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ZEISS Microscopy Knowledge Base

Segmentation Group

Canny Tool

This tool detects edges in a gray scale image using the Canny algorithm, which is the most common, general-purpose edge detection algorithm. The tool enables you to specify how strong the input image is smoothed before edge detection is performed.

Parameter

Description

Sigma

Specifies the extent of smoothing applied to the input image before the edges are detected.

Smoothing reduces noise and thus the chance of obtaining false edges.

Threshold

Specifies the steepness an edge must possess to be recognized by the tool. The steepness is the rate at which the gray values change from bright to dark or vice versa.

  • Lower values: More edges are detected, including shallower and less pronounced ones.
  • Higher values: Only steep and strongly pronounced edges are detected.

Marr Tool

This tool detects edges or regions in an image using the Marr–Hildreth algorithm, which smooths the image using a Gauss filter and then applies a Laplace filter to the smoothed image. The result is a measure for the second derivative of the image brightness at each position. Zero crossings of this second derivative are detected to obtain the edges.

Due to its detection method this tool is very sensitive to any change in image intensity. It is thus prone to detecting false edges, i.e. to detect noise or slight local changes in brightness as edges. Compared to other edge detectors, you might need to apply a larger amount of smoothing to obtain satisfying results.

You can display either the detected edges or the regions enclosed by the edges.

Parameter

Description

Sigma

Specifies the extent of smoothing applied to the input image before the edges or regions are detected.

Smoothing reduces noise and thus the chance of obtaining false edges.

Display Mode

Specifies how the results are displayed:

Edges

The edges around regions of bright pixels are displayed as thin white lines, all other pixels are displayed black.

Regions

The regions of bright pixels are displayed white, the edges and the space between the bright regions are displayed black.

Threshold Tool

This tool splits the image into foreground and background areas. Pixels within a specified range are considered foreground pixels and are not modified. All other pixels are considered background pixels and set to black. You can use an automatic method to determine the range or you can specify it manually. If the input image is a color image, you can set the brightness ranges for each color channel separately.

Automatic range

Parameter

Description

Method

Specifies the algorithm 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.

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 for each value calculates the spread of the pixel intensities of the background and foreground pixels. The threshold is set at the value that minimizes both spreads.

This method is particularly suited to light objects on a dark background.

Maximum Peak

The threshold is set to the pixel value that occurs most frequently.

Dark gray histogram with a tall left peak marked by a blue dotted vertical line and dot, x-axis 0–255, grayscale bar below

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.

Dark histogram with central blue dotted line and double arrow spanning two dotted gray boundaries

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.

Dark histogram with blue dotted diagonal from left peak to rightmost dot, blue marker and vertical dotted guide on a trough

Three Sigma Threshold

The pixel value that occurs most frequently is calculated. 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 peak value and three times the standard deviation.

Manual range

Parameter

Description

Level Low, Level High

Specifies the lower and upper boundaries of the brightness range.

All pixels with gray values outside the brightness range are considered to be background pixels and changed to black.

If the input image is a color image, you can specify the boundaries for each color channel separately.

Create Binary

  • Activated: Pixels within the brightness range are changed to white, background pixels are changed to black.
  • The resulting image is binary and can be used as a mask for subsequent image processing operations.
  • Deactivated: Pixels within the brightness range remain unchanged, background pixels are changed to black.

Invert Result

Reverts the way the pixels are treated.

  • Pixels inside the brightness range: treated as background and changed to black.
  • Pixels outside the brightness range: remain unchanged or changed to white if binary is activated.

Threshold Auto Tool

This tool splits the image into foreground and background areas. Pixels within a specified range are considered foreground pixels and are not modified. All other pixels are considered background pixels and set to black. The brightness range is determined by an automatic method.

Automatic range

Parameter

Description

Method

Specifies the algorithm 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.

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 for each value calculates the spread of the pixel intensities of the background and foreground pixels. The threshold is set at the value that minimizes both spreads.

This method is particularly suited to light objects on a dark background.

Maximum Peak

The threshold is set to the pixel value that occurs most frequently.

Dark gray histogram with a tall left peak marked by a blue dotted vertical line and dot, x-axis 0–255, grayscale bar below

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.

Dark histogram with central blue dotted line and double arrow spanning two dotted gray boundaries

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.

Dark histogram with blue dotted diagonal from left peak to rightmost dot, blue marker and vertical dotted guide on a trough

Three Sigma Threshold

The pixel value that occurs most frequently is calculated. 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 peak value and three times the standard deviation.

Manual range

Parameter

Description

Create Binary

  • Activated: Pixels within the brightness range are changed to white, background pixels are changed to black.
  • The resulting image is binary and can be used as a mask for subsequent image processing operations.
  • Deactivated: Pixels within the brightness range remain unchanged, background pixels are changed to black.

Invert Result

Reverts the way the pixels are treated

  • Pixels inside the brightness range: treated as background and changed to black.
  • Pixels outside the brightness range: remain unchanged or changed to white if binary is activated.

Threshold Dynamic Tool

This tool splits the image into foreground and background pixels by performing an adaptive gray value segmentation. It is particularly suited to the segmentation of small structures on a varying background.

The tool initially applies a low-pass filter and then subtracts this low-pass-filtered image from the input image. The effect largely depends on the size of the filter matrix (Kernel Size).

Parameter

Description

Kernel Size

Specifies the matrix size of the low pass filter in X and Y direction symmetrically around the pixel currently processed and determines the extent of the smoothing effect.

Set a low Kernel Size value to segment small regions or regions with low gray value contrast from the background. Set a higher Kernel Size value to segment larger regions from the background.

As the affected pixel is at the center, the edge length of the filter matrix is always an odd number. If you enter an even number via the keyboard, the value is always set to the next highest odd number.

Threshold

Defines the difference in brightness between the regions to be detected and the background.

Segmented pixels are set to the maximum gray value (white), other pixels are set to zero (black).

Create Binary

  • Activated: Pixels within the brightness range are changed to white, background pixels are changed to black.
  • The resulting image is binary and can be used as a mask for subsequent image processing operations.
  • Deactivated: Pixels within the brightness range remain unchanged, background pixels are changed to black.

Invert Result

Reverts the way the pixels are treated.

  • Pixels inside the brightness range: treated as background and changed to black.
  • Pixels outside the brightness range: remain unchanged or changed to white if binary is activated.

Valleys Tool

This tool detects lines of dark pixels (valleys composed of low gray values) on a bright background. In the output image, the detected lines are displayed white on a uniform black background.

Parameter

Description

Sigma

Specifies the extent of smoothing applied to the input image before the lines of dark pixels are detected.

  • Lower values: Thinner valleys are preserved, more valleys are detected, and more lines are visible in the output image.
  • Higher values: Thinner valleys are removed due to smoothing, fewer valleys are detected, and fewer lines are visible in the output image.

Threshold

Specifies the steepness a valley must possess to be recognized by the tool. The steepness is the rate at which the gray values increase starting from the bottom of a valley.

  • Lower values: More valleys are detected, including shallower and less pronounced ones.
  • Higher values: Only steep and strongly pronounced valleys are detected.
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