Display Mode

Select here, how you want the values of the histogram to be calculated.

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Count

The histogram indicates how many data sets fall into the relevant class, it contains the frequency of the values in the class concerned.

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Count Cumulative

The histogram cumulates the counts of values in each class. Class 1 contains the number of values for class 1, class 2 contains the sum of the values from class 1 and class 2, class 3 contains the sum of the values from class 2 and class 3, etc.

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Percentage

The histogram indicates what percentage of the data sets fall into the relevant class, it therefore contains the percentage share of the values in the class concerned.

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Percentage Cumulative

The histogram cumulates the percentage of the count of values in each class. Class 1 contains the percentage for class 1, class 2 contains the sum of the percentages from class 1 and class 2, class 3 contains the sum of the percentages from class 2 and class 3, etc.
The last class therefore contains 100%.

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Sum

The histogram contains the sum of the numerical values of the data sets that fall into the relevant class, the values of the data sets that fall into the class concerned are therefore added together.

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Sum Cumulative

The histogram cumulates the sums of the values in each class. Class 1 contains the sum of the numerical values from class 1, class 2 contains the sum of the numerical values from class 1 and class 2, class 3 contains the sum of the numerical values from class 2 and class 3, etc.
The last class therefore contains the sum of all individual values.

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Percentage Sum

The histogram indicates the percentage share of the total numerical values in the relevant class.

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Percentage Sum Cumulative

The histogram cumulates the percentage of the sums of values of all data points which belong to the class. Class 1 contains the percentage of the total numerical values from class 1, class 2 contains the sum of the percentages of the total numerical values from class 1 and class 2, class 3 contains the sum of the percentages of the total numerical values from class 2 and class 3, etc.
The last class therefore contains 100%.

Pixel Format

Select the desired pixel format from the dropdown list.

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8 Bit B/W

The output image is a monochrome image, the whole-number gray values of which can lie in the range from 0 to 255.

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16 Bit B/W

The output image is a monochrome image, the whole-number gray values of which can lie in the range from 0 to 65535.

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32 Bit B/W Float

The output image is a monochrome image with real numbers as pixel values.

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2x32 Bit Complex

The output image is a monochrome image with complex numbers (real part and imaginary part) as pixel values. Such images are generally created by means of transformation into the Fourier space.

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24 Bit RGB

The output image is a color image, the whole-number color values of which in the red, green, and blue channels can lie in the range from 0 to 255.

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48 Bit RGB

The output image is a color image, the whole-number color values of which in the red, green, and blue channels can lie in the range from 0 to 65535.

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2x32 Bit RGB Float

The output image is a color image with real numbers as color values in the red, green and blue channels.

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3x64 Bit RGB Complex

The output image is a color image with complex numbers (real part and imaginary part) in the red, green and blue channels. Such images are generally created by means of transformation into the Fourier space.

Output Pixel type

Here you choose the desired output image format, e.g. 24 Bit RGB.

Preserve Scaled Size

Has an effect if the size of the target image is different from the size of the source image:

• Activated: The copied annotations remain unchanged.
• Deactivated: The copied annotations are scaled according to the possible image size difference.

Cross Correlation

If activated, you can correlate two images with each other. Note that the second input image needs to have the same dimensionality and size.

X, Y

Correlates the signal in the X or Y direction.

Z

Correlates the signal in Z. Only available for data sets containing Z-sections.

Time

Correlates the signal in time. Only available for time series data sets.

Pattern

Select the desired pattern for the gray scale image here.

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Uniform

All pixels have an identical gray/color value.

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2D Gray Scale Vertical

The gray scale runs from top to bottom, starting with the gray value selected in parameter Min. Gray Value.

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2D Gray Scale Horizontal

The gray scale runs from left to right, starting with the gray value selected in parameter Min. Gray Value.

Width

Set the desired width of the output image in pixels using the slider or the input field.

Height

Set the desired height of the output image in pixels using the slider or the input field.

Min. Gray Value

Set the minimum gray value of the gray scale using the slider or input field.

