Tissue Optics Modeling in High Resolution Microscopy

Abstract

The optical properties of tissue and cells are of key significance in optical biomedical technology, such as in optical imaging and spectroscopy. In this thesis, scattering by randomly shaped particles has been investigated, to understand better optical properties of biological tissue and cell suspensions. After proposing generation functions for the randomly shaped particles, the T-matrix method and appropriate effective size distribution were applied to compute rigorously phase functions, depending on the physical and geometrical characteristics of the scatterers. The derived phase functions show good agreement with the experimental results. To obtain the high quality optical imaging, an advanced microscopy with high resolution, high optical sectioning ability and deep penetration depth is required. In this thesis, we analyzed the confocal microscopy with divided apertures with diffraction theory. In addition, the optimization of axial resolution with respect to the width of the divider was presented. To suppress the out-of-focus central bright spot in the confocal microscopy with divided apertures, an improvement with serrated divided apertures was reported. The results show an increasing efficiency of the rejection of scattered light. Diffraction analysis also shows that the serrated apertures maintain the optical sectioning strength while attenuating the background coming from far from the focal plane. In addition, the signal to background ratio is also improved. Finally, the focal modulation microscopy (FMM) was introduced to increase the imaging depth into tissue and rejection of background from a thick scattering object. FMM can simultaneously acquire conventional confocal images and FMM images. The application to saturable fluorescence was also discussed. The study on edge enhancement for FMM shows that compared with confocal microscopy, using FMM can result in a sharper image of the edge and the edge gradient can be increased up to 75.4% and 58.9% for thick edge and thin edge, respectively. A further improvement with FMM using annular apertures (AFMM) was also reported. Compared with confocal microscopy, AFMM can simultaneously enhance the axial and transverse resolution. By adjusting the width of the annular objective aperture, AFMM can be adjusted from best spatial resolution performance to highest signal level. In addition, AFMM has the potential to further increase the imaging penetration depth. This research indicated the great potential of FMM in the biological and biomedical system.