Deep-tissue three-dimensional (3D) optical imaging of live mammals with high spatiotemporal resolution in non-invasive manners has been challenging due to light scattering. Here, we developed near-infrared II (NIR-II, 1000–1700 nm) light sheet microscopy (LSM) with excitation and emission up to ~ 1320 nm and ~ 1700 nm respectively for optical sectioning through live tissues at ~ 750-μm penetration depth without any invasive surgery. Suppressed light scattering allowed imaging at ~ 2 mm depth in glycerol-cleared brain tissues. NIR-II LSM in normal and oblique configurations enabled in vivo imaging of live mice through intact tissue, revealing abnormal blood flow and T cell motion in tumor microcirculation and mapping out programmed-death ligand 1 (PD-L1) and programmed cell death protein 1 (PD-1) in tumors with cellular resolution. 3D imaging through intact mouse head resolved vascular channels between skull and brain cortex, and monitored recruitment of macrophages/microglia to traumatic brain injury site post injury.

In the past decade, noticeable progress has been achieved regarding fluorescence imaging in the second near-infrared (NIR-II) window. Fluorescence imaging in the NIR-II window demonstrates superiorities of deep tissue penetration and high spatial and temporal resolution, which are beneficial for profiling physiological processes. Meanwhile, molecular imaging has emerged as an efficient tool to decipher biological activities on the molecular and cellular level. Extending molecular imaging into the NIR-II window would enhance the imaging performance, providing more detailed and accurate information of the biological system. In this progress report, selected achievements made in NIR-II molecular imaging are summarized. The organization of this report is based on strategies underlying rational designs of NIR-II imaging probes and their applications in molecular imaging are highlighted. This progress report may provide guidance and reference for further development of functional NIR-II probes designed for high-performance molecular imaging.

The nanostructures and patterns that exist in nature have inspired researchers to develop revolutionary components for use in modern technologies and our daily lives. The nanoscale imaging of biological samples with sophisticated analytical tools, such as scanning electron microscopy (SEM) and transmission electron microscopy (TEM), has afforded a precise understanding of structures and has helped reveal the mechanisms contributing to the behaviors of the samples but has done so with the loss of photonic properties. Here, we present a new method for printing biocompatible “superlenses” directly on biological objects to observe subdiffraction-limited features under an optical microscope in color. We demonstrate the nanoscale imaging of butterfly wing scales with a super-resolution and larger field-of-view (FOV) than those of previous dielectric microsphere techniques. Our approach creates a fast and flexible path for the direct color observation of nanoscale biological features in the visible range and enables potential optical measurements at the subdiffraction-limited scale.

Noninvasive optical imaging with deep tissue penetration depth and high spatiotemporal resolution is important to longitudinally studying the biology at the single-cell level in live mammals, but has been challenging due to light scattering. Here, we developed near-infrared II (NIR-II) (1,000 to 1,700 nm) structured-illumination light-sheet microscopy (NIR-II SIM) with ultralong excitation and emission wavelengths up to ∼1,540 and ∼1,700 nm, respectively, suppressing light scattering to afford large volumetric three-dimensional (3D) imaging of tissues with deep-axial penetration depths. Integrating structured illumination into NIR-II light-sheet microscopy further diminished background and improved spatial resolution by approximately twofold. In vivo oblique NIR-II SIM was performed noninvasively for 3D volumetric multiplexed molecular imaging of the CT26 tumor microenvironment in mice, longitudinally mapping out CD4, CD8, and OX40 at the single-cell level in response to immunotherapy by cytosine-phosphate-guanine (CpG), a Toll-like receptor 9 (TLR-9) agonist combined with OX40 antibody treatment. NIR-II SIM affords an additional tool for noninvasive volumetric molecular imaging of immune cells in live mammals.

Most NIR-IIb fluorophores are nanoparticle-based probes with long retention (% 1month or longer) in the body. Here,weapplied anovel cross-linked coating to functionalize core/shell lead sulfide/cadmium sulfide quantum dots (PbS/ CdS QDs) emitting at % 1600 nm. The coating was comprised of an amphiphilic polymer followed by three crosslinked amphiphilic polymeric layers (P 3 coating), imparting high biocompatibility and > 90 %excretion of QDs within 2weeks of intravenous administration. The P 3-QDs were conjugated to an engineered anti-CD8 diabody (Cys-diabody) for in vivo molecular imaging of CD8 + cytotoxic Tlymphocytes (CTLs) in response to anti-PD-L1 therapy. Tw o-plex molecular imaging in combination with down-conversion Er nanoparticles (ErNPs) was performed for real-time in vivo monitoring of PD-L1 positive tumor cells and CTLs with cellular resolution by non-invasive NIR-IIb light sheet microscopy. Imaging of angiogenesis in the tumor microenvironment and of lymph nodes deep in the body with asignal-to-background ratio of up to % 170 was also achieved using P 3-QDs.

Significance Surgical removal of tumors has been performed to combat cancer for over a century by surgeons relying on visual inspection and experience to identify margins between malignant and healthy tissues. Herein, we present a rare-earth down-conversion nanoparticle–anti-CD105 conjugate for cancer targeting and a handheld imager capable of concurrent photographic imaging and fluorescence/luminescence imaging. An unprecedented tumor-to-muscle ratio was achieved by near-infrared-IIb (NIR-IIb, 1,500 to 1,700 nm) imaging during surgery, ∼100 times higher than previous organic dyes for unambiguous determination of tumor margin. The sensitivity/biocompatibility/safety of the probes and instrumentation developed here open a paradigm of imaging-guided surgery at the single-cell level, meeting all major requirements for clinical translation to combat cancer and save human lives.

Most microsphere-assisted super-resolution imaging experiments require a high-refractive-index microsphere to be immersed in a liquid to improve the super-resolution. However, samples are inevitably polluted by residuals in the liquid. This Letter presents a novel (to the best of our knowledge) method employing a microsphere lens group (MLG) that can easily achieve high-quality super-resolution imaging in air. The performance of this method is at par or better than that of the high-refractive-index microspheres immersed in liquid. In addition, the MLG generates a real image that is closely related to the photonic nanojet position of the microsphere super-lens. This imaging method is beneficial in microsphere imaging applications where liquids are impractical.

Most NIR-IIb fluorophores are nanoparticle-based probes with long retention (% 1month or longer) in the body. Here,weapplied anovel cross-linked coating to functionalize core/shell lead sulfide/cadmium sulfide quantum dots (PbS/ CdS QDs) emitting at % 1600 nm. The coating was comprised of an amphiphilic polymer followed by three crosslinked amphiphilic polymeric layers (P 3 coating), imparting high biocompatibility and > 90 %excretion of QDs within 2weeks of intravenous administration. The P 3-QDs were conjugated to an engineered anti-CD8 diabody (Cys-diabody) for in vivo molecular imaging of CD8 + cytotoxic Tlymphocytes (CTLs) in response to anti-PD-L1 therapy. Tw o-plex molecular imaging in combination with down-conversion Er nanoparticles (ErNPs) was performed for real-time in vivo monitoring of PD-L1 positive tumor cells and CTLs with cellular resolution by non-invasive NIR-IIb light sheet microscopy. Imaging of angiogenesis in the tumor microenvironment and of lymph nodes deep in the body with asignal-to-background ratio of up to % 170 was also achieved using P 3-QDs.