In vivo imaging of unstained tissues using a compact and flexible multiphoton microendoscope

Cornell University, School of Applied and Engineering Physics, 271 Clark Hall, Ithaca, New York 14853-2501.
Journal of Biomedical Optics (Impact Factor: 2.86). 04/2012; 17(4):040505. DOI: 10.1117/1.JBO.17.4.040505
Source: PubMed


We use a compact and flexible multiphoton microendoscope (MPME) to acquire in vivo images of unstained liver, kidney, and colon from an anesthetized rat. The device delivers femtosecond pulsed 800 nm light from the core of a raster-scanned dual-clad fiber (DCF), which is focused by a miniaturized gradient-index lens assembly into tissue. Intrinsic fluorescence and second-harmonic generation signal from the tissue is epi-collected through the core and inner clad of the same DCF. The MPME has a rigid distal tip of 3 mm in outer diameter and 4 cm in length. The image field-of-view measures 115 μm by 115 μm and was acquired at 4.1 frames/s with 75 mW illumination power at the sample. Organs were imaged after anesthetizing Sprague-Dawley rats with isofluorane gas, accessing tissues via a ventral-midline abdominal incision, and isolating the organs with tongue depressors. In vivo multiphoton images acquired from liver, kidney, and colon using this device show features similar to that of conventional histology slides, without motion artifact, in ~75% of imaged frames. To the best of our knowledge, this is the first demonstration of multiphoton imaging of unstained tissue from a live subject using a compact and flexible MPME device.

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    • "Currently, most of the MPM systems use bulky benchtop setups based on free-space optics, causing some tissue sites difficult to be accessed. Consequently, a fiber-optic MPM micro-endoscope is desirable where light can be delivered in a flexible fiber and signals can be captured using a miniaturized probe [4] [5] [6] [7]. The small footprint and flexibility of such systems have the potential for clinical translation of MPM, as well as new applications in basic scientific research [8]. "
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    ABSTRACT: We report on the development of a compact multiphoton microscopy (MPM) system based on a frequency-doubled, femtosecond erbium-doped fiber laser source at 1.58 μm. By use of periodically poled MgO:LiNbO3, frequency-doubled pulses at 790 nm with average power of 75 mW and pulse width of 130 fs are applied as the excitation source. The fiber laser is optimized for its parameters along with the dispersive properties of the delivery fiber such that the MPM signal is maximized at the sample location. Micro-electro-mechanical system (MEMS) scanner, miniature objective, and multimode fiber are further used to make the MPM system compact. MPM images are obtained from unstained biological samples. The MPM system with a compact, portable, low-cost fiber laser has a great potential to transform the bench-top MPM system to a portable system for in vivo MPM imaging.
    Full-text · Article · Jan 2014 · Proceedings of SPIE - The International Society for Optical Engineering
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    • "In particular, several developments have been made in the field of confocal endoscopy, giving rise to a growing number of research instruments [21,22] and also to a few commercial flexible endoscopes. Very recently, a flexible laser scanning multiphoton endoscope has been developed and successfully used to image native tissue in a living animal [23,24]. Considering that non-linear imaging techniques have already largely demonstrated their capabilities in providing label-free optical tissue biopsies [25–28], in the near future the combination of these optical techniques with endoscopy will represent a powerful clinical tool to be used for both early diagnosis and follow-up of colorectal cancer. "
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    • "To be clinically useful, endoscopic 2P approaches are required. A number of different endoscopes and techniques have been demonstrated in the past [2–9], including in vivo imaging of unstained tissues [10,11]. "
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