27-Hydroxycholesterol Enhances Secretion of Extracellular Vesicles by ROS-Induced Dysregulation of Lysosomes.

Extracellular vesicles (EVs) serve as crucial mediators of cell-to-cell communication in normal physiology as well as in diseased states, and have been largely studied in regard to their role in cancer progression. However, the mechanisms by which their biogenesis and secretion are regulated by metabolic or endocrine factors remain unknown. Here, we delineate a mechanism by which EV secretion is regulated by a cholesterol metabolite, 27-Hydroxycholesterol (27HC), where treatment of myeloid immune cells (RAW 264.7 and J774A.1) with 27HC impairs lysosomal homeostasis, leading to shunting of multivesicular bodies (MVBs) away from lysosomal degradation, towards secretion as EVs. This altered lysosomal function is likely caused by mitochondrial dysfunction and subsequent increase in reactive oxygen species (ROS). Interestingly, cotreatment with a mitochondria-targeted antioxidant rescued the lysosomal impairment and attenuated the 27HC-mediated increase in EV secretion. Overall, our findings establish how a cholesterol metabolite regulates EV secretion and paves the way for the development of strategies to regulate cancer progression by controlling EV secretion.

Cholesterol Metabolite 27-Hydroxycholesterol Enhances the Secretion of Cancer Promoting Extracellular Vesicles by a Mitochondrial ROS-Induced Impairment of Lysosomal Function.

Extracellular vesicles (EVs) serve as crucial mediators of cell-to-cell communication in normal physiology as well as in diseased states, and have been largely studied in regard to their role in cancer progression. However, the mechanisms by which their biogenesis and secretion are regulated by metabolic or endocrine factors remain unknown. Here, we delineate a mechanism by which EV secretion is regulated by a cholesterol metabolite, 27-Hydroxycholesterol (27HC), where treatment of myeloid immune cells (RAW 264.7 and J774A.1) with 27HC impairs lysosomal homeostasis, leading to shunting of multivesicular bodies (MVBs) away from lysosomal degradation, towards secretion as EVs. This impairment of lysosomal function is caused by mitochondrial dysfunction and subsequent increase in reactive oxygen species (ROS). Interestingly, cotreatment with a mitochondria-targeted antioxidant rescued the lysosomal impairment and attenuated the 27HC-mediated increase in EV secretion. Overall, our findings establish how a cholesterol metabolite regulates EV secretion and paves the way for the development of strategies to regulate cancer progression by controlling EV secretion.

Large field-of-view metabolic profiling of murine brain tissue following morphine incubation using label-free multiphoton microscopy.

BACKGROUND: Although the effects on neural activation and glucose consumption caused by opiates such as morphine are known, the metabolic machinery underlying opioid use and misuse is not fully explored. Multiphoton microscopy (MPM) techniques have been developed for optical imaging at high spatial resolution. Despite the increased use of MPM for neural imaging, the use of intrinsic optical contrast has seen minimal use in neuroscience. NEW METHOD: We present a label-free, multimodal microscopy technique for metabolic profiling of murine brain tissue following incubation with morphine sulfate (MSO4). We evaluate two- and three-photon excited autofluorescence, and second and third harmonic generation to determine meaningful intrinsic contrast mechanisms in brain tissue using simultaneous label-free, autofluorescence multi-harmonic (SLAM) microscopy. RESULTS: Regional differences quantified in the cortex, caudate, and thalamus of the brain demonstrate region-specific changes to metabolic profiles measured from FAD intensity, along with brain-wide quantification. While the overall intensity of FAD signal significantly decreased after morphine incubation, this metabolic molecule accumulated near the nucleus accumbens. COMPARISON WITH EXISTING METHODS: Histopathology requires tissue fixation and staining to determine cell type and morphology, lacking information about cellular metabolism. Tools such as fMRI or PET imaging have been widely used, but lack cellular resolution. SLAM microscopy obviates the need for tissue preparation, permitting immediate use and imaging of tissue with subcellular resolution in its native environment. CONCLUSIONS: This study demonstrates the utility of SLAM microscopy for label-free investigations of neural metabolism, especially the intensity changes in FAD autofluorescence and structural morphology from third-harmonic generation.

