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Image Analysis Wizard

This wizard guides you through the setup of an image analysis. It is only available if you have licensed functionality for Image Analysis or 3D Image Analysis. Note that some of the parameters differ depending on whether you set up a 2D or a 3D analysis.

The following basic controls enable you to move through the steps:

Parameter	Description
Next	Moves on to the next step of the wizard.
Back	Moves back to the previous step of the wizard.
Cancel	Cancels the wizard. No changes are applied to your settings.
Finish	Saves the setup and the changes based on your progress and closes the wizard.

Classes

In this step you can define the classes into which the measured objects in the image are divided.

Parameter		Description
Interactive		Activated: The class definition can be changed interactively while the analysis setting is run with Start Interactive Analysis.
Class List		Displays the defined classes. If you create a new analysis setting, a predefined set of classes is created automatically. The classes list differs if you have a 3D analysis setting. For a 2D analysis, each class consists of two entries. The first entry concerns the entirety of all objects belonging to the class. The second entry represents the individual objects. For a 3D analysis, each class consists only of one entry.
Add Class		Adds a new individual class to the list on the base level.
Add Subclass		Adds a new subclass to the selected class.
Remove Class		Deletes the selected class from the list.
Name		Defines the name for the selected class in the list. Note that you must not use the name Root for one of your classes as this a reserved keyword.
Channel		Selects the channel that is used for image segmentation of the selected class in the Classes list.
Color		Selects the color to mark the objects of a class.
Object Color		Only visible if you have selected a class entry for individual object.
–	Random	The objects found by the analysis will be colorized randomly.
–	Fixed	Selects the fixed color to mark the objects of a class.

Frame

In this step you can define one or more measurement frames. Only the area within the measurement frames gets analyzed. You can also define how the analysis treats objects that are cut by the border of the image or the frame.

Parameter		Description
Interactive		Activated: The measurement frame definition can be changed interactively while the analysis setting is run with Start Interactive Analysis.
Select Frame		Enables you to select already created measurement frames. To select a measurement frame, click inside it. To select several measurement frames, press the Ctrl key and click inside the desired measurement frames. Once you have selected a measurement frame, you can change its size.
Draw Rectangle		Enables you to create a rectangle as a measurement frame in the current image.
Draw Circle		Enables you to create a circle as a measurement frame in the current image.
Draw Contour (Polygon)		Enables you to create a contour as a measurement frame in the current image.
Remove All Frames		Removes all drawn-in measurement frames in the current image. To delete single frames, select them and press Del.
Maximize Circle		Only available for 2D settings and only active if you have defined precisely one circle. Activated: Maximizes the drawn-in circle to the full image size. In the case of rectangular images the circle is adjusted to the shorter side.
Center Circle		Only available for 2D settings and only active if you have defined precisely one circle. Activated: Centers the drawn-in circle to the full images size.
Mode		Selects how the measurement frame should be applied. Note that this behavior is only applied when running the analysis (interactively) and not during the setup. The setup always uses the Cut at Frame mode. The following modes are available:
-	Inside Only	Measures only those objects, that are lying completely within the measurement frame. Objects that are touching the frame or are intersected by the frame are not analyzed.
-	Cut at Frame	Measures all objects that are lying within the measurement frame. Objects that are intersected by the measurement frame are measured precisely up to the measurement frame.
The following fields are only active if you have selected a drawn-in graphic element:
Left		Sets the starting point for the frame on the X axis in pixels.
Top		Sets the starting point for the frame on the Y axis in pixels.
Width		Sets the width of the frame in pixels.
Height		Sets the height of the frame in pixels.
Color		Selects the color of the frame.
Show Frame On Analyzed Image		Activated: Displays the frame on the image after the analysis has run.

Automatic Segmentation

In this step you can select the segmentation method that is applied and set parameters for the segmentation of the objects that you want to measure. Note that during the setup of the analysis (via Edit Image Analysis Setting), the segmentation is only performed on the area visible in the viewport. If you enter the analysis wizard via Start Interactive Analysis, the image will be fully segmented.

