Wide-field fluorescent imaging severely suffers low resolution and poor contrast from out-of-focus background to image biological samples. In order to enhance optical sectioning capability, Confocal approach has been developed to filter out-of-focus background using point-to-point detection through a spatial pinhole. Recently, active structured illumination in wide-field fashion has been developed to reduce the transversal scanning cost, but still requires scanning in axial direction. Here, we present a wide-field multi-focal fluorescence microscopy incorporating spatial-spectral volume holographic gratings (MVHGs) with 3D active structured illumination to obtain optically sectioned images without scanning is presented. In contrast to conventional holographic techniques, which in general can not obtain fluorescence images, our approach does not require the formation of a hologram during imaging and is compatible with fluorescence based methods of imaging. Our approach requires pair-wise multi-depth resolved images, one with 3D active illumination, and the other with standard uniform illumination. Our approach is configured such that 3D illuminated planes occur inside the specimen, and also serve as the structured modulation for multiple axial planes imaged by MVHGs and display laterally onto the camera. The system can also be combined with micro-objective and relay systems for endoscopic operation. We demonstrate the proposed system’s ability to simultaneously obtain wide-field, optically sectioned, and multi-depth resolved images of fluorescently labeled tissue structures.
Hsi-Hsun Chen, Chen-Yen Lin, Wei Tang Lin, and Yuan Luo, "Optically sectioned spatial-spectral coded holographic fluorescence microscopy (Conference Presentation)," Proc. SPIE 9691, Endoscopic Microscopy XI; and Optical Techniques in Pulmonary Medicine III, 96910K (Presented at SPIE BiOS: February 15, 2016; Published: 27 April 2016); https://doi.org/10.1117/12.2213937.4828146962001.
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