Label-free nonlinear optical signatures of extracellular vesicles in liquid and tissue biopsies of human breast cancer.

Extracellular vesicles (EVs) have been implicated in metastasis and proposed as cancer biomarkers. However, heterogeneity and small size makes assessments of EVs challenging. Often, EVs are isolated from biofluids, losing spatial and temporal context and thus lacking the ability to access EVs in situ in their native microenvironment. This work examines the capabilities of label-free nonlinear optical microscopy to extract biochemical optical metrics of EVs in ex vivo tissue and EVs isolated from biofluids in cases of human breast cancer, comparing these metrics within and between EV sources. Before surgery, fresh urine and blood serum samples were obtained from human participants scheduled for breast tumor surgery (24 malignant, 6 benign) or healthy participants scheduled for breast reduction surgery (4 control). EVs were directly imaged both in intact ex vivo tissue that was removed during surgery and in samples isolated from biofluids by differential ultracentrifugation. Isolated EVs and freshly excised ex vivo breast tissue samples were imaged with custom nonlinear optical microscopes to extract single-EV optical metabolic signatures of NAD(P)H and FAD autofluorescence. Optical metrics were significantly altered in cases of malignant breast cancer in biofluid-derived EVs and intact tissue EVs compared to control samples. Specifically, urinary isolated EVs showed elevated NAD(P)H fluorescence lifetime in cases of malignant cancer, serum-derived isolated EVs showed decreased optical redox ratio in stage II cancer, but not earlier stages, and ex vivo breast tissue showed an elevated number of EVs in cases of malignant cancer. Results further indicated significant differences in the measured optical metabolic signature based on EV source (urine, serum and tissue) within individuals.

Time-of-flight detection of terahertz phonon-polariton.

A polariton is a fundamental quasiparticle that arises from strong light-matter interaction and as such has attracted wide scientific and practical interest. When light is strongly coupled to the crystal lattice, it gives rise to phonon-polaritons (PPs), which have been proven useful in the dynamical manipulation of quantum materials and the advancement of terahertz technologies. Yet, current detection and characterization methods of polaritons are still limited. Traditional techniques such as Raman or transient grating either rely on fine-tuning of external parameters or complex phase extraction techniques. To overcome these inherent limitations, we propose and demonstrate a technique based on a time-of-flight measurement of PPs. We resonantly launch broadband PPs with intense terahertz fields and measure the time-of-flight of each spectral component with time-resolved second harmonic generation. The time-of-flight information, combined with the PP attenuation, enables us to resolve the real and imaginary parts of the PP dispersion relation. We demonstrate this technique in the van der Waals magnets NiI2 and MnPS3 and reveal a hidden magnon-phonon interaction. We believe that this approach will unlock new opportunities for studying polaritons across diverse material systems and enhance our understanding of strong light-matter interaction.

Simultaneous label-free autofluorescence multi-harmonic microscopy driven by the supercontinuum generated from a bulk nonlinear crystal.

Nonlinear microscopy encompasses several imaging techniques that leverage laser technology to probe intrinsic molecules of biological specimens. These native molecules produce optical fingerprints that allow nonlinear microscopes to reveal the chemical composition and structure of cells and tissues in a label-free and non-destructive fashion, information that enables a plethora of applications, e.g., real-time digital histopathology or image-guided surgery. Because state-of-the-art lasers exhibit either a limited bandwidth or reduced wavelength tunability, nonlinear microscopes lack the spectral support to probe different biomolecules simultaneously, thus losing analytical potential. Therefore, a conventional nonlinear microscope requires multiple or tunable lasers to individually excite endogenous molecules, increasing both the cost and complexity of the system. A solution to this problem is supercontinuum generation, a nonlinear optical phenomenon that supplies broadband femtosecond radiation, granting a wide spectrum for concurrent molecular excitation. This study introduces a source for nonlinear multiphoton microscopy based on the supercontinuum generation from a yttrium aluminum garnet (YAG) crystal, an approach that allows simultaneous label-free autofluorescence multi-harmonic imaging of biological samples and offers a practical and compact alternative for the clinical translation of nonlinear microscopy. While this supercontinuum covered the visible spectrum (550-900 nm) and the near-infrared region (950-1200 nm), the pulses within 1030-1150 nm produced label-free volumetric chemical images of ex vivo chinchilla kidney, thus validating the supercontinuum from bulk crystals as a powerful source for multimodal nonlinear microscopy.