Parameter		Description
Execute		Activated: Sets the defined threshold values when the measurement program is run.
Interactive		Activated: The segmentation can be changed interactively while the analysis setting is run with Start Interactive Analysis.
Class List		Selects the class for which you want to define the segmentation.
Ring Element Class		This additional parameter is only available if you have selected the Zone of Influence (ZOI) method.
-	Ring Distance	Distance from surface of the primary object. Negative values mean that the ring starts at the defined distance within the primary object.
-	Ring Thickness	Defines the thickness of the ring.
ZOI Class		This additional parameter is only available if you have selected the Zone of Influence (ZOI) method.
-	ZOI Width	Allows you to set the distance of border of the ZOI from the border of the ring, or the main object, respectively.
Segmentation Method		Displays and selects a segmentation method for the currently selected class with the dropdown.
-	Explore	Opens the dialog to explore and select an available segmentation method, see Segmentation Method Selection Dialog.

3D Parameters

If you are setting up a 3D analysis, the available parameters are either grouped under 2D Parameters for general (2D optimized) parameters, or under 3D Parameters, if the parameters are specifically optimized for 3D operations.

The visible parameters depend on the selected segmentation method. The following parameters sections can be available:

Smoothing Section

Parameter		Description
Smoothing		Selects how to smooth the image before the threshold values are set. The following methods are available:
-	None	The image is not smoothed.
-	Lowpass	Applies the Lowpass method. The lowpass filter compares the brightness of each pixel to the brightness of its neighboring pixels. If a pixel is brighter than its neighbors, the brightness of this pixel is reduced and the brightness of the neighboring pixels is increased. This suppresses sharp changes in brightness (i.e. contours) and leads to more gradual changes in brightness.
-	Gauss	Applies the Gauss method. Each pixel is replaced by a weighted average of its neighbors. The weighting depends on the sigma value. The Gaussian filter is particularly useful for contour enhancement, which is very sensitive to noise. Using a Gaussian filter before finding contours greatly improves the results.
-	Median	Applies the Median method. Each pixel is replaced by the median of its neighbors. The number of neighboring pixels taken into account depends on the size. In a set of values (in this case the pixel values taken into account), the median is the value for which the number of larger values is equal to the number of smaller values.
Size		Only visible, if you have selected Low Pass or Median. Sets the size of the filter matrix in the X and Y direction, i.e. the number of neighboring pixels taken into account. The size should correspond to the pixel size of the contours to be reduced.
Sigma		Only visible, if you have selected Gauss. Sets the sigma value that defines how much neighboring pixels contribute to the weighting. Larger values broaden the applied Gaussian distribution and lead to reduced noise but also to an increased loss of image information.

Threshold & Histogram Section

Here you can define the threshold values for the selected class in the class list.