Max. Gray Value

Set the maximum gray value of the gray scale using the slider or input field.

Pixel Type

Select the desired pixel type here.

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8 Bit B/W

The output image is a monochrome image whose integer gray values can be in the range of 0 to 255.

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16 Bit B/W

The output image is a monochrome image whose integer gray values can be in the range of 0 to 65535.

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24 Bit RGB

The output image is a color image whose integer color values in the channels Red, Green, Blue can be in the range of 0 to 255.

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48 Bit RGB

The output image is a color image, with integer color values in the color channels Red, Green, Blue can be in the range of 0 to 65535.

Channels

Selects which channels of the input image are used. All channels are selected by default. To deselect a channel, click on the respective channel button.

Z-Position, Time, Block, Scene, Phase

Here you can select which parts of the input image you want to use for the resulting image.

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Extract All

If selected, all parts of the corresponding image are extracted.

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Extract Single

If selected, you can select a single image to be extracted.

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Extract Range

If selected, you can select a certain range of images to be extracted.

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Extract Multiple

If selected, you can select several continuous ranges and individual sections.

Enter one or more sections that you want to select in the input field. To do this, enter the first section, followed by a minus sign, and then the last section. If you want to define an interval, after the last section enter a colon and then the interval. The entry "2-10:2" means that every second section is selected from section 2 to section 10.

Enter a comma after the first section if you want to define another section. You can also select individual sections separated by commas. By entering "2-10:2,14-18,20,23", you select every second section from section 2 to section 10, followed by sections 14 to 18, as well as sections 20 and 23.

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Get current position

Adopts the position from the current display in the image area.

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Interval

Activated: Interval mode is active. The Interval spin box/input field appears.

Enter the desired interval here. E.g. if you enter the value 2 only every 2nd value from the range is considered.

Region

Here you can select if you want to use the entire image or just a region (ROI) of the input image.

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Full

Select this option to use the full image for the new image.

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Rectangle region (ROI)

Select this option to draw in a rectangle region of interest, which will be used for creating a new image.

If a rectangle region was drawn in, you can see and change its coordinates by editing the X/Y/W/H input fields.

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Keep tiles

Only has an effect, if a region (ROI) is defined.

Activated: Extracts the drawn in region including the complete tiles. This setting is recommended when you want to apply DCV processing functions on the resulting image.

Split Dimension

Depends on the loaded image.

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None

The image is not split by any dimension. Only the ranges of the different dimensions defined below will be extracted for the new image.

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Channels (or: Time, Scenes etc.)

Here you can select the dimension for splitting the data set. A new image document opens in ZEN for each element of the selected dimension. The available options depend on the selected image. If your input image contains two channels, split dimension creates two output images, for each channel one.

Channels

Here you can select which channels of the input image you want to be used. All channels are selected by default. To deselect a channel, click on the respective channel button.

Time (or: Z-Position, Rotation, Scene, Illumination, Acquisition block, Mosaic tile, Phase, View)

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Extract All

Activated: All elements of the corresponding dimension are extracted.

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Extract Single

Activated: You can select a single element to be extracted.

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Extract Range

Activated: You can select a certain range of elements to be extracted.

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Extract Multiple

Activated: You can select several continuous ranges and individual sections.

Enter one or more sections that you want to select in the input field. To do this, enter the first section, followed by a minus sign, and then the last section. If you want to define an interval, after the last section enter a colon and then the interval. The entry "2-10:2" means that every second section is selected from section 2 to section 10.

Enter a comma after the first section if you want to define another section. You can also select individual sections separated by commas. By entering "2-10:2,14-18,20,23", you select every second section from section 2 to section10, followed by sections 14 to 18, as well as sections 20 and 23.

Region

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Full

Takes the full region into account.

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Rectangle Region

Takes the rectangle into account that you can draw in the 2D view. After drawing you can modify X, Y coordinates as well as width (W) and height (H) manually.

Keep tiles

Has only an effect, if a region (ROI) is defined.

Activated: Extracts the drawn in region including the complete tiles. This setting is recommended when you want to apply DCV processing functions on the resulting image.