In vivo label-free optical signatures of chemotherapy response in human pancreatic ductal adenocarcinoma patient-derived xenografts.

Pancreatic cancer is a devastating disease often detected at later stages, necessitating swift and effective chemotherapy treatment. However, chemoresistance is common and its mechanisms are poorly understood. Here, label-free multi-modal nonlinear optical microscopy was applied to study microstructural and functional features of pancreatic tumors in vivo to monitor inter- and intra-tumor heterogeneity and treatment response. Patient-derived xenografts with human pancreatic ductal adenocarcinoma were implanted into mice and characterized over five weeks of intraperitoneal chemotherapy (FIRINOX or Gem/NabP) with known responsiveness/resistance. Resistant and responsive tumors exhibited a similar initial metabolic response, but by week 5 the resistant tumor deviated significantly from the responsive tumor, indicating that a representative response may take up to five weeks to appear. This biphasic metabolic response in a chemoresistant tumor reveals the possibility of intra-tumor spatiotemporal heterogeneity of drug responsiveness. These results, though limited by small sample size, suggest the possibility for further work characterizing chemoresistance mechanisms using nonlinear optical microscopy.

Nonlinear Imaging Histopathology: A Pipeline to Correlate Gold-Standard Hematoxylin and Eosin Staining With Modern Nonlinear Microscopy.

Hematoxylin and eosin (H&E) staining, the century-old technique, has been the gold standard tool for pathologists to detect anomalies in tissues and diseases such as cancer. H&E staining is a cumbersome, time-consuming process that delays and wastes precious minutes during an intraoperative diagnosis. However, even in the modern era, real-time label-free imaging techniques such as simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy have delivered several more layers of information to characterize a tissue with high precision. Still, they have yet to translate to the clinic. The slow translation rate can be attributed to the lack of direct comparisons between the old and new techniques. Our approach to solving this problem is to: 1) reduce dimensions by pre-sectioning the tissue in 500 µm slices, and 2) produce fiducial laser markings which appear in both SLAM and histological imaging. High peak-power femtosecond laser pulses enable ablation in a controlled and contained manner. We perform laser marking on a grid of points encompassing the SLAM region of interest. We optimize laser power, numerical aperture, and timing to produce axially extended marking, hence multilayered fiducial markers, with minimal damage to the surrounding tissues. We performed this co-registration over an area of 3 × 3 mm2 of freshly excised mouse kidney and intestine, followed by standard H&E staining. Reduced dimensionality and the use of laser markings provided a comparison of the old and new techniques, giving a wealth of correlative information and elevating the potential of translating nonlinear microscopy to the clinic for rapid pathological assessment.

Weakly supervised identification of microscopic human breast cancer-related optical signatures from normal-appearing breast tissue.

