Fiber-bundle based confocal laser endomicroscopy combined with fluorescent biomarkers has shown promise for high-resolution imaging of tissue microstructure in vivo and in situ. However, limited image acquisition speed and a restriction to single fluorescence agents (due to single channel excitation and fluorescence collection spectral bands) for most existing systems makes simultaneous visualization of multiple morphological and functional features difficult. In this paper, we report the development of a high-speed dual-wavelength line-scan confocal laser endomicroscopy system suitable for multiplexed molecular imaging applications using 488 nm and 660 nm laser sources. The fluorescent confocal images are captured by a rolling-shutter CMOS camera at a constant frame rate of 120 Hz, with the rolling shutter of the CMOS camera acting as a virtual detector slit. Dual-wavelength imaging is achieved by switching between the laser sources for alternate frames, avoiding bleed-through, and providing an effective frame rate of 60 Hz. The two channels are pseudo-coloured and combined, and large area dual-wavelength mosaics are created by registering and stitching the image frames as the probe moves across the tissue. Preliminary images with a resolution of 1.2 µm are presented from fluorescently stained phantoms and ex vivo tissue, demonstrating the clinical feasibility of the technique.
Khushi Vyas, Michael R. Hughes, and Guang-Zhong Yang, "A dual-wavelength line-scan confocal endomicroscopy system for rapid molecular imaging (Conference Presentation)," Proc. SPIE 10470, Endoscopic Microscopy XIII, 104700V (Presented at SPIE BiOS: January 28, 2018; Published: 14 March 2018); https://doi.org/10.1117/12.2288412.5751463372001.
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