| Microscopy is a key means of analyzing biological activity and structures, covering a level accessible by few other approaches. Increasingly, live microscopy is being used as a unique means to study the dynamics, of structure, localization, and motion of processes to develop and test hypotheses of cellular dynamics. However, analysis of sub-micron structures within living specimens often becomes difficult or impossible as the image quality degrades with increasing depth in the sample. This degradation results from aberrations due to the specimen's refractive index properties, which alter image formation at the detector. These aberrations range from a general, depth-dependent-spherical aberration for relatively homogeneous samples, to position-dependent aberrations for more complex samples.; My thesis project focuses on developing computational corrections of live specimen imaging problems encountered with widefield fluorescence microscopy. The main goal of this work is to study the imaging process for widefield microscopy and to develop improved deconvolution approaches based-on-the information gathered. A significant part of this work has been to develop an improved description of an optical microscope, based on techniques developed for astronomy. My project had been subdivided into the following specific steps for further discussion below: (1) Creation of a compact and modular description of the wide field microscope system using phase retrieval; (2) Development and use of depth dependent deconvolution approaches to correct for aberrations from a general sample refractive index mismatch; and (3) estimation of spatially varying PSFs by using ray tracing techniques for the future application in image deconvolution of optically complex samples. |
