Daniel Franke

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.

Continuous injection synthesis of indium arsenide quantum dots emissive in the short-wavelength infrared

With the emergence of applications based on short-wavelength infrared light, indium arsenide quantum dots are promising candidates to address existing shortcomings of other infrared-emissive nanomaterials. However, III–V quantum dots have historically struggled to match the high-quality optical properties of II–VI quantum dots. Here we present an extensive investigation of the kinetics that govern indium arsenide nanocrystal growth. Based on these insights, we design a synthesis of large indium arsenide quantum dots with narrow emission linewidths. We further synthesize indium arsenide-based core-shell-shell nanocrystals with quantum yields up to 82% and improved photo- and long-term storage stability. We then demonstrate non-invasive through-skull fluorescence imaging of the brain vasculature of murine models, and show that our probes exhibit 2–3 orders of magnitude higher quantum yields than commonly employed infrared emitters across the entire infrared camera sensitivity range. We anticipate that these probes will not only enable new biomedical imaging applications, but also improved infrared nanocrystal-LEDs and photon-upconversion technology.

Characterization of Indium Phosphide Quantum Dot Growth Intermediates Using MALDI-TOF Mass Spectrometry

Clusters have been identified as important growth intermediates during group III-V quantum dot (QD) formation. Here we report a one-solvent protocol that integrates synthesis, purification, and mass characterization of indium phosphide (InP) QD growth mixtures. The use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) successfully tracks the evolution of clusters and the formation of QDs throughout the synthesis. Similar clusters are observed during the formation of large particles, suggesting that these clusters serve as a reservoir for QD formation. Combining MALDI and NMR techniques further enables us to extract extinction coefficients and construct sizing curves for cluster-free InP QDs. The use of MALDI MS opens new opportunities for characterization and mechanistic studies of small-sized air-sensitive clusters or QDs.

The Unexpected Influence of Precursor Conversion Rate in the Synthesis of III–V Quantum Dots

Control of quantum dot (QD) precursor chemistry has been expected to help improve the size control and uniformity of III-V QDs such as indium phosphide and indium arsenide. Indeed, experimental results for other QD systems are consistent with the theoretical prediction that the rate of precursor conversion is an important factor controlling QD size and size distribution. We synthesized and characterized the reactivity of a variety of group-V precursors in order to determine if precursor chemistry could be used to improve the quality of III-V QDs. Despite slowing down precursor conversion rate by multiple orders of magnitude, the less reactive precursors do not yield the expected increase in size and improvement in size distribution. This result disproves the widely accepted explanation for the shortcoming of current III-V QD syntheses and points to the need for a new generalizable theoretical picture for the mechanism of QD formation and growth.

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.

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

Significance Imaging in the shortwave IR (SWIR) spectral window allows the observation of processes deep within living animals. Recent studies have shown that SWIR imaging enables unprecedented imaging opportunities, including contact-free monitoring of vital signs, generation of microvasculature blood flow maps, real-time metabolic imaging, and molecularly targeted imaging. Yet, whereas bright SWIR fluorophores have been developed for preclinical research settings, applications in the clinic have been held back by the conventional belief that no clinically approved fluorophore is available. Here, we show that indocyanine green, a clinically approved near-IR dye, exhibits a remarkable amount of SWIR emission, which enables state-of-the-art SWIR imaging with direct translation potential into clinical settings, and even outperforms other commercially available SWIR emitters. 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.

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

Significance Shortwave infrared (SWIR) fluorescence imaging is a tool for visualizing biological processes deep within tissue or living animals. Our study shows that the contrast in a SWIR fluorescence image is primarily mediated by the absorptivity of the tissue, and can therefore be tuned through deliberate selection of imaging wavelength. We show, for example, that, in 3D tissue phantoms and in brain vasculature in vivo in mice, imaging at SWIR wavelengths of the highest water absorptivity results in the greatest fluorescence contrast. We further demonstrate, in microscopy of ex vivo mouse liver tissue, that imaging at wavelengths of high tissue absorptivity can also increase imaging penetration depth, and use a theoretical contrast model to explain this effect. 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.

Near-Infrared Temporal Focusing Microscopy with Quantum Dot Fluorophores

The tissue penetration depth of temporal focusing is increased by utilizing longer excitation wavelengths combined with quantum dots possessing extremely high two-photon cross-sections. Cerebral vasculature is imaged in a mouse brain in vivo.