Line scanning, fiber bundle fluorescence HiLo endomicroscopy with confocal slit detection (Conference Presentation)

Abstract

HiLo technique provides an effective means of eliminating signals from out-of-focus in widefield fluorescence microscopy by synthesizing two images sequentially acquired with uniform and structured illumination. However, light scattering within the sample often deteriorates the optical sectioning effect. Here, we demonstrate that optical sectioning can be improved by combining HiLo technique and confocal slit detection. Light scattering is reduced by using the rolling shutter of a CMOS scanner as a virtual detector slit. Synchronizing the camera rolling shutter with a scanning hybrid-illumination laser line results in a HiLo endomicroscopy with confocal line detection at a high frame rate of 60 fps. In endomicroscopy, an expanded laser beam passes through a beam splitter and is reflected by a spatial light modulator which toggles between two illumination patterns, grid and uniform. The illumination patterns are focused by a cylindrical lens and then delivered through a fiber bundle probe that transfers the laser line to the tissue and collects emitted fluorescence. To check the axial depth sectioning strength, a thin, uniform fluorescent plane is illuminated. The fullwidth half maximum of the axial distance scanning is 19 μm for the proposed HiLo confocal as opposed to 28.5 μm for the line scanning confocal and 51 um for the widefield HiLo. Experiments on imaging phantoms and ex vivo tissues demonstrate that the optical sectioning ability of the HiLo confocal endomicroscopy is improved when compared to its counterparts.