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

ABSTRACT 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 ef fects, will be used to optimize OPTM imaging parameters and triage efforts to further improve the overall system design. Keywords: Computational Modeling, Finite-Difference Time-Domain, Absorption, Refractive Index, Image Formation Theory, Optical Projection Tomographic Microscopy (OPTM), optical CT 1. INTRODUCTION Clinicians generally utilize bright-field microscopy to make qualitative diagnoses of cancer and other diseases by visualizing two-dimensional images. This qualitative procedure leads to reduced sensitivity and specificity when diagnosing disease, such as cancer in situ. In the future, diagnoses could be provided by quantitative assessment of the cellular scattering processes with improved understanding the im pact of refraction and absorptio n on the final image. By better understanding the differences in scattering between cancerous and noncancerous cells, it is possible to improve the early detection of cancer. This type of analysis is especially important for improving the implementation of Optical Projection Tomographic Microscopy (OPTM), which is a novel three-dimensional (3D) imaging technology being developed at the University of Washington, Arizona State University, and Cornell University. OPTM forms isotropic 3D images of single cells embedded in optical gel that flows through a rotating capillary tube. Projections are acquired as cells flow past the objective one-by-one in a rotating cap illary tube where each projection is formed by scanning the focal plane of a high-numerical aperture (NA) objective through the cell. Projections are assimilated from different perspectives around the cell and reconstructed using the filtered backprojection, similar to Computed Tomography (CT). The refractive index and absorptive properties have an impact on each projection, thereby altering the final reconstruction. For individual and isol ated cell analysis, VisionGate Inc. (Pho enix, AZ) is commercializing OPTM as