With the latest advancements in optical bioimaging, rich structural and functional information has been generated from biological samples, which calls for capable computational tools to identify patterns and uncover relationships between optical characteristics and various biomedical conditions. Constrained by the existing knowledge of the novel signals obtained by those bioimaging techniques, precise and accurate ground truth annotations can be difficult to obtain. Here we present a weakly supervised deep learning framework for optical signature discovery based on inexact and incomplete supervision. The framework consists of a multiple instance learning-based classifier for the identification of regions of interest in coarsely labeled images and model interpretation techniques for optical signature discovery. We applied this framework to investigate human breast cancer-related optical signatures based on virtual histopathology enabled by simultaneous label-free autofluorescence multiharmonic microscopy (SLAM), with the goal of exploring unconventional cancer-related optical signatures from normal-appearing breast tissues. The framework has achieved an average area under the curve (AUC) of 0.975 on the cancer diagnosis task. In addition to well-known cancer biomarkers, non-obvious cancer-related patterns were revealed by the framework, including NAD(P)H-rich extracellular vesicles observed in normal-appearing breast cancer tissue, which facilitate new insights into the tumor microenvironment and field cancerization. This framework can be further extended to diverse imaging modalities and optical signature discovery tasks.

Coherent detection of hidden spin-lattice coupling in a van der Waals antiferromagnet.

Strong interactions between different degrees of freedom lead to exotic phases of matter with complex order parameters and emergent collective excitations. Conventional techniques, such as scattering and transport, probe the amplitudes of these excitations, but they are typically insensitive to phase. Therefore, novel methods with phase sensitivity are required to understand ground states with phase modulations and interactions that couple to the phase of collective modes. Here, by performing phase-resolved coherent phonon spectroscopy (CPS), we reveal a hidden spin-lattice coupling in a vdW antiferromagnet FePS3 that eluded other phase-insensitive conventional probes, such as Raman and X-ray scattering. With comparative analysis and analytical calculations, we directly show that the magnetic order in FePS3 selectively couples to the trigonal distortions through partially filled t2g orbitals. This magnetoelastic coupling is linear in magnetic order and lattice parameters, rendering these distortions inaccessible to inelastic scattering techniques. Our results not only capture the elusive spin-lattice coupling in FePS3 but also establish phase-resolved CPS as a tool to investigate hidden interactions.

Label-free optical redox ratio from urinary extracellular vesicles as a screening biomarker for bladder cancer.

Extracellular vesicles (EVs) have been studied for their potential applications in cancer screening, diagnosis, and treatment monitoring. Most studies have focused on the bulk content of EVs; however, it is also informative to investigate their metabolic status, and changes under different physiological and environmental conditions. In this study, noninvasive, multimodal, label-free nonlinear optical microscopy was used to evaluate the optical redox ratio of large EVs (microvesicles) isolated from the urine of 11 dogs in three cohorts (4 healthy, 4 transitional cell carcinoma (TCC) of the bladder, and 3 prostate cancer). The optical redox ratio is a common metric comparing the autofluorescence intensities of metabolic cofactors FAD and NAD(P)H to characterize the metabolic profile of cells and tissues, and has recently been applied to EVs. The optical redox ratio revealed that dogs with TCC of the bladder had a more than 2-fold increase in NAD(P)H-rich urinary EVs (uEVs) when compared to healthy dogs, whereas dogs with prostate cancer had no significant difference. The optical redox ratio values of uEVs kept at -20°C for 48 hours were significantly different from those of freshly isolated uEVs, indicating that this parameter is more reliable when assessing freshly isolated uEVs. These results suggest that the label-free optical redox ratio of uEVs, indicating relative rates of glycolysis and oxidative phosphorylation of parent cells and tissues, may act as a potential screening biomarker for bladder cancer.

Tracking the binding of multi-functional fluorescent tags for Alzheimer's disease using quantitative multiphoton microscopy.

A recent theranostic approach to address Alzheimer's disease (AD) utilizes multifunctional targets that both tag and negate the toxicity of AD biomarkers. These compounds, which emit fluorescence with both an activation and a spectral shift in the presence of Aß, were previously characterized with traditional fluorescence imaging for binary characterization. However, these multifunctional compounds have broad and dynamic emission spectra that are dependent on factors such as the local environment, presence of Aß deposits, etc. Since quantitative multiphoton microscopy is sensitive to the binding dynamics of molecules, we characterized the performance of two such compounds, LS-4 and ZY-12-OMe, using Simultaneous Label-free Autofluorescence Multi-harmonic (SLAM) microscopy and Fast Optical Coherence, Autofluorescence Lifetime imaging and Second harmonic generation (FOCALS) microscopy. This study shows that the combination of quantitative multiphoton imaging with multifunctional tags for AD offers new insights into the interaction of these tags with AD biomarkers and the theranostic mechanisms.

