VIBRANT: spectral profiling for single-cell drug responses.

High-content cell profiling has proven invaluable for single-cell phenotyping in response to chemical perturbations. However, methods with improved throughput, information content and affordability are still needed. We present a new high-content spectral profiling method named vibrational painting (VIBRANT), integrating mid-infrared vibrational imaging, multiplexed vibrational probes and an optimized data analysis pipeline for measuring single-cell drug responses. Three infrared-active vibrational probes were designed to measure distinct essential metabolic activities in human cancer cells. More than 20,000 single-cell drug responses were collected, corresponding to 23 drug treatments. The resulting spectral profile is highly sensitive to phenotypic changes under drug perturbation. Using this property, we built a machine learning classifier to accurately predict drug mechanism of action at single-cell level with minimal batch effects. We further designed an algorithm to discover drug candidates with new mechanisms of action and evaluate drug combinations. Overall, VIBRANT has demonstrated great potential across multiple areas of phenotypic screening.

Enhanced Efflux Activity Facilitates Drug Tolerance in Dormant Bacterial Cells.

Natural variations in gene expression provide a mechanism for multiple phenotypes to arise in an isogenic bacterial population. In particular, a sub-group termed persisters show high tolerance to antibiotics. Previously, their formation has been attributed to cell dormancy. Here we demonstrate that bacterial persisters, under ß-lactam antibiotic treatment, show less cytoplasmic drug accumulation as a result of enhanced efflux activity. Consistently, a number of multi-drug efflux genes, particularly the central component TolC, show higher expression in persisters. Time-lapse imaging and mutagenesis studies further establish a positive correlation between tolC expression and bacterial persistence. The key role of efflux systems, among multiple biological pathways involved in persister formation, indicates that persisters implement a positive defense against antibiotics prior to a passive defense via dormancy. Finally, efflux inhibitors and antibiotics together effectively attenuate persister formation, suggesting a combination strategy to target drug tolerance.

Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy.

Three-dimensional visualization of tissue structures using optical microscopy facilitates the understanding of biological functions. However, optical microscopy is limited in tissue penetration due to severe light scattering. Recently, a series of tissue-clearing techniques have emerged to allow significant depth-extension for fluorescence imaging. Inspired by these advances, we develop a volumetric chemical imaging technique that couples Raman-tailored tissue-clearing with stimulated Raman scattering (SRS) microscopy. Compared with the standard SRS, the clearing-enhanced SRS achieves greater than 10-times depth increase. Based on the extracted spatial distribution of proteins and lipids, our method reveals intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. We further develop volumetric phasor analysis of multispectral SRS images for chemically specific clustering and segmentation in 3D. Moreover, going beyond the conventional label-free paradigm, we demonstrate metabolic volumetric chemical imaging, which allows us to simultaneously map out metabolic activities of protein and lipid synthesis in glioblastoma. Together, these results support volumetric chemical imaging as a valuable tool for elucidating comprehensive 3D structures, compositions, and functions in diverse biological contexts, complementing the prevailing volumetric fluorescence microscopy.

Metabolic Activity Phenotyping of Single Cells with Multiplexed Vibrational Probes.

Quantitative measurements of metabolic activities of individual cells are essential to understanding questions in diverse fields in biology. To address this challenge, we present a method, termed metabolic activity phenotyping (MAP), to probe metabolic fluxes by utilizing multiplexed vibrational metabolic probes. With specifically designed single-whole-cell confocal micro-Raman spectroscopy, quantitative measurement of lipid and protein synthesis activity was achieved with high throughput (several orders of magnitude improvement over a commercial confocal system). In addition, metabolic heterogeneity upon various drug treatments was also revealed and evaluated at the single-cell level. We further demonstrated that MAP was more robust than the label-free Raman methods and was able to make the correct classification among diverse cancer types and breast cancer subtypes by exploring the dimension of metabolism. The capability of MAP to explore metabolic profiles at the single-cell level makes it a valuable tool for basic single-cell studies as well as other screening applications.

