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A Correction to this paper has been published: https://doi.org/10.1038/s41551-020-0569-y.
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Compared to the visible and near-infrared, the short-wave infrared region (SWIR; 1000-2000 nm) has excellent properties for in vivo imaging: low autofluorescence, reduced scattering, and a low-absorption cross-section of blood or tissue. However, the general adoption of SWIR imaging in biomedical research will be enhanced by a broader availability of versatile and bright contrast materials. Quantum dots (QDs) are bright and compact SWIR emitters with narrow size distributions and emission spectra, but their use is limited by the shortcomings of established ligand systems for SWIR QDs. Established ligands often result in SWIR probes with either limited colloidal stability, large size, or broad size distribution or a combination of all three. We present a polymeric QD ligand designed to be compatible with oleate-coated QDs. Our polymeric acid ligand is a copolymer bearing carboxylic acid anchoring groups and PEG-550 chains to solubilize the QD-ligand construct. After a mild and rapid ligand exchange, the resulting constructs are compact (<11 nm hydrodynamic diameter) and have narrow size distribution. Both qualities are preserved for several months in isotonic saline. The constructs are bright in vivo, and to demonstrate their suitability for imaging, we perform whole-body imaging and lymphatic imaging, including visualization of lymphatic flow.
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Ácidos Carboxílicos/química , Corantes Fluorescentes/química , Imagem Óptica/métodos , Pontos Quânticos/química , Alanina/química , Animais , Raios Infravermelhos , Ligantes , Linfonodos/diagnóstico por imagem , Masculino , Metacrilatos/química , Camundongos , Camundongos Nus , Ácido Oleico/química , Polietilenoglicóis/química , Solubilidade , Propriedades de Superfície , ÁguaRESUMO
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.
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Lipofuscina/metabolismo , Hepatopatias/diagnóstico por imagem , Hepatopatias/patologia , Animais , Biomarcadores/metabolismo , Doença Crônica , Progressão da Doença , Feminino , Fluorescência , Humanos , Lipodistrofia/diagnóstico por imagem , Lipodistrofia/metabolismo , Lipodistrofia/patologia , Fígado/diagnóstico por imagem , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/diagnóstico por imagem , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Hepatopatias/metabolismo , Masculino , Camundongos , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Imagem Óptica , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
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.
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Vasos Sanguíneos/diagnóstico por imagem , Meios de Contraste , Corantes Fluorescentes , Raios Infravermelhos , Microscopia Intravital/métodos , Vasos Linfáticos/diagnóstico por imagem , Animais , Bovinos , Meios de Contraste/farmacocinética , Meios de Contraste/farmacologia , Corantes Fluorescentes/farmacocinética , Corantes Fluorescentes/farmacologia , Verde de Indocianina , Camundongos , Microscopia de Fluorescência/métodos , Trastuzumab/farmacocinética , Trastuzumab/farmacologiaRESUMO
Fluorescent analogues of the indole side chain of tryptophan can be useful spectroscopic probes of protein-protein and protein-DNA interactions. Here we present linear dichroism and solvent-dependent spectroscopic studies of two fluorescent analogues of indole, in which the organic CâC unit is substituted with the isosteric inorganic B-N unit. We studied the so-called "external" BN indole, which has C2v symmetry, and the "fused" BN indole with Cs symmetry. We performed a combination of absorption and fluorescence spectroscopy, ultraviolet linear dichroism (UV-LD) in stretched poly(ethylene) (PE) films, and quantum chemical calculations on both BN indole compounds. Our measurements allowed us to characterize the degree of alignment for both molecules in stretched PE films. We thus determined the orientations and magnitudes of the two lowest energy electric dipole transition moments (EDTMs) for external BN indole, and the two lowest energy EDTMs for fused BN indole within the 30â¯000-45â¯000 cm(-1) spectral range. We compared our experimental results to those of quantum chemical calculations using standard density functional theory (DFT). Our theoretical predictions for the low-energy EDTMs are in good agreement with our experimental data. The absorption and fluorescence spectra of the external and the fused BN indoles are sensitive to solvent polarity. Our results indicate that the fused BN indole experiences much greater solvation interactions with polar solvents than does the external BN indole.
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Boro/química , Indóis/química , Nitrogênio/química , Solventes/química , Modelos Químicos , Estrutura Molecular , Polietileno/química , Teoria Quântica , Espectrometria de FluorescênciaRESUMO
Mutations near the fluorescing chromophore of the green fluorescent protein (GFP) have direct effects on the absorption and emission spectra. Some mutants have significant band shifts and most of the mutants exhibit a loss of fluorescence intensity. In this study we continue our investigation of the factors controlling the excited state proton transfer (PT) process of GFP, in particular to study the effects of modifications to the key side chain Ser205 in wt-GFP, proposed to participate in the proton wire. To this aim we combined mutagenesis, X-ray crystallography, steady-state spectroscopy, time-resolved emission spectroscopy and all-atom explicit molecular dynamics (MD) simulations to study the double mutant T203V/S205A. Our results show that while in the previously described GFP double mutant T203V/S205V the PT process does not occur, in the T203V/S205A mutant the PT process does occur, but with a 350 times slower rate than in wild-type GFP (wt-GFP). Furthermore, the kinetic isotope effect in the GFP double mutant T203V/S205A is twice smaller than in the wt-GFP and in the GFP single mutant S205V, which forms a novel PT pathway. On the other hand, the crystal structure of GFP T203V/S205A does not reveal a viable proton transfer pathway. To explain PT in GFP T203V/S205A, we argue on the basis of the MD simulations for an alternative, novel proton-wire pathway which involves the phenol group of the chromophore and water molecules infrequently entering from the bulk. This alternative pathway may explain the dramatically slow PT in the GFP double mutant T203V/S205A compared to wt-GFP.