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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. Keywords: shortwave infrared; biomedical imaging; fluorescence imaging green; near infrared; indocyanine, United States. Department of Energy. Office of Basic Energy Sciences (Award DE-FG02-07ER46454)

For in vivo imaging, the short-wavelength infrared region (SWIR; 1,000-2,000 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 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, multicolour imaging and fast acquisition speed afforded by the SWIR quantum dots, 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., National Institutes of Health (U.S.) (Grant 5-U54-CA151884), National Institutes of Health (U.S.) (Grant P01-CA080124), Massachusetts Institute of Technology. Institute for Soldier Nanotechnologies (Contract W911NF-13-D-0001), National Science Foundation (U.S.) (Grant ECCS-1449291), United States. Department of Energy. Office of Basic Energy Sciences (Grant DE-SC0001088)

Medical imaging is routine in the diagnosis and staging of a wide range of medical conditions. In particular, magnetic resonance imaging (MRI) is critical for visualizing soft tissue and organs, with over 60 million MRI procedures performed each year worldwide. About one-third of these procedures are contrast-enhanced MRI, and gadolinium-based contrast agents (GBCAs) are the mainstream MRI contrast agents used in the clinic. GBCAs have shown efficacy and are safe to use with most patients; however, some GBCAs have a small risk of adverse effects, including nephrogenic systemic fibrosis (NSF), the untreatable condition recently linked to gadolinium (Gd) exposure during MRI with contrast. In addition, Gd deposition in the human brain has been reported following contrast, and this is now under investigation by the US Food and Drug Administration (FDA). To address a perceived need for a Gd-free contrast agent with pharmacokinetic and imaging properties comparable to GBCAs, we have designed and developed zwitterion-coated exceedingly small superparamagnetic iron oxide nanoparticles (ZES-SPIONs) consisting of ∼3-nm inorganic cores and ∼1-nm ultrathin hydrophilic shell. These ZES-SPIONs are free of Gd and show a high T1 contrast power. We demonstrate the potential of ZES-SPIONs in preclinical MRI and magnetic resonance angiography., National Institutes of Health (U.S.) (Grant 1U54-CA119349), National Institutes of Health (U.S.) (Grant 9-P41-EB015871-26A1), United States. Army Research Office (Grant W911NF-07-D-0004), National Institutes of Health (U.S.) (R01-MH103160), National Institutes of Health (U.S.) (R01-DA028299)

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., National Science Foundation (U.S.) (EECS-1449291), National Institutes of Health (U.S.) (Massachusetts Institute of Technology. Laser Biomedical Research Center. 9-P41-EB015871-26A1), Massachusetts Institute of Technology. Institute for Soldier Nanotechnologies (W911NF-13-D-0001), Boehringer Ingelheim Fonds, European Molecular Biology Organization (Long-term Fellowship), National Science Foundation (U.S.). Graduate Research Fellowship Program, American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowship, United States. Dept. of Energy. Center for Excitonics (DE- SC0001088).

Micelles have been employed to encapsulate the supramolecular assembly of quantum dots with palladium(II) porphyrins for the quantification of O₂ levels in aqueous media and in vivo. Förster resonance energy transfer from the quantum dot (QD) to the palladium porphyrin provides a means for signal transduction under both one- and two-photon excitation. The palladium porphyrins are sensitive to O₂ concentrations in the range of 0–160 Torr. The micelle-encapsulated QD-porphyrin assemblies have been employed for in vivo multiphoton imaging and lifetime-based oxygen measurements in mice with chronic dorsal skinfold chambers or cranial windows. Our results establish the utility of the QD-micelle approach for in vivo biological sensing applications., National Cancer Institute (U.S.) (R01- CA126642), International Society for Neurochemistry (W911NF-07-D-0004), United States. Dept. of Energy. Office of Basic Energy Sciences (DE-SC0009758)

Magneto-fluorescent particles have been recognized as an emerging class of materials that exhibit great potential in advanced applications. However, synthesizing such magneto-fluorescent nanomaterials that simultaneously exhibit uniform and tunable sizes, high magnetic content loading, maximized fluorophore coverage at the surface and a versatile surface functionality has proven challenging. Here we report a simple approach for co-assembling magnetic nanoparticles with fluorescent quantum dots to form colloidal magneto-fluorescent supernanoparticles. Importantly, these supernanoparticles exhibit a superstructure consisting of a close-packed magnetic nanoparticle ‘core’, which is fully surrounded by a ‘shell’ of fluorescent quantum dots. A thin layer of ​silica coating provides high colloidal stability and biocompatibility, and a versatile surface functionality. We demonstrate that after surface pegylation, these ​silica-coated magneto-fluorescent supernanoparticles can be magnetically manipulated inside living cells while being optically tracked. Moreover, our ​silica-coated magneto-fluorescent supernanoparticles can also serve as an in vivo multi-photon and magnetic resonance dual-modal imaging probe., National Institutes of Health (U.S.) (5-U54-CA151884), National Institutes of Health (U.S.) (R01-CA126642), Massachusetts Institute of Technology. Institute for Soldier Nanotechnologies (W911NF-13-D-0001)), United States. Dept. of Energy (DE-FG02-07ER46454), National Institutes of Health (U.S.) (Massachusetts Institute of Technology. Laser Biomedical Research Center 9-P41-EB015871-26A1), United States. Dept. of Energy. Office of Basic Energy Sciences (Award DE-SC0001088), United States. Dept. of Defense (Breast Cancer Research Innovator Award W81XWH-10-1-0016)), Human Frontier Science Program (Strasbourg, France) (Grant RGP0005/2007), National Institutes of Health (U.S.) (R01-DA028299), National Institutes of Health (U.S.) (R01-NS076462), European Molecular Biology Organization (Long-term Fellowship)

