Abstract
In vivo fluorescent cellular imaging of deep internal organs is highly challenging, because the excitation needs to penetrate through strong scattering tissue and the emission signal is degraded significantly by photon diffusion induced by tissue-scattering. We report that by combining two-photon Bessel light-sheet microscopy with nonlinear structured illumination microscopy (SIM), live samples up to 600 microns wide can be imaged by light-sheet microscopy with 500 microns penetration depth, and diffused background in deep tissue light-sheet imaging can be reduced to obtain clear images at cellular resolution in depth beyond 200 microns. We demonstrate in vivo two-color imaging of pronephric glomeruli and vasculature of zebrafish kidney, whose cellular structures located at the center of the fish body are revealed in high clarity by two-color two-photon Bessel light-sheet SIM.
Highlights
In vivo cellular imaging of organs in live animals is highly challenging because it requires a technique that penetrates thick surrounding tissue layers while still maintaining cellular resolution
The above limitations make light-sheet imaging of large or deep organs in live animals a challenging task, because: first, it is difficult to effectively illuminate deep structures; second, the emission image contrast and resolution are degraded by photon diffusion; a limited field-of-view may not be enough for imaging large organs
In this paper we address these limitations by combining two-photon Bessel light-sheet imaging with nonlinear structured illumination microscopy
Summary
In vivo cellular imaging of organs in live animals is highly challenging because it requires a technique that penetrates thick surrounding tissue layers while still maintaining cellular resolution. Images from light-sheet microscopy are affected by tissue scattering of emitted fluorescence photons, which degrades the lateral image resolution in deep layers.
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