Dramatic Impact of Materials Combinations on the Chemical Organization of Core-Shell Nanocrystals: Boosting the Tm3+ Emission above 1600 nm.

This article represents the first foray into investigating the consequences of various material combinations on the short-wave infrared (SWIR, 1000-2000 nm) performance of Tm-based core-shell nanocrystals (NCs) above 1600 nm. In total, six different material combinations involving two different types of SWIR-emitting core NCs (α-NaTmF4 and LiTmF4) combined with three different protecting shell materials (α-NaYF4, CaF2, and LiYF4) have been synthesized. All corresponding homo- and heterostructured NCs have been meticulously characterized by powder X-ray diffraction and electron microscopy techniques. The latter revealed that out of the six investigated combinations, only one led to the formation of a true core-shell structure with well-segregated core and shell domains. The direct correlation between the downshifting performance and the spatial localization of Tm3+ ions within the final homo- and heterostructured NCs is established. Interestingly, to achieve the best SWIR performance, the formation of an abrupt interface is not a prerequisite, while the existence of a pure (even thin) protective shell is vital. Remarkably, although all homo- and heterostructured NCs have been synthesized under the exact same experimental conditions, Tm3+ SWIR emission is either fully quenched or highly efficient depending on the type of material combination. The most efficient combination (LiTmF4/LiYF4) achieved a high photoluminescence quantum yield of 39% for SWIR emission above 1600 nm (excitation power density in the range 0.5-3 W/cm2) despite significant intermixing. From now on, highly efficient SWIR-emitting probes with an emission above 1600 nm are within reach to unlock the full potential of in vivo SWIR imaging.

Preventing cation intermixing enables 50% quantum yield in sub-15 nm short-wave infrared-emitting rare-earth based core-shell nanocrystals.

Short-wave infrared (SWIR) fluorescence could become the new gold standard in optical imaging for biomedical applications due to important advantages such as lack of autofluorescence, weak photon absorption by blood and tissues, and reduced photon scattering coefficient. Therefore, contrary to the visible and NIR regions, tissues become translucent in the SWIR region. Nevertheless, the lack of bright and biocompatible probes is a key challenge that must be overcome to unlock the full potential of SWIR fluorescence. Although rare-earth-based core-shell nanocrystals appeared as promising SWIR probes, they suffer from limited photoluminescence quantum yield (PLQY). The lack of control over the atomic scale organization of such complex materials is one of the main barriers limiting their optical performance. Here, the growth of either homogeneous (α-NaYF4) or heterogeneous (CaF2) shell domains on optically-active α-NaYF4:Yb:Er (with and without Ce3+ co-doping) core nanocrystals is reported. The atomic scale organization can be controlled by preventing cation intermixing only in heterogeneous core-shell nanocrystals with a dramatic impact on the PLQY. The latter reached 50% at 60 mW/cm2; one of the highest reported PLQY values for sub-15 nm nanocrystals. The most efficient nanocrystals were utilized for in vivo imaging above 1450 nm.

Shortwave infrared fluorescence imaging of peripheral organs in awake and freely moving mice.

Extracting biological information from awake and unrestrained mice is imperative to in vivo basic and pre-clinical research. Accordingly, imaging methods which preclude invasiveness, anesthesia, and/or physical restraint enable more physiologically relevant biological data extraction by eliminating these extrinsic confounders. In this article, we discuss the recent development of shortwave infrared (SWIR) fluorescent imaging to visualize peripheral organs in freely-behaving mice, as well as propose potential applications of this imaging modality in the neurosciences.

Development of a shortwave infrared sinuscope for the detection of cerebrospinal fluid leaks.

