Measuring point spread functions and calibrating (STED) confocal microscopes

Abstract

In this thesis the point spread function (PSF) used to characterize a confocal microscope was determined for different configurations. The PSF was determined for a sample with a refractive index matched to the refractive index that the objective was designed for, which is in this case n = 1.518 and n = 1.451 [1], and it was investigated how a mismatch in refractive index affects the shape and size of the PSF. Also the PSF of stimulated emission depletion (STED) confocal microscopy was determined to investigate the advantages of STED over conventional confocal microscopy. The alignment of the STED and the excitation laser of the confocal microscope was checked by imaging the reflection of the STED and excitation laser with a gold core nanoparticle. For the calibration of the distance measurement in a sample with a mismatch in refractive index a colloidal crystal was grown to compare the interlayer distance determined by using FTIR spectroscopy and the interlayer distance determined by imaging the crystal with the confocal microscope.