For creating experimental point spread functions from a Z-stack of subresolution fluorescent beads please use the function PSF Wizard which is available together with the Deconvolution module and offers a guided procedure starting with a stack of many beads and includes the Create PSF functionality.

Prerequisite for the Create PSF function here is, that bead averaging has already been done. It is available only for legacy reasons.

This function creates a PSF (Point Spread Function) image from a Z-stack image of a bead acquired for PSF measurement. Please observe the instructions for optimal acquisition here: Using beads for PSF measurement.

The result is a so-called PSF image. For advanced settings and options, please use the specific control elements on the PSF Display tab.

Parameters

Parameter

Description

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.