Significance: Cerebrospinal fluid (CSF) rhinorrhea (leakage of brain fluid from the nose) can be difficult to identify and currently requires invasive procedures, such as intrathecal fluorescein, which requires a lumbar drain placement. Fluorescein is also known to have rare but significant side effects including seizures and death. As the number of endonasal skull base cases increases, the number of CSF leaks has also increased for which an alternative diagnostic method would be highly advantageous to patients. Aim: We aim to develop an instrument to identify CSF leaks based on water absorption in the shortwave infrared (SWIR) without the need of intrathecal contrast agents. This device needed to be adapted to the anatomy of the human nasal cavity while maintaining low weight and ergonomic characteristics of current surgical instruments. Approach: Absorption spectra of CSF and artificial CSF were obtained to characterize the absorption peaks that could be targeted with SWIR light. Different illumination systems were tested and refined prior to adapting them into a portable endoscope for testing in 3D-printed models and cadavers for feasibility. Results: We identified CSF to have an identical absorption profile as water. In our testing, a narrowband laser source at 1480 nm proved superior to using a broad 1450 nm LED. Using a SWIR enabling endoscope set up, we tested the ability to detect artificial CSF in a cadaver model. Conclusions: An endoscopic system based on SWIR narrowband imaging can provide an alternative in the future to invasive methods of CSF leak detection.

Shortwave infrared (SWIR) fluorescence imaging of peripheral organs in awake and freely moving mice.

Extracting biological information from awake and unrestrained mice is imperative to in vivo basic and pre-clinical research. Accordingly, imaging methods which preclude invasiveness, anesthesia, and/or physical restraint enable more physiologically relevant biological data extraction by eliminating these extrinsic confounders. In this article we discuss the recent development of shortwave infrared (SWIR) fluorescent imaging to visualize peripheral organs in freely-behaving mice, as well as propose potential applications of this imaging modality in the neurosciences.

Targeted multicolor in vivo imaging over 1,000 nm enabled by nonamethine cyanines.

Recent progress has shown that using wavelengths between 1,000 and 2,000 nm, referred to as the shortwave-infrared or near-infrared (NIR)-II range, can enable high-resolution in vivo imaging at depths not possible with conventional optical wavelengths. However, few bioconjugatable probes of the type that have proven invaluable for multiplexed imaging in the visible and NIR range are available for imaging these wavelengths. Using rational design, we have generated persulfonated indocyanine dyes with absorbance maxima at 872 and 1,072 nm through catechol-ring and aryl-ring fusion, respectively, onto the nonamethine scaffold. Multiplexed two-color and three-color in vivo imaging using monoclonal antibody and dextran conjugates in several tumor models illustrate the benefits of concurrent labeling of the tumor and healthy surrounding tissue and lymphatics. These efforts are enabled by complementary advances in a custom-built NIR/shortwave-infrared imaging setup and software package for multicolor real-time imaging.

Bright Chromenylium Polymethine Dyes Enable Fast, Four-Color In Vivo Imaging with Shortwave Infrared Detection.

Optical imaging within the shortwave infrared (SWIR, 1000-2000 nm) region of the electromagnetic spectrum has enabled high-resolution and high-contrast imaging in mice, non-invasively. Polymethine dyes, with their narrow absorption spectra and high absorption coefficients, are optimal probes for fast and multiplexed SWIR imaging. Here, we expand upon the multiplexing capabilities in SWIR imaging by obtaining brighter polymethine dyes with varied excitation wavelengths spaced throughout the near-infrared (700-1000 nm) region. Building on the flavylium polymethine dye scaffold, we explored derivatives with functional group substitution at the 2-position, deemed chromenylium polymethine dyes. The reported dyes have reduced nonradiative rates and enhanced emissive properties, enabling non-invasive imaging in mice in a single color at 300 fps and in three colors at 100 fps. Combined with polymethine dyes containing a red-shifted julolidine flavylium heterocycle and indocyanine green, distinct channels with well-separated excitation wavelengths provide non-invasive video-rate in vivo imaging in four colors.

Lysosomal lipoprotein processing in endothelial cells stimulates adipose tissue thermogenic adaptation.