Deactivated: Drawn ROI will cut through mosaic tiles.

Propagate ROI

Has only an effect, if a region (ROI) is defined in multi-scene images.

Activated: Applies the defined region (ROI) to all scenes.

Split Dimension

Depends on the loaded image.

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None

The image is not split by any dimension. Only the ranges of the different dimensions defined below will be extracted for the new image.

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Channels (or: Time, Scenes, etc.)

Here you can select the dimension for splitting the data set. A new file will be created in the target folder for each element of the selected dimension. The available options depend on the selected image. If your input image contains two channels, split dimension creates two output images for each channel.

Channels

Here you can select which channels of the input image you want to be used. All channels are selected by default. To deselect a channel, click on the respective channel button.

Time (or: Z-Position, Rotation, Scene, Illumination, Acquisition block, Mosaic tile, Phase, View)

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Extract All

Activated: All elements of the corresponding dimension are extracted.

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Extract Single

Activated: You can select a single element to be extracted.

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Extract Range

Activated: You can select a certain range of elements to be extracted.

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Extract Multiple

Activated: You can select several continuous ranges and individual sections.

Enter one or more sections that you want to select in the input field. To do this, enter the first section, followed by a minus sign, and then the last section. If you want to define an interval, after the last section enter a colon and then the interval. The entry "2-10:2" means that every second section is selected from section 2 to section 10.

Enter a comma after the first section if you want to define another section. You can also select individual sections separated by commas. By entering "2-10:2,14-18,20,23", you select every second section from section 2 to section10, followed by sections 14 to 18, as well as sections 20 and 23.

Region

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Full

Takes the full region into account.

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Rectangle Region

Takes the rectangle into account that you can draw in the 2D view. After drawing you can modify X, Y coordinates as well as width (W) and height (H) manually.

Keep tiles

Has only an effect, if a region (ROI) is defined.

Activated: Extracts the drawn in region including the complete tiles. This setting is recommended when you want to apply DCV processing functions on the resulting image.

Deactivated: Drawn ROI will cut through mosaic tiles.

Propagate ROI

Has only an effect, if a region (ROI) is defined in multi-scene images.

Activated: Applies the defined region (ROI) to all scenes.

Target Folder

Selects the folder on the disk where the images are to be saved.

Overwrite existing files

Overwrites image files.

Compression

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Original

The output image has the same compression as the original image.

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Compression

JPEG XR compression is applied to the output images.

Defaults

Sets the values back to default, if they have been changed.

Z-Stack Correction

Activated: Performs background correction of the Z-stack before the processing.

Circular Average

Activated: Forces a PSF with lateral symmetry. This option should not usually be activated as lateral asymmetries correspond better to the real situation. Circular averaging is only recommended when a measured PSF is used with the Fast Iterative method.

Threshold Cropping

Activated: The PSF is restricted to gray value ranges up to 0.25% of the brightest voxel present. If the value is reduced or the option is deactivated, the PSF may be larger. This increases the calculation time. However, it is also possible to achieve slightly better results in this case. This option is deactivated by default.

Threshold

By using this slider and input field, you can set the percentage from which the PSF is clipped if the Volume Clipping option has been selected.

Iterative Restoration

Activated: If Z-stack images of beads with diameters greater than the microscope's resolution limit are used to generate the PSF, this option must be selected. The bead diameter used can be entered using the slider and input field.

In Place

Activated: The changes are applied to the original image and no new image document is opened as output.
Deactivated: The way the changes are applied is defined by the Output tool.

Subset

Contains the description of how the input image was created as a subset.

Shows which areas have been selected in generating the subset image for each dimension (channels, Z-stack, time series), as well as for the defined image section.

Example:
The entry "Z (1-8: 2) | T (2-7)" means that the sub-image consists of sections 1,3,5,7 at the intervals of 2 to 7 of the input image.

Background

Specifies which value is assigned to invalid pixels. Note that this background color is not visible in the viewer such as the 2D view, it is merely a value for the invalid pixels.