Metastasis formation is the major reason for the extremely poor prognosis in small cell lung cancer (SCLC) patients. The molecular interaction partners regulating metastasis formation in SCLC are largely unidentified, however, from other tumor entities it is known that tumor cells use the adhesion molecules of the leukocyte adhesion cascade to attach to the endothelium at the site of the future metastasis. Using the human OH-1 SCLC line as a model, we found that these cells expressed E- and P-selectin binding sites, which could be in part attributed to the selectin binding carbohydrate motif sialyl Lewis A. In addition, protein backbones known to carry these glycotopes in other cell lines including PSGL-1, CD44 and CEA could be detected in in vitro and in vivo grown OH1 SCLC cells. By intravital microscopy of murine mesenterial vasculature we could capture SCLC cells while rolling along vessel walls demonstrating that SCLC cells mimic leukocyte rolling behavior in terms of selectin and selectin ligand interaction in vivo indicating that this mechanism might indeed be important for SCLC cells to seed distant metastases. Accordingly, formation of spontaneous distant metastases was reduced by 50% when OH-1 cells were xenografted into E-/P-selectin-deficient mice compared with wild type mice (p = 0.0181). However, as metastasis formation was not completely abrogated in selectin deficient mice, we concluded that this adhesion cascade is redundant and that other molecules of this cascade mediate metastasis formation as well. Using several of these adhesion molecules as interaction partners presumably make SCLC cells so highly metastatic., Bundesministerium für Wissenschaft und Forschung (TOMCAT, grant number 01EZ0824; http://www.bmbf.de/), Landesexzellenzinitiative Hamburg (Nanotechnology in Medicine – NAME)

We have recently developed compact and water-soluble zwitterionic dopamine sulfonate (ZDS) ligand coated superparamagnetic iron oxide nanoparticles (SPIONs) for use in various biomedical applications. The defining characteristics of ZDS-coated SPIONs are small hydrodynamic diameters, low non-specific interactions with fetal bovine serum, the opportunity for specific labeling, and stability with respect to time, pH, and salinity. We report here on the magnetic characterization of ZDS-coated SPIONs and their in vitro and in vivo performance relative to non-specific interactions with HeLa cells and in mice, respectively. ZDS-coated SPIONs retained the superparamagnetism and saturation magnetization (M[subscript s]) of as-synthesized hydrophobic SPIONs, with M[subscript s] = 74 emu g[superscript −1] [Fe]. Moreover, ZDS-coated SPIONs showed only small non-specific uptake into HeLa cancer cells in vitro and low non-specific binding to serum proteins in vivo in mice., National Institutes of Health (U.S.) (MIT-Harvard Center for Cancer Nanotechnology Excellence Grant 1U54-CA119349), National Cancer Institute (U.S.) (Grant R01-CA126642), Massachusetts Institute of Technology. Institute for Soldier Nanotechnologies (W911NF-07-D-0004), National Science Foundation (U.S.) (Collaborative Research in Chemistry Program CHE-0714189)

Members of the carcinoembryonic antigen cell adhesion molecules (CEACAMs) family are the prototype of tumour markers. Classically they are used as serum markers, however, CEACAMs could serve as targets for molecular imaging as well. In order to test the anti CEACAM monoclonal antibody T84.1 for imaging purposes, CEACAM expression was analysed using this antibody. Twelve human cancer cell lines from different entities were screened for their CEACAM expression using qPCR, Western Blot and FACS analysis. In addition, CEACAM expression was analyzed in primary tumour xenografts of these cells. Nine of 12 tumour cell lines expressed CEACAM mRNA and protein when grown in vitro. Pancreatic and colon cancer cell lines showed the highest expression levels with good correlation of mRNA and protein level. However, when grown in vivo, the CEACAM expression was generally downregulated except for the melanoma cell lines. As the CEACAM expression showed pronounced expression in FemX-1 primary tumours, this model system was used for further experiments. As the accessibility of the antibody after i.v. application is critical for its use in molecular imaging, the binding of the T84.1 monoclonal antibody was assessed after i.v. injection into SCID mice harbouring a FemX-1 primary tumour. When applied i.v., the CEACAM specific T84.1 antibody bound to tumour cells in the vicinity of blood vessels. This binding pattern was particularly pronounced in the periphery of the tumour xenograft, however, some antibody binding was also observed in the central areas of the tumour around blood vessels. Still, a general penetration of the tumour by i.v. application of the anti CEACAM antibody could not be achieved despite homogenous CEACAM expression of all melanoma cells when analysed in tissue sections. This lack of penetration is probably due to the increased interstitial fluid pressure in tumours caused by the absence of functional lymphatic vessels., Germany. Bundesministerium für Bildung und Forschung (TOMCAT, grant number 01EZ0824)