In response to cold exposure, thermogenic adipocytes internalize large amounts of fatty acids after lipoprotein lipase-mediated hydrolysis of triglyceride-rich lipoproteins (TRL) in the capillary lumen of brown adipose tissue (BAT) and white adipose tissue (WAT). Here, we show that in cold-exposed mice, vascular endothelial cells in adipose tissues endocytose substantial amounts of entire TRL particles. These lipoproteins subsequently follow the endosomal-lysosomal pathway, where they undergo lysosomal acid lipase (LAL)-mediated processing. Endothelial cell-specific LAL deficiency results in impaired thermogenic capacity as a consequence of reduced recruitment of brown and brite/beige adipocytes. Mechanistically, TRL processing by LAL induces proliferation of endothelial cells and adipocyte precursors via beta-oxidation-dependent production of reactive oxygen species, which in turn stimulates hypoxia-inducible factor-1α-dependent proliferative responses. In conclusion, this study demonstrates a physiological role for TRL particle uptake into BAT and WAT and establishes endothelial lipoprotein processing as an important determinant of adipose tissue remodeling during thermogenic adaptation.

Author Correction: Non-invasive monitoring of chronic liver disease via near-infrared and shortwave-infrared imaging of endogenous lipofuscin.

A Correction to this paper has been published: https://doi.org/10.1038/s41551-020-0569-y.

Shortwave infrared polymethine fluorophores matched to excitation lasers enable non-invasive, multicolour in vivo imaging in real time.

High-resolution, multiplexed experiments are a staple in cellular imaging. Analogous experiments in animals are challenging, however, due to substantial scattering and autofluorescence in tissue at visible (350-700 nm) and near-infrared (700-1,000 nm) wavelengths. Here, we enable real-time, non-invasive multicolour imaging experiments in animals through the design of optical contrast agents for the shortwave infrared (SWIR, 1,000-2,000 nm) region and complementary advances in imaging technologies. We developed tunable, SWIR-emissive flavylium polymethine dyes and established relationships between structure and photophysical properties for this class of bright SWIR contrast agents. In parallel, we designed an imaging system with variable near-infrared/SWIR excitation and single-channel detection, facilitating video-rate multicolour SWIR imaging for optically guided surgery and imaging of awake and moving mice with multiplexed detection. Optimized dyes matched to 980 nm and 1,064 nm lasers, combined with the clinically approved indocyanine green, enabled real-time, three-colour imaging with high temporal and spatial resolutions.

Non-invasive monitoring of chronic liver disease via near-infrared and shortwave-infrared imaging of endogenous lipofuscin.

Monitoring the progression of non-alcoholic fatty liver disease is hindered by a lack of suitable non-invasive imaging methods. Here, we show that the endogenous pigment lipofuscin displays strong near-infrared and shortwave-infrared fluorescence when excited at 808 nm, enabling label-free imaging of liver injury in mice and the discrimination of pathological processes from normal liver processes with high specificity and sensitivity. We also show that the near-infrared and shortwave-infrared fluorescence of lipofuscin can be used to monitor the progression and regression of liver necroinflammation and fibrosis in mouse models of non-alcoholic fatty liver disease and advanced fibrosis, as well as to detect non-alcoholic steatohepatitis and cirrhosis in biopsied samples of human liver tissue.

Shortwave Infrared Imaging with J-Aggregates Stabilized in Hollow Mesoporous Silica Nanoparticles.

Tissue is translucent to shortwave infrared (SWIR) light, rendering optical imaging superior in this region. However, the widespread use of optical SWIR imaging has been limited, in part, by the lack of bright, biocompatible contrast agents that absorb and emit light above 1000 nm. J-Aggregation offers a means to transform stable, near-infrared (NIR) fluorophores into red-shifted SWIR contrast agents. Here we demonstrate that J-aggregates of NIR fluorophore IR-140 can be prepared inside hollow mesoporous silica nanoparticles (HMSNs) to result in nanomaterials that absorb and emit SWIR light. The J-aggregates inside PEGylated HMSNs are stable for multiple weeks in buffer and enable high resolution imaging in vivo with 980 nm excitation.

Increasing the penetration depth of temporal focusing multiphoton microscopy for neurobiological applications.