Parameter		Description
Threshold		Sets the brightness boundaries between which pixels are considered.
–	Reset	Clears the upper and lower thresholds. No pixels are considered.
–	Undo	Undoes the last change made to the threshold values.
–	Redo	Restores the last undone change to the threshold values.
Color Model		Only visible if the image is a color image, see Color Model.
-	RGB	In RGB mode you can define the threshold values for the red, green and blue color channels.
-	HLS	In HLS mode you can define the threshold values for hue, saturation and lightness.
–	Low	Sets the lower threshold. Only pixel values above this value are considered. The range of possible values depends on the bit depth of the image.
–	High	Sets the upper threshold. Only pixel values below this value are considered. The range of possible values depends on the bit depth of the image.
–	Invert	Only pixels outside the threshold boundaries are considered, i.e. those pixels below the lower threshold and above the higher threshold.
–	Full Range	Sets the lower threshold to 0 and the upper threshold to the highest value (depending on bit depth). The entire range of pixel values is considered.
Histogram		In the histogram you can change the lower and upper threshold value for the activated value. Drag the lower or upper adjustment handle or shift the entire highlighted area between the lower and upper threshold value.
Click		Click in the image on the regions that you want to define as objects. The threshold values are adapted according to the pixel intensities at the clicked position in the image.
Automatic		The threshold values are determined automatically from the histogram. During setup only the part of the image displayed in the viewport is taken for the calculation of the threshold. After the automatic calculation of the threshold values you can further modify the threshold values found interactively by selecting Click for threshold value definition.
Pick Behavior		Only visible, if you have selected Click.
-	+	Enables you to expand the currently segmented regions by the gray values/colors of the objects subsequently clicked on.
-	-	Enables you to reduce the currently segmented regions by the gray values/colors of the objects subsequently clicked on.
Tolerance		Only visible, if you have selected Click. Specifies how many additional pixel values are included in the selection based on their brightness. A higher value means that more pixel values similar to the selected one are included. A lower value means that only the exact pixel value selected is included.
Neighborhood		Only visible, if you have selected Click. Specifies how many additional pixel values are included in the selection based on their physical proximity to the selected pixel. A higher value means that more pixels surrounding the selected pixel are included. The threshold boundaries are adapted so that all the pixel values of these neighboring pixels are included. A lower value means that the boundaries are adapted based on only the pixels directly next to the selected pixel.
Method		Only visible, if you have selected Automatic. Selects the algorithm that is used to automatically detect the threshold boundaries. The most suitable algorithm depends on your precise requirements. The value also depends on the bit depth of the image. The following methods are available:
-	Otsu	The pixel values below the threshold are designated as background and those above the threshold as foreground. It iterates through all possible threshold values and calculates the variance of the pixel intensities of the background and foreground pixels for each value. The threshold is set at the value that minimizes both variances. This method is particularly suited to light objects on a dark background.
-	Maximum Peak	Separates background and foreground pixels at the maximum value of the histogram.
-	Iso-Data	The pixel values below the threshold are designated as background and those above the threshold as foreground. An initial threshold value is chosen, and the mean pixel intensity of the foreground and background pixels is calculated. These two mean values are averaged and the result serves as the input threshold for the next calculation. The process is repeated until the threshold value no longer changes.
-	Triangle Threshold	The algorithm constructs a line between the peak of the highest frequency pixel intensity and the lowest pixel intensity. The distance between the line and the histogram is computed for all values along the line. The pixel intensity where the line is longest is used as the threshold. This method is particularly suited when the foreground pixels only have a weak peak in the histogram.
-	Three Sigma Threshold	Calculates the pixel value that occurs most frequently. The standard deviation of the values in the peak is calculated. The threshold is set to the pixel intensity that is the sum of the average peak value and three times the standard deviation.

Variance Section

This section is only visible if Variance-Based Thresholding is selected.

Parameter		Description
Kernel Size		Sets the kernel size used to calculate the variance value of one pixel with its neighboring pixels.
Variance		Defines the lower and upper threshold for the variance.

Subtract BG Section

Parameter		Description
Subtract BG		Only visible if Segmentation with Background Subtraction is selected. Selects which kind of background subtraction is performed.
-	None	No background subtraction is performed.
-	Rolling ball	The rolling ball background subtraction is performed.

Object Size & Hole Section

Parameter		Description
Min. Object Size		Sets the minimum size in pixels that an object must have in order to be segmented.
Min. Hole Size		Sets the minimum size in pixels that a hole must have in order to be recognized for segmentation. This input is synchronized with the input for Min. Object Size, which must not be smaller than Min. Hole Size.
Fill all Holes		Specifies how holes in detected objects are treated.
–	On	Fills holes in segmented objects ().
–	Off	Does not fill the holes in segmented objects ().

Binary Section

Parameter		Description
Binary		Selects which morphological operations are performed on the segmented (binary) image.
-	None	No operation is performed.
-	Open	Performs first erosion and then dilation. The effect is smoothing and removing of isolated pixels.
-	Close	Performs first dilation and then erosion. The effect is smoothing of the objects and filling of small holes.
-	Dilate	Enlarges the boundaries of segmented regions. Areas grow in size and holes within the regions become smaller.
-	Erode	Erodes boundaries of the segmented regions. The areas shrink in size and holes within the areas become larger.
Count		Sets how often the selected binary operation is performed with the slider or input field.

