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Extracellular vesicles (EVs), including exosomes, are circulating nanoscale particles heavily implicated in cell signaling and can be isolated in vast numbers from human biofluids. Study of their molecular profiling and materials properties is currently underway for purposes of describing a variety of biological functions and diseases. However, the large, and as yet largely unquantified, variety of EV subpopulations differing in composition, size, and likely function necessitates characterization schemes capable of measuring single vesicles. Here we describe the first application of multispectral optical tweezers (MS-OTs) to single vesicles for molecular fingerprinting of EV subpopulations. This versatile imaging platform allows for sensitive measurement of Raman chemical composition (e.g., variation in protein, lipid, cholesterol, nucleic acids), coupled with discrimination by fluorescence markers. For exosomes isolated by ultracentrifugation, we use MS-OTs to interrogate the CD9-positive subpopulations via antibody fluorescence labeling and Raman spectra measurement. We report that the CD9-positive exosome subset exhibits reduced component concentration per vesicle and reduced chemical heterogeneity compared to the total purified EV population. We observed that specific vesicle subpopulations are present across exosomes isolated from cell culture supernatant of several clonal varieties of mesenchymal stromal cells and also from plasma and ascites isolated from human ovarian cancer patients.

The aim of this study is to evaluate the capability of femtosecond Laser Induced Breakdown Spectroscopy (fsLIBS) to discriminate between normal and cancerous bone, with implications to femtosecond laser surgery procedures. The main advantage of using femtosecond lasers for surgery is that the same laser that is being used to ablate can also be used for a feedback system to prevent ablation of certain tissues. For bone tumor removal, this technique has the potential to reduce the number of repeat surgeries that currently must be performed due to incomplete removal of the tumor mass. In this paper, we performed fsLIBS on primary bone tumor, secondary tumor in bone, and normal bone. These tissues were excised from consenting patients and processed through the UC Davis Cancer Center Biorepository. For comparison, each tumor sample had a matched normal bone sample. fsLIBS was performed to characterize the spectral signatures of each tissue type. A minimum of 20 spectra were acquired for each sample. We did not detect significant differences between the fsLIBS spectra of secondary bone tumors and their matched normal bone samples, likely due to the heterogeneous nature of secondary bone tumors, with normal and cancerous tissue intermingling. However, we did observe an increase in the fsLIBS magnesium peak intensity relative to the calcium peak intensity for the primary bone tumor samples compared to the normal bone samples. These results show the potential of using femtosecond lasers for both ablation and a real-time feedback control system for treatment of primary bone tumors.

Current analysis of exosomes focuses primarily on bulk analysis, where exosome-to-exosome variability cannot be assessed. In this study, we used Raman spectroscopy to study the chemical composition of single exosomes. We measured spectra of individual exosomes from 8 cell lines. Cell-line-averaged spectra varied considerably, reflecting the variation in total exosomal protein, lipid, genetic, and cytosolic content. Unexpectedly, single exosomes isolated from the same cell type also exhibited high spectral variability. Subsequent spectral analysis revealed clustering of single exosomes into 4 distinct groups that were not cell-line specific. Each group contained exosomes from multiple cell lines, and most cell lines had exosomes in multiple groups. The differences between these groups are related to chemical differences primarily due to differing membrane composition. Through a principal components analysis, we identified that the major sources of spectral variation among the exosomes were in cholesterol content, relative expression of phospholipids to cholesterol, and surface protein expression. For example, exosomes derived from cancerous versus non-cancerous cell lines can be largely separated based on their relative expression of cholesterol and phospholipids. We are the first to indicate that exosome subpopulations are shared among cell types, suggesting distributed exosome functionality. The origins of these differences are likely related to the specific role of extracellular vesicle subpopulations in both normal cell function and carcinogenesis, and they may provide diagnostic potential at the single exosome level.

