Optical Sectioning of Live Mammal with Near-Infrared Light Sheet

Abstract

Deep-tissue three-dimensional optical imaging of live mammals in vivo with high spatiotemporal resolution in non-invasive manners has been challenging due to light scattering. Here, we developed near-infrared (NIR) light sheet microscopy (LSM) with optical excitation and emission wavelengths up to ~ 1320 nm and ~ 1700 nm respectively, far into the NIR-II (1000-1700 nm) region for 3D optical sectioning through live tissues. Suppressed scattering of both excitation and emission photons allowed one-photon optical sectioning at ~ 2 mm depth in highly scattering brain tissues. NIR-II LSM enabled non-invasive in vivo imaging of live mice, revealing never-before-seen dynamic processes such as highly abnormal tumor microcirculation, and 3D molecular imaging of an important immune checkpoint protein, programmed-death ligand 1 (PD-L1) receptors at the single cell scale in tumors. In vivo two-color near-infrared light sheet sectioning enabled simultaneous volumetric imaging of tumor vasculatures and PD-L1 proteins in live mammals.Optical imaging of biological systems capable of high spatiotemporal resolution in vivo and ex vivo has revolutionized biology and medicine for visualization and understanding of structures, functions and dynamic processes at the cellular and even molecular scale 1,2 . To circumvent light scattering by tissues, in vivo 3D imaging by nonlinear two-photon fluorescence microscopy (670-1070 nm excitation) 3-5 or three-photon microscopy (1300-1700 nm excitation) 6-9 has reached penetration depths ~ 0.7-1.5 mm, benefiting from increased scattering mean free path of the nearinfrared (NIR) excitation employed 6 . Light sheet microscopy (LSM) uses orthogonally arranged planar illumination and wide-field detection, capable of high speed 3D optical sectioning, low photo-damage 10,11 and volumetrically imaging/tracking with subcellular resolution 2 . Currently the excitation and emission of LSM are mostly in the visible except for two photon excitation in the NIR at ~ 940 nm 12 or three photon excitation at 1000 nm 13 . Light scattering has limited LSM to imaging small transparent animals, organisms (zebrafish larvae, drosophila larvae, Medaka embryo, C. elegans, etc.), mammalian tissue samples after chemical clearing 11,14,15 , and mouse brain at a depth of ~ 200 μm after craniotomy 16 .

Near‐Infrared‐II Molecular Dyes for Cancer Imaging and Surgery

“…There are also several home‐built fluorescence platforms for high resolution and high contrast live imaging . We summarize two key NIR‐II microscopy setups and their advantages in visualizing the tumor microenvironment with subcellular resolution.…”

“…They further built a NIR‐II light sheet microscopy (LSM) with optical excitation and detection wavelengths up to 1320 and 1700 nm respectively. The frame‐to‐frame imaging route afforded a much‐improved scanning rate and can visualize never‐before‐seen dynamic biological processes …”

“…[18] There are also several home-built fluorescence platforms for high resolution and high contrast live imaging. [18,[132][133][134] We summarize two key NIR-II microscopy setups and their advantages in visualizing the tumor microenvironment with subcellular resolution. Super-resolved fluorescence microscopy, two-photon microscopy, light-sheet microscopy, and NIR-II confocal microscopy are also expected to be combined with suitable NIR dyes, providing the possibility to observe the whole tumor heterogeneity.…”

“…The frame-to-frame imaging route afforded a much-improved scanning rate and can visualize never-before-seen dynamic biological processes. [133] Observing deep tissue information in vivo at a (sub)cellular resolution to explore the molecular signals and cell behaviors is a critically important direction for both oncology and other biological processes. Intravital imaging, affording quantitative and dynamic visualization/mapping into tumor biology and immunology, has realized this goal, [135] with the help of the long-term imaging windows (cranial window, dorsal skinfold chamber, the mammary window, as well as abdominal window).…”