Separate Section

Parameter		Description
Separate		Selects whether you want to process the image further after segmentation. Objects that are touching one another can be separated using different methods.
-	None	Objects are not separated.
-	Morphology	Separates objects by first reducing and then enlarging them, making sure that once objects have been separated they do not merge together again.
-	Watersheds	Separates objects that are roughly the same shape. The result is two shapes separated by a thin 1-pixel boundary. The rest of the shape perimeter remains unchanged. This method may however result in the splitting of elongated objects.
Count		Sets the count value, which is similar to a Sigma for Gauss applied to a binary image.

3D Preview Section

Only visible if you set up a 3D analysis setting. With this section you can manipulate the preview in 3D.

Parameter	Description
Z-Position	Selects the z-position around which the preview is shown.
Thickness	Sets the preview thickness around the Z-Position.

Segmentation Method Selection Dialog

With this dialog you can select the method that is used for segmenting the class currently selected in the class list.

Parameter		Description
Global Thresholding		Applies a fixed threshold across the entire image to segment objects of interest. Use it for images with consistent object intensity like fluorescence images.
AI Instance Segmentation		Uses a deep learning model trained on arivis Cloud to distinguish and separate individual objects, even if touching or overlapping. Use it for precise identification in complex images. You need the Docker Desktop software running on your PC and have a suitable model available, see Machine Learning and AI Functionalities in ZEN.
AI Semantic Segmentation		Uses a semantic machine learning model trained with Intellesis in ZEN or arivis Cloud for classifying regions. Use it for labeling image regions by predefined classes, using random forest or deep learning. For semantic segmentation models, you need an installation of the 3rd party Python Tools.
Background Subtraction		Uses a rolling ball algorithm to subtract uneven background, then applies a global threshold to segment objects. Use it for images with varying background intensity.
Variance-Based Thresholding		Segments based on intensity changes. Use it for brightfield images where objects are distinguished by variance rather than consistent intensity.
Dynamic Thresholding		Applies local thresholds to different regions to handle inhomogeneous backgrounds. Use it for images with uneven illumination.
Don't show this automatically		Activated: The dialog is not opened automatically anymore for all users and can only be opened with the Explore button. Deactivated: The dialog opens automatically when entering the respective step of the image analysis.
OK		Uses the selected segmentation method for the class and closes the dialog.

Region Filter

Settings based on image view

Note that during the setup of the analysis (via Edit Image Analysis Setting), the segmentation is only performed on the area visible in the viewport. Therefore, if you adapt the region filter by clicking on the objects displayed in the viewport, and select objects that are cut by the current viewport, these objects will only be segmented partially. The region filter will only be adapted based on the part of the object that is in the viewport. In case you want to select objects that exceed the viewport, we suggest to adapt the region filter values manually.

If you enter the analysis wizard via Start Interactive Analysis, the image will be fully segmented during the segmentation step, and therefore it is possible to adapt the region filters by clicking on the objects in the image.

In this step you can define the region filter conditions under which you want an object to be measured.

Parameter	Description
Execute	Activated: Uses the region filters when the measurement program is run.
Interactive	Activated: The region filters can be changed while the analysis setting is run with Start Interactive Analysis.
Classes	Selects the class for which you want to define the conditions.
Select Features	Opens the editor to select region filters.
Copy to All	Copies the defined region filters to all classes.
Region Filters	If you have defined one or more blocks with region filters in the Region Filter Editor, you can select the block for which you want to set the filter. Select the relevant block and set the maximum/minimum values either by clicking on the objects in the image you want to include in the measurement, or by entering the maximum/minimum values separately.
Undo	Undoes the last change made to the condition.
Redo	Restores the last undone change to the condition.
Reset	Resets all settings for the conditions.

Region Filter Editor

All features in the list of selected features are calculated during image analysis. The results are displayed in the results table for all detected objects of the same class. The columns of the features are sorted according to the order they appear in the Selected Features list.

The results of the settings you set here are displayed in the last step of the wizard in the table with results on the right side.

For a detailed description of the individual features, see Measurement Features.