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Auto

Sets the value for the invalid pixels automatically based on the document type, i.e. white for brightfield images and black for fluorescence images.

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Black

Sets the value for the invalid pixels to black.

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White

Sets the value for the invalid pixels to white.

Create Mask

Activated: Creates a pixel mask for valid pixels.
Deactivated: Creates no pixel mask and there is no way to distinguish between valid and invalid pixels, i.e. pixels which contain real data or not. Operations using the (pyramid) tiles could deliver erroneous results (such as visual artifacts in the viewer).

Downsampling Filter

Selects a filter which is applied when generating the pyramid steps.

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None

No filtering is applied.

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Blur

Applies a 2x2 blur kernel before decimation (i.e. an average is calculated).

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Binomial 3x3

Applies a binomial 3x3 filter before decimation.

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Binomial 5x5

Applies a binomial 5x5 filter before decimation.

Channel Input 1

Here you can select whether you want to use an individual channel or all channels of the first input image for the calculation.

Channel Input 2

Here you can select whether you want to use an individual channel or all channels of the second input image for the calculation.

First Images

Activated: For the second input image uses only the first time points of a time lapse image for the calculation. This allows you, for example, to normalize a time lapse image to the intensity values of the first time points.

Enter the number of images that you want to be used for the calculation using the input field.

Formula

Enter the calculation formula here using the keyboard and numeric keypad. Use "S1" as a placeholder for the first input image and "S2" for the second input image.

Input 1

Inserts the placeholder for the first input image into the Formula input field at the current cursor position.

Input 2

Inserts the placeholder for the second input image into the Formula input field at the current cursor position.

Absolute Intensities

Activate this radio button if input image 1 and input image 2 have the same pixel type.

Normalize Intensities 0..1

Activate this radio button if input image 1 and input image 2 have different pixel types. To allow such images to be combined, the intensity values of the two images are normalized to the value range from 0 to 1 before the calculation.

Operators...

Opens a list of all available operators. Here you can select the operator that you want. If you double-click on a list entry, it is inserted into the Formula input field at the current cursor position.

Delete

Deletes the contents of the Formula input field.

Undo

Undoes the last entry in the Formula input field.

Width

Width in x of the image.

Height

Height in y of the image.

Z Slices

Number of z slices of the image. If the value is > 1, it will become a Z-stack image.

Channels

Number of channels of the image, if value is > 1, it will become a multi-channel image.

Time Slices

Number of time slices, if value is > 1, it will become a time series image.

Min. Gray Value

Minimum Gray Value for generation.

Max Gray Value

Maximum Gray Value for generation.

Pixel Type

Specifies the pixel type of the image.

Pattern

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Uniform

All pixels of the image have identical Min. Gray Value.

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2D Gray Scale Vertical

The image shows a gray scale with values between Min. Gray Value and Max. Gray Value from top to bottom.

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2D Gray Scale Horizontal

The image shows a gray scale with values between Min. Gray Value and Max. Gray Value from left to right.

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Ramp

The image shows a ramp with values between Min. Gray Value and Max. Gray Value starting from each corner of the image to the center.

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Gaussian

The image shows a Gaussian shaped grayscale with values between Min. Gray Value and Max. Gray Value starting from the borders of the image to the center.

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Checkerboard

The image shows a checkerboard where the “dark” fields have Min. Gray Value and the “bright” fields have Max. Gray Value.

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Cosine Checkerboard

The image shows a checkerboard where the “dark” fields have Min. Gray Value and the “bright” fields have Max. Gray Value overlaid with a cosine modulation.

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Chirp Cosine

The image shows a cosine pattern where the “dark” fields have Min. Gray Value and the “bright” fields have Max. Gray Value overlaid with a chirp modulation.

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Chirp Checker

The image shows a checkerboard where the “dark” fields have Min. Gray Value and the “bright” fields have Max. Gray Value overlaid with a chirp modulation.

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Random Spheres

a 3D (Z stack) image is created which contains Number of Spheres spheres with Sphere Diameter diameter which are randomly distributed in the image.