Feral Mallards (Anas platyrhynchos) habitat use patterns and behavior are little documented. We quantified activities and habitat use by feral Mallards around urban ponds in Tampa Florida. We recorded 201 activity budgets of 20 min, sampled at 15-sec intervals, during daytime hours in 2019-2021. Male and female feral Mallards spent most time in grass (39.5%), followed by shoreline (27.1%), water (23.7%), tree-shrub (8.2%), and urban (1.4%). A use-availability analysis indicated that feral Mallards selected for water and tree cover but against urban land. The findings have implications for the conservation and management of both nuisance and native waterfowl in urban areas. [ABSTRACT FROM AUTHOR]

Raman hyperspectral imaging is an effective method for label-free imaging with chemical specificity, yet the weak signals and correspondingly long integration times have hindered its wide adoption as a routine analytical method. Recently, low resolution Raman imaging has been proposed to improve the spectral signal-to-noise ratio, which significantly improves the speed of Raman imaging. In this paper, low resolution Raman spectroscopy is combined with "context-aware" matrix completion, where regions of the sample that are not of interest are skipped, and the regions that are measured are under-sampled, then reconstructed with a low-rank constraint. Both simulations and experiment show that low-resolution Raman boosts the speed and image quality of the computationally-reconstructed Raman images, allowing deeper sub-sampling, reduced exposure time, and an overall >10-fold improvement in imaging speed, without sacrificing chemical specificity or spatial image quality. As the method utilizes traditional point-scan imaging, it retains full confocality and is "backwards-compatible" with pre-existing traditional Raman instruments, broadening the potential scope of Raman imaging applications. [ABSTRACT FROM AUTHOR]

The weak signal strength of Raman imaging leads to long imaging times. To increase the speed of Raman imaging, line scanning and compressed Raman imaging methods have been proposed. Here we combine both line scanning and compressed sensing to further increase the speed. However, the direct combination leads to poor reconstruction results due to the missed coverage of the sample. To avoid this issue, "full-coverage" Compressed Line-scan Raman Imaging (FC-CLRI) is proposed, where line positions are random but constrained to measure each line position of the sample at least once. In proof-of-concept studies of polymer beads and yeast cells, FC-CLRI achieved reasonable image quality while making only 20–40% of the measurements of a fully-sampled line-scan image, achieving 640 μm2 FOV imaging in <2 min with 1.5 mW μm−2 laser power. Furthermore, we critically evaluate the CLRI method through comparison with simple downsampling, and have found that FC-CLRI preserves spatial resolution better while naïve downsampling provides an overall higher image quality for complex samples. [ABSTRACT FROM AUTHOR]

A microscopy system has been constructed that is capable of simultaneously acquiring both Raman spectra and angle-resolved elastic-scattering patterns in either epi- or transillumination modes with a 7 [micro]m spot size. The benefits and drawbacks of the epi- and transillumination modalities are discussed. Validation studies have been performed on single beads of a few micrometers in size, as well as on ensembles of submicrometer particles. In addition, transilluminated Raman and elastic-scattering spectra were obtained from single granulocytes and peripheral blood monocytes. Both the Raman- and the elastic-scattering channels show clear differences between the two types of immune cells. OCIS codes: 170.5660, 290.4020.

The article argues that artificial intelligence (AI) techniques violate the pretext doctrine in administrative law, applying the U.S. Supreme Court's rationale to AI techniques and articulation of the pretext doctrine in the case Department of Commerce v. New York.

Dark‐field microscopy is known to offer both high resolution and direct visualization of thin samples. However, its performance and optimization on thick samples is under‐explored and so far, only meso‐scale information from whole organisms has been demonstrated. In this work, we carefully investigate the difference between trans‐ and epi‐illumination configurations. Our findings suggest that the epi‐illumination configuration is superior in both contrast and fidelity compared to trans‐illumination, while having the added advantage of experimental simplicity and an "open top" for experimental intervention. Guided by the theoretical analysis, we constructed an epi‐illumination dark‐field microscope with measured lateral and axial resolutions of 260 nm and 520 nm, respectively. Subcellular structures in whole organisms were directly visualized without the need for image reconstruction, and further confirmed via simultaneous fluorescence imaging. With an imaging speed of 20 to 50 fps, we visualize fast dynamic processes such as cell division and pharyngeal pumping in Caenorhabditis elegans. [ABSTRACT FROM AUTHOR]