Parameter		Description
Selected Features		Displays the features that you have selected block by block. All features in a block are And-linked, i.e. an object is only measured if the values of each individual feature fall within the defined range.
Add Block		Adds an Or block. If several Or blocks are defined, an object is measured if it meets the condition in at least one block.
Clear Block		Deletes all features in an Or block.
Remove Block		Deletes the selected Or block.
Remove All		Deletes all Or blocks.
Available Features		Displays the list with all available features. Double-click on the feature or to add the feature to the list of selected features on the left.
Search Field		Searches for features by name.
Filter Dropdown		Filters the list of displayed features.
-	All	Lists all available features.
-	Geometric Features	Lists only the subset of geometric features.
-	Intensity Features	Lists only the subset of intensity features.
-	Image Features	Lists only the subset of image features.
-	Position Features	Lists only the subset of position features.
-	Geometric Features Unscaled	Lists only the subset of unscaled geometric features.
Add		Adds the selected feature to the list of selected features on the left.

Features

Analysis Setting for Experiment Feedback

Features you have defined are available from within the Feedback Experiment. Any time you change the image analysis settings, you need to reload the *.czias file in the feedback script editor to activate the changes. The image analysis settings are typically saved in the ProgramData folder on your hard drive, e.g. C:\ProgramData\Carl Zeiss\ZEN\Users\XXXX\Documents\Image Analysis Settings.

In this step you select the measurement features.

Parameter		Description
Interactive		Activated: The features can be changed interactively while the analysis setting is run with Start Interactive Analysis.
Classes		Selects the class for which you want to define measurement features. For a 2D analysis setting, the tree view has two entries for each class for which you can define features. The first entry ("Classes") concerns the collection of the objects belonging to the class. The second entry ("Class") represents all individual objects belonging to the class. The typical features of "Classes" are statistical values of the measurement feature over all single objects of that class. For example, Mean Intensity channel 1 gives you the mean intensity of all single objects. Area gives you the sum of all areas (i.e. the total area) of the individual objects. Fo a 3D analysis, each class has only one entry in the tree view for which you can define features for individual regions of the class and features for all regions of the respective class.
Features of Individual Regions/Features of All Regions		Displays the selected features for the currently selected class and allows you to select additional features. Depending on the class selection, you can select and see the features for individual regions or all regions.
-	Select	Opens the dialog for feature selection.
-	Copy to All	Copies the defined features to all other classes.
-	Feature	Displays the feature name(s) added for the currently selected class.
-	Display	If you activate Display in the feature selection dialog for a feature, the result of the measurement is displayed next to the corresponding object in the analyzed image.
Annotations		Only visible for 2D analysis settings and if in the tree view a "Class"-node (i.e. the node concerning the individual regions) is selected. Displays the selected annotations for the currently selected class and allows you to select features which will be shown as annotations in the analyzed image. An example is the center of each region.
-	Select	Opens the dialog for feature selection.
-	Copy to All	Copies the annotations to all other classes.
Annotation Options		Only visible for 2D analysis settings.
-	Color	Activated: Allows you to select the color for the region annotations.
Custom Feature		Opens the editor to define a custom feature, see Creating Custom Features.
Feature list		Displays a list of all created custom features.

Select Features of Individual Regions Dialog

For a description of individual measurement features, see Measurement Features.

Parameter		Description
Selected Features		Displays all selected features that are calculated for the object during image analysis.
-	Name	Displays the name of the respective feature.
-	Display	Activated: The value of the feature for each object is displayed in the analyzed image.
-	Copy	Only visible for Classes (collection of objects) and if more than one class exists. Selects where the feature is copied to. If the Copy column is empty, the selected feature is not copied to any result table. For a description of the possible copy operations, see Copy Operations for Features.
-	Delete	Deletes the currently selected feature from the list.
-	Move Up	Moves the currently selected feature one position up in the list.
-	Move Down	Moves the currently selected feature one position down in the list.
Available Features		Displays the list with all available features. Double-click on the feature to add the feature to the list of selected features on the left.
Search Features		Here you can enter parts of the name of the feature that you are looking for. The features in which the entered character string occurs are listed. Select a type of feature according to which you want the features to be filtered from the dropdown list.
-	All	All features are listed.
-	Geometric Features	All geometric features are listed.
-	Intensity Features	All features that analyze intensity values are listed.
-	Image Features	All features that contain meta information about the measured image are listed.
-	Position Features	All features that describe the position are listed.
-	Geometric Features Unscaled	All features that describe unscaled geometric features are listed.
-	Position Features Unscaled	All features that describe unscaled positions are listed are listed.
-	Polygon-based Features	All polygon-based features are listed.
-	Statistical Features	All features that can be used for plotting in a heatmap (i.e. that provide statistical values suitable for heatmap plotting) are listed.
Add		Adds the selected feature to the list of selected features on the left.