Label-free multimodal nonlinear optical imaging of needle biopsy cores for intraoperative cancer diagnosis.

SIGNIFICANCE: Needle biopsy (NB) procedures are important for the initial diagnosis of many types of cancer. However, the possibility of NB specimens being unable to provide diagnostic information, (i.e., non-diagnostic sampling) and the time-consuming histological evaluation process can cause delays in diagnoses that affect patient care. AIM: We aim to demonstrate the advantages of this label-free multimodal nonlinear optical imaging (NLOI) technique as a non-destructive point-of-procedure evaluation method for NB tissue cores, for the visualization and characterization of the tissue microenvironment. APPROACH: A portable, label-free, multimodal NLOI system combined second-harmonic generation (SHG) and third-harmonic generation and two- and three-photon autofluorescence (2PF, 3PF) microscopy. It was used for intraoperative imaging of fresh NB tissue cores acquired during canine cancer surgeries, which involved liver, lung, and mammary tumors as well as soft-tissue sarcoma; in total, eight canine patients were recruited. An added tissue culture chamber enabled the use of this NLOI system for longitudinal imaging of fresh NB tissue cores taken from an induced rat mammary tumor and healthy mouse livers. RESULTS: The intraoperative NLOI system was used to assess fresh canine NB specimens during veterinary cancer surgeries. Histology-like morphological features were visualized by the combination of four NLOI modalities at the point-of-procedure. The NLOI results provided quantitative information on the tissue microenvironment such as the collagen fiber orientation using Fourier-domain SHG analysis and metabolic profiling by optical redox ratio (ORR) defined by 2PF/(2PF + 3PF). The analyses showed that the canine mammary tumor had more randomly oriented collagen fibers compared to the tumor margin, and hepatocarcinoma had a wider distribution of ORR with a lower mean value compared to the liver fibrosis and the normal-appearing liver. Moreover, the loss of metabolic information during tissue degradation of fresh murine NB specimens was shown by overall intensity decreases in all channels and an increase of mean ORR from 0.94 (standard deviation 0.099) to 0.97 (standard deviation 0.077) during 1-h longitudinal imaging of a rat mammary tumor NB specimen. The tissue response to staurosporine (STS), an apoptotic inducer, from fresh murine liver NB specimens was also observed. The mean ORR decreased from 0.86 to 0.74 in the first 40 min and then increased to 0.8 during the rest of the hour of imaging, compared to the imaging results without the addition of STS, which showed a continuous increase of ORR from 0.72 to 0.75. CONCLUSIONS: A label-free, multimodal NLOI platform reveals microstructural and metabolic information of the fresh NB cores during intraoperative cancer imaging. This system has been demonstrated on animal models to show its potential to provide a more comprehensive histological assessment and a better understanding of the unperturbed tumor microenvironment. Considering tissue degradation, or loss of viability upon fixation, this intraoperative NLOI system has the advantage of immediate assessment of freshly excised tissue specimens at the point of procedure.

Label-Free Multimodal Multiphoton Intravital Imaging.

Label-free intravital optical imaging is an emergent visualization tool that is not only useful for basic biological research, but also for preclinical research with potential translational clinical applications. The complete absence of exogenous labeling or genetic alterations avoids plausible harmful perturbation to biological processes and the pristine physiological environment, as the endogenous biomolecules enable intrinsic imaging contrasts to interrogate various live multicellular organisms of interest. This tool has evolved from single-modality, single-photon imaging into multimodal multiphoton imaging, in order to gain different contrasts simultaneously during imaging sessions, and permit long-term time-lapse studies that have begun to spawn more diverse applications.

