Analyzing the effect of absorption and refractive index on image formation in high numerical aperture transmission microscopy of single cells

Transmission bright-field microscopy is the clinical mainstay for disease diagnosis where image contrast is provided by absorptive and refractive index differences between tissue and the surrounding media. Different microscopy techniques often assume one of the two contrast mechanisms is negligible as a means to better understand the tissue scattering processes. This particular work provides better understanding of the role of refractive index and absorption within Optical Projection Tomographic Microscopy (OPTM) through the development of a generalized computational model based upon the Finite-Difference Time-Domain method. The model mimics OPTM by simulating axial scanning of the objective focal plane through the cell to produce projection images. These projection images, acquired from circumferential positions around the cell, are reconstructed into isometric three-dimensional images using the filtered backprojection normally employed in Computed Tomography (CT). The model provides a platform to analyze all aspects of bright-field microscopes, such as the degree of refractive index matching and the numerical aperture, which can be varied from air-immersion to high NA oil-immersion. In this preliminary work, the model is used to understand the effects of absorption and refraction on image formation using micro-shells and idealized nuclei. Analysis of absorption and refractive index separately provides the opportunity to better assess their role together as a complex refractive index for improved interpretation of bright-field scattering, essential for OPTM image reconstruction. This simulation, as well as ones in the future looking at other effects, will be used to optimize OPTM imaging parameters and triage efforts to further improve the overall system design.