Annotations Dialog

Parameter		Description
Selected Features		Displays all selected features that are calculated for the object during image analysis.
-	Name	Displays the name of the respective feature.
-	Delete	Deletes the currently selected feature from the list.
-	Move Up	Moves the currently selected feature one position up in the list.
-	Move Down	Moves the currently selected feature one position down in the list.
Available Features		Displays the list with all available features. Double-click on the feature to add the feature to the list of selected features on the left.
Search Features		Here you can enter parts of the name of the feature that you are looking for. The features in which the entered character string occurs are listed. Select a type of feature according to which you want the features to be filtered from the dropdown list.
-	All	All features are listed.
-	Geometric Features	All geometric features are listed.

Custom Feature Editor

Parameter		Description
Custom Features		Displays a list of the created custom features.
–	Name	Displays the name of the created custom feature.
–	Add	Adds a new custom feature that can be defined with the options on the right side.
–	Delete	Deletes the currently selected feature.
Define Custom Feature
–	Name	Defines the name of the feature.
–	Unit	Specifies the unit of the feature as free text input. This input is optional.
Define Operands		Displays and defines the operands used for the calculation of the custom feature.
–	Operand	Displays the name of the operand.
–	Class	Selects the class that is used for the definition of the operand. For a single region class, also the classes of the children can be selected.
–	Feature	Displays all available predefined measurement features for the selected class and selects which measurement feature should be used for the definition of the current operand. If you activate the checkbox behind the selection dropdown, the selected feature is also set visible in the result table.
–	Expression	Displays the expression of the Operand(s) defined by the Class and Feature selection.
–	Add	Adds a new operand to the list.
–	Delete	Deletes the currently selected operand.
Define Custom Expression		Defines the mathematical calculation of the feature, using the Operands and mathematical operators, e.g. *100(a/b+Math.Pow(c,2))**.
–	+	Adds the mathematical operator for summation to the calculation.
–	-	Adds the mathematical operator for subtraction to the calculation.
–	*	Adds the mathematical operator for multiplication to the calculation.
–	/	Adds the mathematical operator for division to the calculation.
–	Pow	Adds the mathematical operator to calculate the power of a certain base value. Note that this operator requires two input values (the base and power value) separated by a comma, e.g. Math.Pow (2,3), which corresponds to 2³.
–	Sqrt	Adds the mathematical operator to calculate the square root of a value.
–	Abs	Adds the mathematical operator to return the absolute number, i.e. non negative values.
Verify Expression		Checks the syntax of the Expression.
OK		Adds the defined custom features and closes the dialog.
Cancel		Closes the dialog without saving the changes.

Statistics

In this step you can define custom statistical features for your regions or objects. Note that this step is not available for 3D Image Analysis. When an image analysis using custom statistical features is successfully performed, the resulting values are available in the heatmap graph when the base-node of the classes tree is selected and if well plate or sample carrier information is present.

Parameter		Description
Interactive		Activated: The custom feature definition can be changed interactively while the analysis setting is run with Start Interactive Analysis.
Class List		Selects the class for which you want to define the custom statistical feature(s).
Define Custom Feature		Opens the editor to define a custom statistic feature, see Creating Custom Statistical Features.
Custom Statistic Feature List		Displays the created custom statistic features.
–	Name	Displays the name of the custom statistic feature.

Image Analysis Wizard

Classes

Frame

Automatic Segmentation

Smoothing Section

Threshold & Histogram Section

Variance Section

Subtract BG Section

Object Size & Hole Section

Binary Section

Separate Section

3D Preview Section

Segmentation Method Selection Dialog

See also

Region Filter

See also

Region Filter Editor

Features

See also

Select Features of Individual Regions Dialog

Annotations Dialog

Custom Feature Editor

See also

Statistics

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