Multiphoton Endoscopy
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Multiphoton microscopy has the potential to be a versatile tool for clinical disease diagnosis and treatment, providing numerous benefits over current practices involving the histopathological analysis of biopsies. Specifically, multiphoton excitation has the unique ability to generate fluorescence from intrinsic molecules (e.g., NADH, flavins) and produce harmonic generations (e.g., second harmonic generation) within tissues, thereby allowing for real-time images of unprocessed, unstained tissues with resolution and details comparable to gold standard histopathology. Therefore, multiphoton endoscopes (MPEs) that enter the body via a natural orifice or a small surgical incision and are capable of providing real-time, cellular level images from unstained tissues would be valuable clinical tools. These endoscopes could be used clinically to provide real-time margin assessment during tumor resection as well as guide or replace conventional biopsy procedures. A challenge faced in the realization of a clinically useful MPE is achieving multiple imaging parameters (e.g., high resolution, fast frame rates, large field of view (FOV), axial sectioning) within a compact device size. To date we have developed the first compact and flexible MPE capable of acquiring sub-micron resolution, in vivo images of unstained tissues. The device consists of a miniaturized resonant/nonresonant fiber raster scanner and a gradient index lens assembly that are packaged into a 3-mm outer diameter, 4-cm rigid length tube. Our MPE acquires 110 [MICRO SIGN]m x 110 [MICRO SIGN]m multiphoton images with high scan uniformity at 4 frames/s (512 x 512 pixels), and achieves lateral and axial resolutions for two-photon imaging of 0.8 and 10 [MICRO SIGN]m, respectively. To help translate our endoscopes into the clinic, we also present new techniques that will improve upon the capabilities of our initial device. To achieve instantaneous axial-sectioning and faster frame rates without sacrificing the signal-to-noise (SNR) ratio per frame we have integrated parallel image acquisition into our original MPE, thereby creating a multifocal multiphoton endoscope. Furthermore, to achieve a larger image FOV, while maintaining high optical resolution, we integrated a lensed fiber into our miniaturized scanner. We believe that the integration of these techniques is essential for translating the benefits of multiphoton microscopy into the clinic.
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Weiss, Robert S.