Grating imager systems for fluorescence optical-sectioning microscopy

Abstract

In fluorescence microscopy, optical sectioning is defined as the attenuation or removal of out-of-focus features from an image, and it is a prerequisite for quantitative analysis of three-dimensional structure or function within the specimen. Optical sectioning is most commonly performed by confocal scanning fluorescence microscopy or two-photon scanning fluorescence microscopy. However, structured illumination can be used in conventional fluorescence microscopes to obtain optical sectioning performance, and, in advanced systems, 3D superresolution. The simplest structured-illumination system uses a Ronchi grating as a mask to project parallel stripes within the sharp depth-of-focus of the objective to encode in-focus specimen features differently from out-of-focus features. By shifting the grating, the in-focus image component can be discriminated and separated by elementary image processing operations. This implementation of structured illumination, the fluorescence grating imager, uses a conventional light source, is compatible with all high-quality fluorescence filter sets, and provides high optical-sectioning performance when used to image specimens in which (1) the out-of-focus image component is not much brighter than the in-focus features and (2) there is no significant movement in the specimen during the grating shift and image capture process.