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Sphere Array

a 3D (Z stack) image is created which contains Number of Spheres spheres with Sphere Diameter diameter which are equally distributed in the image.

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Single Sphere

a 3D (Z stack) image is created which contains a single sphere with Sphere Diameter diameter which is positioned in the center of the image.

Sphere Diameter

Diameter of the created spheres.

Number of Spheres

Number of spheres which are generated in the 3D image.

Import Reference Spectra

For the unmixing process previously generated emission spectra of ideally pure dyes can be loaded and used for unmixing. This function is mutually exclusive to the Automatic Component Extraction function.

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Import from

Allows you to select and import reference spectra by clicking on .

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Spectra List

Displays the list of imported spectra with an ID, the File Name and the Channel.

Automatic Component Extraction

Use this function if no reference spectra are available. Indicates the number of components the system should be looking for in the image. The number of components cannot be higher than the number of channels. It will only work if each of the emission signals is present in an area of the image without overlap of another emission signal. Otherwise, ACE cannot produce a reliable result.

Components

Adjust the number of spectrally distinguishable fluorescent components within the imaged sample. The number of extractable components cannot be higher than the number of acquired channels. The maximum possible value is 10 components.

Weighted Unmixing

If activated, spectral channels with high noise contribute less to the unmixing result. This option includes a statistical analysis of the signal-related (Poisson-) noise and weighs the respective contribution for the fitting with the combination of reference spectra to the experimental data.
Note: This option involves a more sophisticated unmixing algorithm and therefore takes longer than the basic unmixing analysis. Weighted unmixing generates improved unmixing results when acquisition channels are not so well balanced but still have a good signal-to-noise ratio.

Autoscale

Activated: Balances the intensity of the unmixed channels to equal levels.

Calculate Residuals

Activated: Generates an additional channel in which the intensity values represent the difference between the acquired spectral data and the fitted linear combination of the reference spectra. In essence, the residual value is the biggest remaining "residual" from the least square fit routine. The residuals are a general measure for how good the fit of the algorithm has performed. The higher the intensity in this additional channel, the worse is the fit of the spectra to the data set.

Widefield Crosstalk Removal

Not visible for LSM images. This option allows Unmixing to be performed on multichannel fluorescence images created without a spectral or confocal detector. Typically, this would be multichannel images acquired with a filter based multichannel microscope system. In this case, the function will automatically create the same number and type of channels present in the input image for the output image of the Unmixing function.

Activated: Removes the crosstalk of widefield channels and ensures that the channel information (emission and excitation wavelength) and metadata of the input image are copied to the output image.
Note that the number of spectra must be equal to the number of channels in the image. The function tries to automatically identify which channel matches which spectrum. If this fails, the function assumes that the order of spectra matches the sequence of the channels.

Auto Filter

Activated: A suitable Super Resolution parameter for the LSM Plus processing is automatically determined for the selected data set.
Deactivated: Displays the Super Resolution slider and input field.

Super Resolution

Only visible if Auto Filter is deactivated.
Sets the parameter for super resolution manually.

Strength

Only visible if Auto Filter is activated.
Sets the strength of the automatically assigned filter value for super resolution.

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Low

Uses a decreased strength for the automatically determined value.

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Standard

Uses the standard strength for the automatically determined value.

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High

Uses an increased strength for the automatically determined value.

Output Pixel type

Here you choose the desired output image format, e.g. 8 Bit B/W.

Split Mode

Choose the mode how to split the multiblock image.

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Homogeneous groups

Splits the multiblock image into the single dimensions. The blocks will remain.

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Single blocks

Splits the multiblock image into single blocks.

Display field

The path of the destination folder is displayed automatically in the display field. To change the folder, click on the button to the right of the display field.

Output Folder

Displays and sets the path of the output folder. To change the folder, click on the right of the input field.

Include Scene Information in Generated File Name

Activated: Includes the scene information in the file name of the separate image.

Overwrite existing files

Activated: All files in the target folder are deleted if the function is applied again.

Compression

Selects the type of compression, e.g. Original (no compression) or Compression (JPEG XR).