Background-free imaging of chemical bonds by a simple and robust frequency-modulated stimulated Raman scattering microscopy.

Being able to image chemical bonds with high sensitivity and speed, stimulated Raman scattering (SRS) microscopy has made a major impact in biomedical optics. However, it is well known that the standard SRS microscopy suffers from various backgrounds, limiting the achievable contrast, quantification and sensitivity. While many frequency-modulation (FM) SRS schemes have been demonstrated to retrieve the sharp vibrational contrast, they often require customized laser systems and/or complicated laser pulse shaping or introduce additional noise, thereby hindering wide adoption. Herein we report a simple but robust strategy for FM-SRS microscopy based on a popular commercial laser system and regular optics. Harnessing self-phase modulation induced self-balanced spectral splitting of picosecond Stokes beam propagating in standard single-mode silica fibers, a high-performance FM-SRS system is constructed without introducing any additional signal noise. Our strategy enables adaptive spectral resolution for background-free SRS imaging of Raman modes with different linewidths. The generality of our method is demonstrated on a variety of Raman modes with effective suppressing of backgrounds including non-resonant cross phase modulation and electronic background from two-photon absorption or pump-probe process. As such, our method is promising to be adopted by the SRS microscopy community for background-free chemical imaging.

Metabolic activity induces membrane phase separation in endoplasmic reticulum.

Membrane phase behavior has been well characterized in model membranes in vitro under thermodynamic equilibrium state. However, the widely observed differences between biological membranes and their in vitro counterparts are placing more emphasis on nonequilibrium factors, including influx and efflux of lipid molecules. The endoplasmic reticulum (ER) is the largest cellular membrane system and also the most metabolically active organelle responsible for lipid synthesis. However, how the nonequilibrium metabolic activity modulates ER membrane phase has not been investigated. Here, we studied the phase behavior of functional ER in the context of lipid metabolism. Utilizing advanced vibrational imaging technique, that is, stimulated Raman scattering microscopy, we discovered that metabolism of palmitate, a prevalent saturated fatty acid (SFA), could drive solid-like domain separation from the presumably uniformly fluidic ER membrane, a previously unknown phenomenon. The potential of various fatty acids to induce solid phase can be predicted by the transition temperatures of their major metabolites. Interplay between saturated and unsaturated fatty acids is also observed. Hence, our study sheds light on cellular membrane biophysics by underscoring the nonequilibrium metabolic status of living cell.

Applications of vibrational tags in biological imaging by Raman microscopy.

As a superb tool to visualize and study the spatial-temporal distribution of chemicals, Raman microscopy has made a big impact in many disciplines of science. While label-free imaging has been the prevailing strategy in Raman microscopy, recent development and applications of vibrational/Raman tags, particularly when coupled with stimulated Raman scattering (SRS) microscopy, have generated intense excitement in biomedical imaging. SRS imaging of vibrational tags has enabled researchers to study a wide range of small biomolecules with high specificity, sensitivity and multiplex capability, at a single live cell level, tissue level or even in vivo. As reviewed in this article, this platform has facilitated imaging distribution and dynamics of small molecules such as glucose, lipids, amino acids, nucleic acids, and drugs that are otherwise difficult to monitor with other means. As both the vibrational tags and Raman instrumental development progress rapidly and synergistically, we anticipate that this technique will shed light onto an even broader spectrum of biomedical problems.

Probing the structure of water in individual living cells.

Water regulates or even governs a wide range of biological processes. Despite its fundamental importance, surprisingly little is known about the structure of intracellular water. Herein we employ a Raman micro-spectroscopy technique to uncover the composition, abundance and vibrational spectra of intracellular water in individual living cells. In three different cell types, we show a small but consistent population (~3%) of non-bulk-like water. It exhibits a weakened hydrogen-bonded network and a more disordered tetrahedral structure. We attribute this population to biointerfacial water located in the vicinity of biomolecules. Moreover, our whole-cell modeling suggests that all soluble (globular) proteins inside cells are surrounded by, on average, one full molecular layer (about 2.6 Angstrom) of biointerfacial water. Furthermore, relative invariance of biointerfacial water is observed among different single cells. Overall, our study not only opens up experimental possibilities of interrogating water structure in vivo but also provides insights into water in life.

