Angular Gating and Biological Scattering in Optical Microscopy

Abstract

The main purpose of my work is to precisely and effectively explore biological phenomenon in vivo by using optical method. To achieve this aim, my major work focuses on two aspects: one is to solve the fundamental problems of lack of precise optical scattering models for biological tissue and cells, and the other is to establish a high performance optical microscopy. For the first aspect, we developed a random nonspherical model and a fractal model for the biological tissue and cells. These two models are introduced based on different fundamentals and have different applications. The power spectrum of the contrast phase images is investigated. The phase function, the anisotropy factor of scattering, and the reduced scattering coefficient are derived. The effect of different size distributions is also discussed. The theoretical results show good agreement with experimental data. The application of this model in phase contrast microscopy is in process. For the second aspect, we discuss the confocal microscopy with angular gating techniques (divided apertures) and investigate the performance of focal modulation microscopy (FMM), which modifies a confocal microscopy by a combination of angular gating technique with modulation and demodulation techniques. We analytically derived the three-dimensional coherent transfer function (CTF) for reflection-mode confocal scanning microscopy with angular techniques under the paraxial approximation and also analyzed the three-dimensional incoherent transfer function (OTF) for fluorescence confocal scanning microscopy with angular gating techniques. The effects on different aperture shapes such as off-axis apertures, elliptical apertures, and Schwartz apertures are investigated. FMM was introduced to increase imaging depth into tissue and rejection of background from a thick scattering object. A theory for image formation in one-photon FMM is presented, and the effects of detecting the in-phase modulated fluorescence signal are discussed. Two different non-overlapping apertures of D-shaped and quadrant apertures are studies. Two-photon FMM was proposed by us at the first time. The enhanced depth penetration permitted by two-photon excitation with the near-infrared photons is particularly attractive for deep-tissue imaging. The investigation of the imaging depth in an extension of single-photon FMM to two-photon FMM (2PFMM) allows the penetration depth to be three-fold of that in convention two-photon microscopy (2PM). This result suggests that 2PFMM may hold great promise for non-invasive detection of cancer and pre-cancer, treatment planning, and may also server as a research tool for small animal whole body imaging. The effects of different apodization conditions and polarization distributions on imaging in 4Pi microscopy are also discussed. With radially polarized illumination, the transverse resolution in the 4Pi mode can be increased by about 18%, but at the expense of axial resolution.