The first ever demonstration of temporal focusing with short wave infrared (SWIR) excitation and emission is demonstrated, achieving a penetration depth of 500 µm in brain tissue. This is substantially deeper than the highest previously-reported values for temporal focusing imaging in brain tissue, and demonstrates the value of these optimized wavelengths for neurobiological applications.

Absorption by water increases fluorescence image contrast of biological tissue in the shortwave infrared.

Recent technology developments have expanded the wavelength window for biological fluorescence imaging into the shortwave infrared. We show here a mechanistic understanding of how drastic changes in fluorescence imaging contrast can arise from slight changes of imaging wavelength in the shortwave infrared. We demonstrate, in 3D tissue phantoms and in vivo in mice, that light absorption by water within biological tissue increases image contrast due to attenuation of background and highly scattered light. Wavelengths of strong tissue absorption have conventionally been avoided in fluorescence imaging to maximize photon penetration depth and photon collection, yet we demonstrate that imaging at the peak absorbance of water (near 1,450 nm) results in the highest image contrast in the shortwave infrared. Furthermore, we show, through microscopy of highly labeled ex vivo biological tissue, that the contrast improvement from water absorption enables resolution of deeper structures, resulting in a higher imaging penetration depth. We then illustrate these findings in a theoretical model. Our results suggest that the wavelength-dependent absorptivity of water is the dominant optical property contributing to image contrast, and is therefore crucial for determining the optimal imaging window in the infrared.

Shortwave infrared fluorescence imaging with the clinically approved near-infrared dye indocyanine green.

Fluorescence imaging is a method of real-time molecular tracking in vivo that has enabled many clinical technologies. Imaging in the shortwave IR (SWIR; 1,000-2,000 nm) promises higher contrast, sensitivity, and penetration depths compared with conventional visible and near-IR (NIR) fluorescence imaging. However, adoption of SWIR imaging in clinical settings has been limited, partially due to the absence of US Food and Drug Administration (FDA)-approved fluorophores with peak emission in the SWIR. Here, we show that commercially available NIR dyes, including the FDA-approved contrast agent indocyanine green (ICG), exhibit optical properties suitable for in vivo SWIR fluorescence imaging. Even though their emission spectra peak in the NIR, these dyes outperform commercial SWIR fluorophores and can be imaged in the SWIR, even beyond 1,500 nm. We show real-time fluorescence imaging using ICG at clinically relevant doses, including intravital microscopy, noninvasive imaging in blood and lymph vessels, and imaging of hepatobiliary clearance, and show increased contrast compared with NIR fluorescence imaging. Furthermore, we show tumor-targeted SWIR imaging with IRDye 800CW-labeled trastuzumab, an NIR dye being tested in multiple clinical trials. Our findings suggest that high-contrast SWIR fluorescence imaging can be implemented alongside existing imaging modalities by switching the detection of conventional NIR fluorescence systems from silicon-based NIR cameras to emerging indium gallium arsenide-based SWIR cameras. Using ICG in particular opens the possibility of translating SWIR fluorescence imaging to human clinical applications. Indeed, our findings suggest that emerging SWIR-fluorescent in vivo contrast agents should be benchmarked against the SWIR emission of ICG in blood.

Brown adipose tissue thermogenic adaptation requires Nrf1-mediated proteasomal activity.

Adipocytes possess remarkable adaptive capacity to respond to nutrient excess, fasting or cold exposure, and they are thus an important cell type for the maintenance of proper metabolic health. Although the endoplasmic reticulum (ER) is a critical organelle for cellular homeostasis, the mechanisms that mediate adaptation of the ER to metabolic challenges in adipocytes are unclear. Here we show that brown adipose tissue (BAT) thermogenic function requires an adaptive increase in proteasomal activity to secure cellular protein quality control, and we identify the ER-localized transcription factor nuclear factor erythroid 2-like 1 (Nfe2l1, also known as Nrf1) as a critical driver of this process. We show that cold adaptation induces Nrf1 in BAT to increase proteasomal activity and that this is crucial for maintaining ER homeostasis and cellular integrity, specifically when the cells are in a state of high thermogenic activity. In mice, under thermogenic conditions, brown-adipocyte-specific deletion of Nfe2l1 (Nrf1) resulted in ER stress, tissue inflammation, markedly diminished mitochondrial function and whitening of the BAT. In mouse models of both genetic and dietary obesity, stimulation of proteasomal activity by exogenously expressing Nrf1 or by treatment with the proteasome activator PA28α in BAT resulted in improved insulin sensitivity. In conclusion, Nrf1 emerges as a novel guardian of brown adipocyte function, providing increased proteometabolic quality control for adapting to cold or to obesity.

