Quantifying optical sectioning in reflection microscopy with patterned illumination

Abstract

Sectioning techniques based on patterned illumination have been widely used to obtain well-contrasted images of thick samples using widefield imaging setups. While their application to fluorescence microscopy has been extensively demonstrated and studied, their application to reflection imaging is scarcer and their performance has only been partly characterized. In this paper, we study numerically and analytically two such sectioning techniques, line confocal (LC) and structured illumination microscopy (SIM), in the context of their application to coherent reflection imaging. We derive approximate analytical equations to relate the performance of sectioning to the optical setup parameters, allowing straightforward understanding of their influence on the image intensity and depth of focus, and we systematically compare our predictions with experimental data. Finally, we quantify the precision and accuracy of each method in typical practical cases, providing guidelines to choose the most appropriate (LC, SIM, or a simple background subtraction on a widefield image) for the sample under study. We illustrate optical sectioning in the particular case of reflection interference contrast (RIC) microscopy, an imaging technique widely used in soft matter and biophysics studies to monitor object-surface interactions, or quantify surface functionalization. Our derivation, however, should also prove useful for other reflection methods such as optical coherence tomography (OCT) or flood illumination ophtalmoscopy.