A block-based forward imaging model for improved sample volume representation in computational optical sectioning microscopy


In typical fluorescence imaging systems the refractive index (RI) variability between the immersion medium of the objective lens, the coverslip, and the specimen, changes the spherical wave-front of the emitted light and introduces spherical aberrations (SA) in the acquired 3D image. In existing computational optical sectioning algorithms (COSM) to simplify the complexity of the problem, the specimen is either assumed to be thin or in the case of depth-variant algorithms to have a constant RI which is an invalid assumption for biological samples. Accurate modeling of biological samples requires a space variant (SV) imaging system i.e. a different point spread functions (PSF) for each pixel. To reduce the computational load an approximate block-based forward model is introduced in this study. The entire object space is divided into a collection of small 3D blocks where the PSFs at the faces of the blocks are known. An optimized combination of overlap-save and overlap-add methods of interpolation are used to obtain the final SV (axially and laterally variant) image. Simulated SV images using the new imaging model, of a numerical object comprising of similar structures dispersed in a medium with spatially variant RI are discussed. Images of fluorescent microspheres (6-μm in diameter) dispersed in a controlled sample with two distinct RIs are compared to simulated images of a numerical object subjected to the same imaging condition, to evaluate the new model. The accuracy of the block-based forward model to model the effect of space variance within a specimen was assessed using intensity profiles through the microspheres. The qualitative similarities in the appearance of the experimental and simulated image indicate the validity of the blockbased forward model to appropriately model samples with lateral variability in RI.

Publication Title

Progress in Biomedical Optics and Imaging - Proceedings of SPIE