Towards Mapping Mouse Metabolic Tissue Atlas by Mid-Infrared Imaging with Heavy Water Labeling.

Understanding metabolism is of great significance to decipher various physiological and pathogenic processes. While great progress has been made to profile gene expression, how to capture organ-, tissue-, and cell-type-specific metabolic profile (i.e., metabolic tissue atlas) in complex mammalian systems is lagging behind, largely owing to the lack of metabolic imaging tools with high resolution and high throughput. Here, the authors applied mid-infrared imaging coupled with heavy water (D2 O) metabolic labeling to a scope of mouse organs and tissues. The premise is that, as D2 O participates in the biosynthesis of various macromolecules, the resulting broad C-D vibrational spectrum should interrogate a wide range of metabolic pathways. Applying multivariate analysis to the C-D spectrum, the authors successfully identified both inter-organ and intra-tissue metabolic signatures of mice. A large-scale metabolic atlas map between different organs from the same mice is thus generated. Moreover, leveraging the power of unsupervised clustering methods, spatially-resolved metabolic signatures of brain tissues are discovered, revealing tissue and cell-type specific metabolic profile in situ. As a demonstration of this technique, the authors captured metabolic changes during brain development and characterized intratumoral metabolic heterogeneity of glioblastoma. Altogether, the integrated platform paves a way to map the metabolic tissue atlas for complex mammalian systems.

Multiplexed live-cell profiling with Raman probes.

Single-cell multiparameter measurement has been increasingly recognized as a key technology toward systematic understandings of complex molecular and cellular functions in biological systems. Despite extensive efforts in analytical techniques, it is still generally challenging for existing methods to decipher a large number of phenotypes in a single living cell. Herein we devise a multiplexed Raman probe panel with sharp and mutually resolvable Raman peaks to simultaneously quantify cell surface proteins, endocytosis activities, and metabolic dynamics of an individual live cell. When coupling it to whole-cell spontaneous Raman micro-spectroscopy, we demonstrate the utility of this technique in 14-plexed live-cell profiling and phenotyping under various drug perturbations. In particular, single-cell multiparameter measurement enables powerful clustering, correlation, and network analysis with biological insights. This profiling platform is compatible with live-cell cytometry, of low instrument complexity and capable of highly multiplexed measurement in a robust and straightforward manner, thereby contributing a valuable tool for both basic single-cell biology and translation applications such as high-content cell sorting and drug discovery.

Super-resolution vibrational microscopy by stimulated Raman excited fluorescence.

Inspired by the revolutionary impact of super-resolution fluorescence microscopy, super-resolution Raman imaging has been long pursued because of its much higher chemical specificity than the fluorescence counterpart. However, vibrational contrasts are intrinsically less sensitive compared with fluorescence, resulting in only mild resolution enhancement beyond the diffraction limit even with strong laser excitation power. As such, it is still a great challenge to achieve biocompatible super-resolution vibrational imaging in the optical far-field. In 2019 Stimulated Raman Excited Fluorescence (SREF) was discovered as an ultrasensitive vibrational spectroscopy that combines the high chemical specificity of Raman scattering and the superb sensitivity of fluorescence detection. Herein we developed a novel super-resolution vibrational imaging method by harnessing SREF as the contrast mechanism. We first identified the undesired role of anti-Stokes fluorescence background in preventing direct adoption of super-resolution fluorescence technique. We then devised a frequency-modulation (FM) strategy to remove the broadband backgrounds and achieved high-contrast SREF imaging. Assisted by newly synthesized SREF dyes, we realized multicolor FM-SREF imaging with nanometer spectral resolution. Finally, by integrating stimulated emission depletion (STED) with background-free FM-SREF, we accomplished high-contrast super-resolution vibrational imaging with STED-FM-SREF whose spatial resolution is only determined by the signal-to-noise ratio. In our proof-of-principle demonstration, more than two times of resolution improvement is achieved in biological systems with moderate laser excitation power, which shall be further refined with optimized instrumentation and imaging probes. With its super resolution, high sensitivity, vibrational contrast, and mild laser excitation power, STED-FM-SREF microscopy is envisioned to aid a wide variety of applications.

