Fiber bundle fluorescence endomicroscopy is an effective method for in vivo imaging of biological tissue samples. Line-scanning confocal laser endomicroscopy realizes confocal imaging at a much higher frame rate compared to the point scanning system, but with reduced optical sectioning. To address this problem, we describe a fiber bundle endomicroscopy system that utilizes the HiLo technique to enhance the optical sectioning while still maintaining high image acquisition rates. Confocal HiLo endomicroscopy is achieved by synchronizing the scanning hybrid-illumination laser line with the rolling shutter of a CMOS camera. An evident improvement of axial sectioning is achieved as compared to the line-scanning confocal endomicroscopy without the HiLo technique. Comparisons are also made with epifluorescence endomicroscopy with and without HiLo. The optical sectioning enhancement is demonstrated on lens tissue as well as porcine kidney tissue.
We report a compact rigid instrument capable of delivering en-face optical coherence tomography (OCT) images alongside (epi)-fluorescence endomicroscopy (FEM) images by means of a robotic scanning device. Two working imaging channels are included: one for a one-dimensional scanning, forward-viewing OCT probe and another for a fiber bundle used for the FEM system. The robotic scanning system provides the second axis of scanning for the OCT channel while allowing the field of view (FoV) of the FEM channel to be increased by mosaicking. The OCT channel has resolutions of 25 / 60 μm (axial/lateral) and can provide en-face images with an FoV of 1.6 × 2.7 mm2. The FEM channel has a lateral resolution of better than 8 μm and can generate an FoV of 0.53 × 3.25 mm2 through mosaicking. The reproducibility of the scanning was determined using phantoms to be better than the lateral resolution of the OCT channel. Combined OCT and FEM imaging were validated with ex-vivo ovine and porcine tissues, with the instrument mounted on an arm to ensure constant contact of the probe with the tissue. The OCT imaging system alone was validated for in-vivo human dermal imaging with the handheld instrument. In both cases, the instrument was capable of resolving fine features such as the sweat glands in human dermal tissue and the alveoli in porcine lung tissue.
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.
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