The Cholesterol Metabolite 27HC Increases Secretion of Extracellular Vesicles Which Promote Breast Cancer Progression.

Cholesterol has been implicated in the clinical progression of breast cancer, a disease that continues to be the most commonly diagnosed cancer in women. Previous work has identified the cholesterol metabolite 27-hydroxycholesterol (27HC) as a major mediator of the effects of cholesterol on breast tumor growth and progression. 27HC can act as an estrogen receptor (ER) modulator to promote the growth of ERα+ tumors, and as a liver X receptor (LXR) ligand in myeloid immune cells to establish an immune-suppressive program. In fact, the metastatic properties of 27HC require the presence of myeloid cells with neutrophils (polymorphonuclear neutrophils; PMNs) being essential for the increase in lung metastasis in murine models. In an effort to further elucidate the mechanisms by which 27HC alters breast cancer progression, we made the striking finding that 27HC promoted the secretion of extracellular vesicles (EVs), a diverse assortment of membrane bound particles that includes exosomes. The resulting EVs had a size distribution that was skewed slightly larger than EVs generated by treating cells with vehicle. The increase in EV secretion and size was consistent across 3 different subtypes: primary murine PMNs, RAW264.7 monocytic cells, and 4T1 murine mammary cancer cells. Label-free analysis of 27HC-EVs indicated that they had a different metabolite composition to those from vehicle-treated cells. Importantly, 27HC-EVs from primary PMNs promoted tumor growth and metastasis in 2 different syngeneic models, demonstrating the potential role of 27HC-induced EVs in the progression of breast cancer. EVs from PMNs were taken up by cancer cells, macrophages, and PMNs, but not T cells. Since EVs did not alter proliferation of cancer cells, it is likely that their protumor effects are mediated through interactions with myeloid cells. Interestingly, RNA-seq analysis of tumors from 27HC-EV-treated mice do not display significantly altered transcriptomes, suggesting that the effects of 27HC-EVs occur early on in tumor establishment and growth. Future work will be required to elucidate the mechanisms by which 27HC increases EV secretion, and how these EVs promote breast cancer progression. Collectively, however, our data indicate that EV secretion and content can be regulated by a cholesterol metabolite, which may have detrimental effects in terms of disease progression, important findings given the prevalence of both breast cancer and hypercholesterolemia.

Intraoperative Label-Free Multimodal Nonlinear Optical Imaging for Point-of-Procedure Cancer Diagnostics.

Intraoperative imaging in surgical oncology can provide information about the tumor microenvironment as well as information about the tumor margin. Visualizing microstructural features and molecular and functional dynamics may provide important diagnostic and prognostic information, especially when obtained in real-time at the point-of-procedure. A majority of current intraoperative optical techniques are based on the use of the labels, such as fluorescent dyes. However, these exogenous agents disrupt the natural microenvironment, perturb biological processes, and alter the endogenous optical signatures that cells and the microenvironment can provide. Portable nonlinear imaging systems have enabled intraoperative imaging for real-time detection and diagnosis of tissue. We review the development of a label-free multimodal nonlinear optical imaging technique that was adapted into a portable imaging system for intraoperative optical assessment of resected human breast tissue. New developments have applied this technology to assessing needle-biopsy specimens. Needle-biopsy procedures most always precede surgical resection and serve as the first sampling of suspicious masses for diagnosis. We demonstrate the diagnostic feasibility of imaging core needle-biopsy specimens during veterinary cancer surgeries. This intraoperative label-free multimodal nonlinear optical imaging technique can potentially provide a powerful tool to assist in cancer diagnosis at the point-of-procedure.

Dual size-exclusion chromatography for efficient isolation of extracellular vesicles from bone marrow derived human plasma.