Ultra-bright Raman dots for multiplexed optical imaging.

Imaging the spatial distribution of biomolecules is at the core of modern biology. The development of fluorescence techniques has enabled researchers to investigate subcellular structures with nanometer precision. However, multiplexed imaging, i.e. observing complex biological networks and interactions, is mainly limited by the fundamental 'spectral crowding' of fluorescent materials. Raman spectroscopy-based methods, on the other hand, have a much greater spectral resolution, but often lack the required sensitivity for practical imaging of biomarkers. Addressing the pressing need for new Raman probes, herein we present a series of Raman-active  nanoparticles (Rdots) that exhibit the combined advantages of ultra-brightness and compact sizes (~20 nm). When coupled with the emerging stimulated Raman scattering (SRS) microscopy, these Rdots are brighter than previously reported Raman-active organic probes by two to three orders of magnitude. We further obtain evidence supporting for SRS imaging of Rdots at single particle level. The compact size and ultra-brightness of Rdots allows immunostaining of specific protein targets (including cytoskeleton and low-abundant surface proteins) in mammalian cells and tissue slices with high imaging contrast. These Rdots thus offer a promising tool for a large range of studies on complex biological networks.

[Application of three-dimensional endoanal and endorectal ultrasound in the diagnosis of anorectal fistula].

OBJECTIVE: To evaluate the efficacy of three-dimensional anal and endorectal ultrasound in identifying the internal opening and tracing the tract of the anorectal fistula. METHODS: From November 2008 to January 2010, 127 patients suffering anorectal fistula were managed with three-dimensional endoanal and endorectal ultrasound. The internal opening, the tract of the fistula and fistula trace were identified by the ultrasonography with three-dimensional imaging. All results were confirmed and compared with findings from the operation. RESULTS: The internal opening of the fistula was specified in 116 patients, the accuracy rate was 91.3% (116/127). The internal opening of the fistula was located above the dentate line in 112 patients, and located in rectal ampulla in 4 patients. The main fistula tract was identified in all the patients, the accuracy rate was 100%. In this group, the fistula tunneled as follows: trans-sphincteric in 47 patients, intersphincteric in 75 cases, supra sphincteric in 2 cases, extra sphincteric in 3 patients. Secondary extension was found in 37 patients, the accuracy rate was 100% (37/37). CONCLUSIONS: Three-dimensional anal and endorectal ultrasound is an effective way for localizing the internal opening and the tract of anorectal fistula. It can provide valuable information for curative operation.

Stimulated Raman Excited Fluorescence Spectroscopy of Visible Dyes.

Fluorescence spectroscopy and Raman spectroscopy are two major classes of spectroscopy methods in physical chemistry. Very recently, stimulated Raman excited fluorescence (SREF) has been demonstrated ( Xiong, H.; et al. Nature Photonics , 2019 , 13 , 412 - 417 ) as a new hybrid spectroscopy that combines the vibrational specificity of Raman spectroscopy with the superb sensitivity of fluorescence spectroscopy (down to the single-molecule level). However, this proof-of-concept study was limited by both the tunability of the commercial laser source and the availability of the excitable molecules in the near-infrared. As a result, the generality of SREF spectroscopy remains unaddressed, and the understanding of the critical electronic preresonance condition is lacking. In this work, we built a modified excitation source to explore SREF spectroscopy in the visible region. Harnessing a large palette of red dyes, we have systematically studied SREF spectroscopy on a dozen different cases with a fine spectral interval of several nanometers. The results not only establish the generality of SREF spectroscopy for a wide range of molecules but also reveal a tight window of proper electronic preresonance for the stimulated Raman pumping process. Our theoretical modeling and further experiments on newly synthesized dyes also support the obtained insights, which would be valuable in designing and optimizing future SREF experiments for single-molecule vibrational spectroscopy and supermultiplex vibrational imaging.