Wide-field three-photon excitation in biological samples.

Three-photon wide-field depth-resolved excitation is used to overcome some of the limitations in conventional point-scanning two- and three-photon microscopy. Excitation of chromophores as diverse as channelrhodopsins and quantum dots is shown, and a penetration depth of more than 700 µm into fixed scattering brain tissue is achieved, approximately twice as deep as that achieved using two-photon wide-field excitation. Compatibility with live animal experiments is confirmed by imaging the cerebral vasculature of an anesthetized mouse; a complete focal stack was obtained without any evidence of photodamage. As an additional validation of the utility of wide-field three-photon excitation, functional excitation is demonstrated by performing three-photon optogenetic stimulation of cultured mouse hippocampal neurons expressing a channelrhodopsin; action potentials could reliably be excited without causing photodamage.

Next-generation in vivo optical imaging with short-wave infrared quantum dots.

For in vivo imaging, the short-wavelength infrared region (SWIR; 1000-2000 nm) provides several advantages over the visible and near-infrared regions: general lack of autofluorescence, low light absorption by blood and tissue, and reduced scattering. However, the lack of versatile and functional SWIR emitters has prevented the general adoption of SWIR imaging by the biomedical research community. Here, we introduce a class of high-quality SWIR-emissive indium-arsenide-based quantum dots (QDs) that are readily modifiable for various functional imaging applications, and that exhibit narrow and size-tunable emission and a dramatically higher emission quantum yield than previously described SWIR probes. To demonstrate the unprecedented combination of deep penetration, high spatial resolution, multicolor imaging and fast-acquisition-speed afforded by the SWIR QDs, we quantified, in mice, the metabolic turnover rates of lipoproteins in several organs simultaneously and in real time as well as heartbeat and breathing rates in awake and unrestrained animals, and generated detailed three-dimensional quantitative flow maps of the mouse brain vasculature.

Shortwave Infrared in Vivo Imaging with Gold Nanoclusters.

The use of visible/NIR-emitting gold nanoclusters (Au NCs), previously proposed for in vivo imaging, has been limited to some extent by low quantum yields (QYs) and the limited penetration of visible light in tissue. Here we report short wavelength infrared (SWIR, λ = 1-2 µm) emitting Au NCs with a good photoluminescence QY for this wavelength range (0.6% to 3.8% for λem = 1000 to 900 nm) and excellent stability under physiological conditions. We show that surface ligand chemistry is critical to achieving these properties. We demonstrate the potential of these SWIR-emitting Au NCs for in vivo imaging in mice. The Au NCs have a hydrodynamic diameter that is small (∼5 nm) enough that they exhibit a rapid renal clearance, and images taken in the SWIR region show better resolution of the blood vessels than in the NIR region.

Flavylium Polymethine Fluorophores for Near- and Shortwave Infrared Imaging.

Bright fluorophores in the near-infrared and shortwave infrared (SWIR) regions of the electromagnetic spectrum are essential for optical imaging in vivo. In this work, we utilized a 7-dimethylamino flavylium heterocycle to construct a panel of novel red-shifted polymethine dyes, with emission wavelengths from 680 to 1045 nm. Photophysical characterization revealed that the 1- and 3-methine dyes display enhanced photostability and the 5- and 7-methine dyes exhibit exceptional brightness for their respective spectral regions. A micelle formulation of the 7-methine facilitated SWIR imaging in mice. This report presents the first polymethine dye designed and synthesized for SWIR in vivo imaging.