Fluorescence microscopy point spread function model accounting for aberrations due to refractive index variability within a specimen

Sreya Ghosh; Chrysanthe Preza

doi:10.1117/1.JBO.20.7.075003

7 July 2015 Fluorescence microscopy point spread function model accounting for aberrations due to refractive index variability within a specimen

Sreya Ghosh, Chrysanthe Preza

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Journal of Biomedical Optics, Vol. 20, Issue 7, 075003 (July 2015). https://doi.org/10.1117/1.JBO.20.7.075003

Abstract

A three-dimensional (3-D) point spread function (PSF) model for wide-field fluorescence microscopy, suitable for imaging samples with variable refractive index (RI) in multilayered media, is presented. This PSF model is a key component for accurate 3-D image restoration of thick biological samples, such as lung tissue. Microscope- and specimen-derived parameters are combined with a rigorous vectorial formulation to obtain a new PSF model that accounts for additional aberrations due to specimen RI variability. Experimental evaluation and verification of the PSF model was accomplished using images from 175-nm fluorescent beads in a controlled test sample. Fundamental experimental validation of the advantage of using improved PSFs in depth-variant restoration was accomplished by restoring experimental data from beads (6 μm in diameter) mounted in a sample with RI variation. In the investigated study, improvement in restoration accuracy in the range of 18 to 35% was observed when PSFs from the proposed model were used over restoration using PSFs from an existing model. The new PSF model was further validated by showing that its prediction compares to an experimental PSF (determined from 175-nm beads located below a thick rat lung slice) with a 42% improved accuracy over the current PSF model prediction.

CC BY: © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

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Sreya Ghosh and Chrysanthe Preza "Fluorescence microscopy point spread function model accounting for aberrations due to refractive index variability within a specimen," Journal of Biomedical Optics 20(7), 075003 (7 July 2015). https://doi.org/10.1117/1.JBO.20.7.075003

Published: 7 July 2015

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