Fluorescence Nanoscopy in Whole Cells by Asynchronous Localization of Photoswitching Emitters

We demonstrate nanoscale resolution in far-field fluorescence microscopy using reversible photoswitching and localization of individual fluorophores at comparatively fast recording speeds and from the interior of intact cells. These advancements have become possible by asynchronously recording the photon bursts of individual molecular switching cycles. We present images from the microtubular network of an intact mammalian cell with a resolution of 40 nm

Deep tissue volumetric optoacoustic tracking of individual circulating tumor cells in an intracardially perfused mouse model

Widespread metastasis is the major cause of death from melanoma and other types of cancer. At present, the dynamic aspects of the metastatic cascade remain enigmatic. The feasibility to track circulating melanoma cells deep within living intact organisms can greatly impact our knowledge on tumor metastasis, but existing imaging approaches lack the sensitivity, spatio-temporal resolution or penetration depth to capture flowing tumor cells over large fields of view within optically-opaque biological tissues. Vast progress with the development of optoacoustic tomography technologies has recently enabled two- and three-dimensional imaging at unprecedented frame rates in the order of hundreds of Hertz, effectively mapping up to a million image voxels within a single volumetric snapshot. Herein, we employ volumetric optoacoustic tomography for real-time visualization of passage and trapping of individual B16 melanoma cells in the whole mouse brain. Detection of individual circulating melanoma cells was facilitated by substituting blood with an artificial cerebrospinal fluid that removes the strong absorption background in the optoacoustic images. The approach can provide new opportunities for studying trafficking and accumulation of metastatic melanoma cells in different organs.ISSN:1522-8002ISSN:1476-558

Functional multispectral optoacoustic tomography imaging of hepatic steatosis development in mice

Abstract The increasing worldwide prevalence of obesity, fatty liver diseases and the emerging understanding of the important roles lipids play in various other diseases is generating significant interest in lipid research. Lipid visualization in particular can play a critical role in understanding functional relations in lipid metabolism. We investigated the potential of multispectral optoacoustic tomography (MSOT) as a novel modality to non‐invasively visualize lipids in laboratory mice around the 930nm spectral range. Using an obesity‐induced non‐alcoholic fatty liver disease (NAFLD) mouse model, we examined whether MSOT could detect and differentiate different grades of hepatic steatosis and monitor the accumulation of lipids in the liver quantitatively over time, without the use of contrast agents, i.e. in label‐free mode. Moreover, we demonstrate the efficacy of using the real‐time clearance kinetics of indocyanine green (ICG) in the liver, monitored by MSOT, as a biomarker to evaluate the organ’s function and assess the severity of NAFLD. This study establishes MSOT as an efficient imaging tool for lipid visualization in preclinical studies, particularly for the assessment of NAFLD

Fully Biogenic Near-Infrared Phosphors: Phycobiliproteins and Cellulose at Force Toward Highly Efficient and Stable Bio-Hybrid Light-Emitting Diodes

<p>Stable/efficient low-energy emitters for photon down-conversion in bio-hybrid light-emitting diodes (Bio-HLEDs) are still challenging, as the archetypal fluorescent protein (FP) mCherry has led to the best deep-red Bio-HLEDs with poor stabilities: 3 h (on-chip)/160 h (remote). Capitalizing on the excellent refolding under temperature/pH/chemical stress, high brightness, and high compatibility with polysaccharides of phycobiliproteins (smURFP), first-class low-energy emitting Bio-HLEDs are achieved. They outperform those with mCherry regardless of using reference polyethylene oxide (on-chip: 24 h vs. 3 h) and new biopolymer hydroxypropyl cellulose (HPC; on-chip: 44 h vs. 3 h) coatings. Fine optimization of smURFP-HPC-coatings leads to stable record devices (on-chip: 2600 h/108 days) compared to champion devices with perylene diimides (on-chip: <700 h) and artificial FPs (on-chip: 35 h). Finally, spectroscopy/computational/thermal assays confirm that device degradation is related to the photo-induced reduction of biliverdin to bilirubin. Overall, this study pinpoints a new family of biogenic emitters toward superior protein-based lighting.</p&gt

Bacterial outer membrane vesicles as cationic dye carriers for optoacoustics-guided phototherapy of cancer

Abstract Background Cationic dyes are widely used as biomarkers for optical imaging. However, most of these are hydrophobic and cannot be employed in vivo without chemical conjugation or modification. Herein, we report for the first time the use of bacterial outer membrane vesicles (OMVs) as nanocarriers of cationic dyes for cancer theranostics. Results We demonstrate that cationic dyes (IR780, Cy7, and Cy7.5) form stable complexes with negatively charged bacterial-OMVs, improving the dyes’ in vivo circulation and optoacoustic properties. Such OMV-Dye complexes are biodegradable and safe for in vivo applications. Importantly, this method of cationic dye loading is faster and easier than synthetic chemistry approaches, and the efficient tumor accumulation of OMV-Dyes enables sensitive tumor detection using optoacoustic technology. As a proof-of-concept, we generated OMV-IR780 for optoacoustics-guided in vivo tumor phototherapy in a mouse model. Conclusions Our results demonstrate cationic dye-bound OMVs as promising novel nanoagents for tumor theranostics

Generation of Monomeric Reversibly Switchable Red Fluorescent Proteins for Far-Field Fluorescence Nanoscopy

Reversibly switchable fluorescent proteins (RSFPs) are GFP-like proteins that may be repeatedly switched by irradiation with light from a fluorescent to a nonfluorescent state, and vice versa. They can be utilized as genetically encodable probes and bear large potential for a wide array of applications, in particular for new protein tracking schemes and subdiffraction resolution microscopy. However, the currently described monomeric RSFPs emit only blue-green or green fluorescence; the spectral window for their use is thus rather limited. Using a semirational engineering approach based on the crystal structure of the monomeric nonswitchable red fluorescent protein mCherry, we generated rsCherry and rsCherryRev. These two novel red fluorescent RSFPs exhibit fluorescence emission maxima at ∼610 nm. They display antagonistic switching modes, i.e., in rsCherry irradiation with yellow light induces the off-to-on transition and blue light the on-to-off transition, whereas in rsCherryRev the effects of the switching wavelengths are reversed. We demonstrate time-lapse live-cell subdiffraction microscopy by imaging rsCherryRev targeted to the endoplasmic reticulum utilizing the switching and localization of single molecules