High-resolution multimodal flexible coherent Raman endoscope

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(4.2mm outer diameter,71mm 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 320μm 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.

Repeated photoporation with graphene quantum dots enables homogeneous labeling of live cells with extrinsic markers for fluorescence microscopy

In the replacement of genetic probes,there is increasing interest in labeling living cells with high-quality extrinsic labels,which avoid over-expression artifacts and are available in a wide spectral range.This calls for a broadly applicable technology that can deliver such labels unambiguously to the cytosol of living cells.Here,we demonstrate that nanoparticle-sensitized photoporation can be used to this end as an emerging intracellular delivery technique.We replace the traditionally used gold nanoparticles with graphene nanoparticles as photothermal sensitizers to permeabilize the cell membrane upon laser irradiation.We demonstrate that the enhanced thermal stability of graphene quantum dots allows the formation of multiple vapor nanobubbles upon irradiation with short laser pulses,allowing the delivery of a variety of extrinsic cell labels efficiently and homogeneously into live cells.We demonstrate high-quality time-lapse imaging with confocal,total internal reflection fluorescence(TIRF),and Airyscan superresolution microscopy.As the entire procedure is readily compatible with fluorescence(super resolution)microscopy,photoporation with graphene quantum dots has the potential to become the long-awaited generic platform for controlled intracellular delivery of fluorescent labels for live-cell imaging.