Abstract
Coherent Raman scattering microscopy is a fast, label-free, and chemically specific imaging technique that shows high potential for future in vivo optical histology. However, the imaging depth in tissues is limited to the sub-millimeter range because of absorption and scattering. Realization of coherent Raman imaging using a fiber endoscope system is a crucial step towards imaging deep inside living tissues and providing information that is inaccessible with current microscopy tools. Until now, the development of coherent Raman endoscopy has been hampered by several issues, mainly related to the fiber delivery of the excitation pulses and signal collection. Here, we present a flexible, compact, coherent Raman, and multimodal nonlinear endoscope (4.2 mm outer diameter, 71 mm rigid length) based on a resonantly scanned hollow-core Kagomé-lattice double-clad fiber. The fiber design enables distortion-less, background-free delivery of femtosecond excitation pulses and back-collection of nonlinear signals through the same fiber. Sub-micrometer spatial resolution over a large field of view is obtained by combination of a miniature objective lens with a silica microsphere lens inserted into the fiber core. We demonstrate high-resolution, high-contrast coherent anti-Stokes Raman scattering, and second harmonic generation endoscopic imaging of biological tissues over a field of view of 320 µm at a rate of 0.8 frames per second. These results pave the way for intraoperative label-free imaging applied to real-time histopathology diagnosis and surgery guidance.
Highlights
Identification of cancer tumors is generally carried out ex vivo by histological inspection of tissue biopsies
The background between the beads is remarkably dark, which demonstrates coherent anti-Stokes Raman scattering (CARS) imaging free of a fiber four wave mixing (FWM) background
We checked that the point spread function (PSF) is not significantly altered when the field of view (FoV) is increased up to 350 μm (Fig. S9)
Summary
Identification of cancer tumors is generally carried out ex vivo by histological inspection of tissue biopsies This process requires several steps, such as tissue resection, sectioning, and staining, and is time-consuming and labor-intensive. CARS/SRS endoscopy is more demanding than two-photon excited fluorescence (TPEF) or second harmonic generation (SHG)[16,17,18] as two spatially and temporally overlapping excitation beams are required. When these two pulses copropagate in a fiber, a strong parasitic background arises due to the occurrence of nonlinear four wave mixing (FWM) in the fiber core[9]. The complexity introduced by these designs leads to bulky endoscope heads and/or a loss in optical resolution and contrast, which are not compatible with in vivo histopathology applications
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