Extended Depth of Focus

Method

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Wavelets

Uses a wavelet transformation to detect the sharpest areas in the images.

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Contrast

For this method, the value is the difference between the brightest and the darkest pixel value within the “Kernel”.

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Maximum Projection

Images with the brightest and darkest pixels are generated first. Of these images the image with the higher variance is used as the resulting image. Usually, best suited for fluorescence images.

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Variance

Calculates the variance of the pixel values within the “Kernel”. Usually, best suited for bright field images.

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Fast EDF

Uses a method for fast EDF to detect the sharpest areas in the images.

Z-Stack Alignment

This parameter should not be used for processing whole slide image scans. It is only required when processing images stemming from a stereo microscope.

Not visible if you have selected Maximum Projection as Method.
Selects if the z-stack image should be aligned before the calculation and with what quality level. If you acquire images with a stereo microscope, the images are displaced against each other. This displacement can be corrected. The higher the selected quality of alignment, the longer takes the calculation. Select No Alignment, if you acquire images with a compound microscope.

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No Alignment

The z-stack image is not aligned before the calculation. Select this setting if the z-stack image has not been acquired using a stereo microscope.

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Normal

Uses an alignment with high speed for normal image quality.

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High

Uses an alignment with lower speed for high image quality.

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Highest

Uses an alignment with the lowest speed with best image quality.

Process Tiles Separately

Only visible if you have selected Maximum Projection as Method.
Activated: Processes each tile region separately.

Contrast Lengthscale

Only visible if Contrast or Variance is selected as Method.
Sets the length scale (pixel units) at which the contrast for every slice is calculated. For images with small and sharp structures, a small contrast length scale provides good results. For images with larger structures with smoother edges, a larger contrast length scale is required.

Smoothing

Only visible if Contrast, Variance, or Fast EDF is selected as Method.
Sets a value for smoothing. This parameter helps you to bridge smaller regions with missing contrast information, i.e. image regions with no structures.

Reconstruction

Only visible if Contrast or Variance is selected as Method.
Defines the amount of image area with little contrast that needs to be reconstructed by an algorithm. If an image contains larger regions with little or no contrast information, the algorithm has to interpolate image information at a larger extent.

Decay Flicker Correction

Only visible if Contrast or Variance is selected as Method.
Activated: Equalizes the brightness and intensity of pixels over the entire z-stack. During the acquisition of fluorescence images, bleaching can lead to a difference in pixel brightness, also called decay. When acquiring an image with a lamp, flickering can lead to fluctuations in pixel intensity. This option tries to correct both effects through the equalization over the entire stack.

Pixelwise Iteration

Only visible if Fast EDF is selected.
Larger values lead to a smoother heightmap. Use the slider to adjust the desired value.

Default values (depending on the selected preset):

• Preset = Default: 2
• Preset = Small Structures: 2
• Preset = Medium Structures: 3
• Preset = Large Structures: 3

Depthwise Kernel Size

Only visible if Fast EDF is selected.
Larger values lead to a more robust heightmap/image, especially for areas with low contrast. Should only be used by small depth of focus step sizes.

Default values (depending on the selected preset):

• Preset = Default: 1
• Preset = Small Structures: 0
• Preset = Medium Structures: 2
• Preset = Large Structures: 2

Resize Ratio

Only visible if Fast EDF is selected.
Sets the scaling factor. Typically, smaller values are faster.

Default Value: 0.25

• 0.25
• 0.5
• 1.0