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Measurement and modeling of point spread function for improving the spatial resolution in microscopy

Tzardis Evaggelos

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Summary

Due to the widespread use of light microscope as a diagnostic tool for many scientific fields like medicine, biology, chemistry as well as for industrial applications, light microscopy has been undergoing vast and continuing innovations both regarding the hardware and software domain. The optical resolution of light microscopes is physically constrained by the phenomenon of diffraction. Out-of-focus light and light originated from adjacent areas of a sample are superposed degrading the quality of the image of the object under study. In this degradation, the Point Spread Function (PSF) of the optical system is the main culprit and it is the one that determines the optical resolution. This degradation effect can be eased by sophisticated and expensive confocal microscopy systems or reversed to some degree by much cheaper widefield deconvolution microscopy methods. Deconvolution processes need a PSF as much as accurate it can be in order to provide satisfactory and realistic results. The description of the PSF can be done either by mathematical models or by experimental measurements. Experiments for this purpose include measurements of fluorescent microbeads as well as estimation of the Modulation Transfer Function (MTF) of the optical systems which finally yields the PSF. The present diploma thesis deals with the mathematical modelling of the PSF in comparison with its experimental measurement via a method that uses the general MTF estimation process, with application on optical microscopes. This evaluation is done with the use of quantitative metrics that describe the quality of the deconvolved images aiming at an objective assessment of them. Results show a superiority of the experimental PSF as far as the metrics are concerned. As for the visual perception, in the majority of the occasions, deconvolution with a modelled PSF seems to produce results of higher contrast. Contrast enhancement is therefore not in agreement necessarily with the improvement of images that approaches the real optical information . In addition, several standard contrast enhancement techniques are used for extra comparison. This work is integrated in a graphical user interface which additionally allows quantitative comparison on user-imported images.

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