Stimulated Raman Excited Fluorescence Spectroscopy and Imaging.

Powerful optical tools have revolutionized science and technology. The prevalent fluorescence detection offers superb sensitivity down to single molecules but lacks sufficient chemical information1-3. In contrast, Raman-based vibrational spectroscopy provides exquisite chemical specificity about molecular structure, dynamics and coupling, but is notoriously insensitive3-5. Here we report a hybrid technique of Stimulated Raman Excited Fluorescence (SREF) that integrates superb detection sensitivity and fine chemical specificity. Through stimulated Raman pumping to an intermediate vibrational eigenstate followed by an upconversion to an electronic fluorescent state, SREF encodes vibrational resonance into the excitation spectrum of fluorescence emission. By harnessing narrow vibrational linewidth, we demonstrated multiplexed SREF imaging in cells, breaking the "color barrier" of fluorescence. By leveraging superb sensitivity of SREF, we achieved all-far-field single-molecule Raman spectroscopy and imaging without plasmonic enhancement, a long-sought-after goal in photonics. Thus, through merging Raman and fluorescence spectroscopy, SERF would be a valuable tool for chemistry and biology.

Aberration of X chromosome in liver neoplasm detected by fluorescence in situ hybridization.

BACKGROUND: A diverse range of cytogenetic alterations of autosomal chromosomes has been reported to date. However, few studies have addressed the abnormalities of X chromosome in hepatocellular carcinoma (HCC) except sporadic reports on the deletion of band F1 in X chromosome, and the clonal analysis of methylation pattern of the X chromosome-linked human androgen receptor gene. Identification of specific X chromosome alterations during the course of neoplastic development would be essential to defining the genetic basis of HCC. Therefore, we studied the regularity of aberration of X chromosome in liver cancer. METHODS: Hepatocarcinoma cellular lines and tumor tissues were detected respectively through DNA probes of X chromosome after fluorescence in situ hybridization (FISH). RESULTS: Increased copies of X chromosome were observed in all samples, and four signals of hybridization were of the major type. CONCLUSIONS: Increased copy number of X chromosome frequently occur in liver cancer. The relationship between copy number of X chromosome and liver cancer genesis needs further investigation. This study is the first of its kind determining the copy number of X chromosome in liver cancer by using FISH.

[Expression of Fas, FasL and IFN-gamma in gastric cancer].

OBJECTIVE: To study the expression of Fas, Fas ligand (FasL) and IFN-gamma in gastric cancer and its possible significance. METHODS: Fifty-eight gastric paraffin wax embedded cancer tissues and fifty-three normal tissues adjacent gastric cancer were tested for the expression of Fas and FasL protein by immunohistochemistry and IFN-gamma mRNA by in situ hubridisation respectively. RESULTS: The positive rate of Fas in cancer cells of gastric cancer tissues was significantly lower than that in gastric epithelial cells of the tissues adjacent cancer(19.0% and 64.2%, respectively; chi 2 = 23.46, P = 0.00). The positive rate of FasL in cancer cells of gastric cancer tissues was significantly higher than that in gastric epithelial cells of the tissues adjacent cancer(63.8% and 45.3%, respectively; chi 2 = 3.83, P = 0.05). The positive rate of IFN-gamma in cancer cells of gastric cancer tissues was significantly lower than that in gastric epithelial cells of the tissues adjacent cancer(0.0% and 49.1%, respectively; chi 2 = 37.16, P = 0.00). CONCLUSION: The Fas-FasL system was unbalanced, and the expression of IFN-gamma was low in gastric cancer cells in this study. These may be related to the carcinogenesis of gastric epithelial cells and might be responsible for the immune escape of these cells.