Isolation of pure extracellular vesicles (EVs), especially from blood, has been a major challenge in the field of EV research. The presence of lipoproteins and soluble proteins often hinders the isolation of high purity EVs upon utilization of conventional separation methods. To circumvent such problems, we designed a single-step dual size-exclusion chromatography (dSEC) column for effective isolation of highly pure EVs from bone marrow derived human plasma. With an aim to select appropriate column design parameters, we analyzed the physiochemical properties of the major substances in bone marrow derived plasma, which include EVs, lipoproteins, and soluble proteins. Based on these findings, we devised a novel dSEC column with two different types of porous beads sequentially stacked each other for efficient separation of EVs from other contaminants. The newly developed dSEC columns exhibited better performance in isolating highly pure EVs from AML plasma in comparison to conventional isolation methods.

Fluorescent nanodiamonds for characterization of nonlinear microscopy systems.

Characterizing the performance of fluorescence microscopy and nonlinear imaging systems is an essential step required for imaging system optimization and quality control during longitudinal experiments. Emerging multimodal nonlinear imaging techniques require a new generation of microscopy calibration targets that are not susceptible to bleaching and can provide a contrast across the multiple modalities. Here, we present a nanodiamond-based calibration target for microscopy, designed for facilitating reproducible measurements at the object plane. The target is designed to support day-to-day instrumentation development efforts in microscopy laboratories. The images of a phantom contain information about the imaging performance of a microscopy system across multiple spectral windows and modalities. Since fluorescent nanodiamonds are not prone to bleaching, the proposed imaging target can serve as a standard, shelf-stable sample to provide rapid reference measurements for ensuring consistent performance of microscopy systems in microscopy laboratories and imaging facilities.

Real-time intraoperative diagnosis by deep neural network driven multiphoton virtual histology.

Recent advances in label-free virtual histology promise a new era for real-time molecular diagnosis in the operating room and during biopsy procedures. To take full advantage of the rich, multidimensional information provided by these technologies, reproducible and reliable computational tools that could facilitate the diagnosis are in great demand. In this study, we developed a deep-learning-based framework to recognize cancer versus normal human breast tissue from real-time label-free virtual histology images, with a tile-level AUC (area under receiver operating curve) of 95% and slide-level AUC of 100% on unseen samples. Furthermore, models trained on a high-quality laboratory-generated dataset can generalize to independent datasets acquired from a portable intraoperative version of the imaging technology with a physics-based adapted design. Classification activation maps and final feature visualization revealed discriminative patterns, such as tumor cells and tumor-associated vesicles, that are highly associated with cancer status. These results demonstrate that through the combination of real-time virtual histopathology and a deep-learning framework, accurate real-time diagnosis could be achieved in point-of-procedure clinical applications.

Label-free visualization and characterization of extracellular vesicles in breast cancer.

Despite extensive interest, extracellular vesicle (EV) research remains technically challenging. One of the unexplored gaps in EV research has been the inability to characterize the spatially and functionally heterogeneous populations of EVs based on their metabolic profile. In this paper, we utilize the intrinsic optical metabolic and structural contrast of EVs and demonstrate in vivo/in situ characterization of EVs in a variety of unprocessed (pre)clinical samples. With a pixel-level segmentation mask provided by the deep neural network, individual EVs can be analyzed in terms of their optical signature in the context of their spatial distribution. Quantitative analysis of living tumor-bearing animals and fresh excised human breast tissue revealed abundance of NAD(P)H-rich EVs within the tumor, near the tumor boundary, and around vessel structures. Furthermore, the percentage of NAD(P)H-rich EVs is highly correlated with human breast cancer diagnosis, which emphasizes the important role of metabolic imaging for EV characterization as well as its potential for clinical applications. In addition to the characterization of EV properties, we also demonstrate label-free monitoring of EV dynamics (uptake, release, and movement) in live cells and animals. The in situ metabolic profiling capacity of the proposed method together with the finding of increasing NAD(P)H-rich EV subpopulations in breast cancer have the potential for empowering applications in basic science and enhancing our understanding of the active metabolic roles that EVs play in cancer progression.