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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.

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.

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., Raman-based imaging of biomarkers is often challenging due to low sensitivity. Here, the authors use a swelling-diffusion approach to develop a series of Raman probes that are both ultra-bright and compact in size, and demonstrate multiplexed imaging of specific protein targets in cells and tissue slices.

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.

Powerful optical tools have revolutionized science and technology. The prevalent fluorescence detection offers superb sensitivity down to single molecules but lacks sufficient chemical information1. In contrast, Raman-based vibrational spectroscopy provides exquisite chemical specificity about molecular structure, dynamics and coupling, but is notoriously insensitive. 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 the narrow vibrational linewidth, we demonstrated multiplexed SREF imaging in cells, breaking the ‘colour barrier’ of fluorescence. By leveraging the 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, SREF would be a valuable tool for chemistry and biology.

Significance Membranes can adopt distinct phases. The endoplasmic reticulum (ER) is the largest membrane system inside cells and also harbors the richest metabolic activity including lipid synthesis. Unlike plasma membrane where separated “lipid raft” domains have been predicted and observed, ER membrane is thought to be uniformly fluidic. However, such understanding is based on biophysical studies of model membrane under thermodynamic equilibrium. It remains unclear whether and how lipid synthesis activity perturbs the equilibrium and promotes phase segregation in ER membrane. Herein, we utilized coherent Raman imaging technique to track lipid synthesis and surprisingly revealed solid-like domains emerging from liquid ER membrane. Interestingly, this phenomenon can be tuned by the incoming nutrient source, demonstrating the susceptibility of ER membrane to nonequilibrium modulation., 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.

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.

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.

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.

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.

Although bacterial persistence was first observed in 1944, its underlying mechanism is just beginning to be understood and many fundamental questions remain. In this review, we summarize studies in order to chart the full map of bacterial persistence. Because persistence significantly contributes to disease recalcitrance, we also elucidate the probable relationships between bacterial persistence and prolonged chronic infections, with some comments on future research directions and therapeutic strategies.

To study the expression of Fas, Fas ligand (FasL) and IFN-gamma in gastric cancer and its possible significance.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.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).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.