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Validation of 3D-PSF models

Raphael Frey
Section Microtechnique, EPFL

Semester project
February 2006

The main objectives of this semester project were the creation of a 3D-point-spread function model, and the comparison of this model with real images of fluorescent beads taken with a Zeiss Axiovert microscope. Parameters like numerical aperture (NA), defocus (z_offset) and spherical aberrations (W040) are determinant for the behaviour of the 3D-PSF model. The minimisation of the quadratic error of the difference between the real PSF-images and different PSF-models will provide parameters for a PSF-model which fittest the most with the real microscope images. But also other minimisation methods can be applied to find an optimal PSF-model. Here are typical cuts in x-z (sagital plane) of a 3D-PSF model which satisfy a minimum-criteria and a real 3D-PSF:

Figure 1 Zeiss: real PSF-images with a nominal NA=1.4, model: PSF-model with NA=1, W040=-0.6µm and z_offset = 1.54µm.