Quantitative Real-Time Analysis of Living Materials by Stimulated Raman Scattering Microscopy.

Composite materials built in part from living organisms have the potential to exhibit useful autonomous, adaptive, and self-healing behavior. The physicochemical, biological, and mechanical properties of such materials can be engineered through the genetic manipulation of their living components. Successful development of living materials will require not only new methods for design and preparation but also new analytical tools that are capable of real-time noninvasive mapping of chemical compositions. Here, we establish a strategy based on stimulated Raman scattering microscopy to monitor phosphatase-catalyzed mineralization of engineered bacterial films in situ. Real-time label-free imaging elucidates the mineralization process, quantifies both the organic and inorganic components of the material as functions of time, and reveals spatial heterogeneity at multiple scales. In addition, we correlate the mechanical performance of films with the extent of mineralization. This work introduces a promising strategy for quantitatively analyzing living materials, which should contribute to the accelerated development of such materials in the future.

Visualization of Modified Bisarylbutadiyne-Tagged Small Molecules in Live-Cell Nuclei by Stimulated Raman Scattering Microscopy.

Visualizing the distribution of small-molecule drugs in living cells is an important strategy for developing specific, effective, and minimally toxic drugs. As an alternative to fluorescence imaging using bulky fluorophores or cell fixation, stimulated Raman scattering (SRS) imaging combined with bisarylbutadiyne (BADY) tagging enables the observation of small molecules closer to their native intracellular state. However, there is evidence that the physicochemical properties of BADY-tagged analogues of small-molecule drugs differ significantly from those of their parent drugs, potentially affecting their intracellular distribution. Herein, we developed a modified BADY to reduce deviations in physicochemical properties (in particular, lipophilicity and membrane permeability) between tagged and parent drugs, while maintaining high Raman activity in live-cell SRS imaging. We highlight the practical application of this approach by revealing the nuclear distribution of a modified BADY-tagged analogue of JQ1, a bromodomain and extra-terminal motif inhibitor with applications in targeted cancer therapy, in living HeLa cells. The modified BADY, methoxypyridazyl pyrimidyl butadiyne (MPDY), revealed intranuclear JQ1, while BADY-tagged JQ1 did not show a clear nuclear signal. We anticipate that the present approach combining MPDY tagging with live-cell SRS imaging provides important insight into the behavior of intracellular drugs and represents a promising avenue for improving drug development.

Multi-molecular hyperspectral PRM-SRS microscopy.

Lipids play crucial roles in many biological processes. Mapping spatial distributions and examining the metabolic dynamics of different lipid subtypes in cells and tissues are critical to better understanding their roles in aging and diseases. Commonly used imaging methods (such as mass spectrometry-based, fluorescence labeling, conventional optical imaging) can disrupt the native environment of cells/tissues, have limited spatial or spectral resolution, or cannot distinguish different lipid subtypes. Here we present a hyperspectral imaging platform that integrates a Penalized Reference Matching algorithm with Stimulated Raman Scattering (PRM-SRS) microscopy. Using this platform, we visualize and identify high density lipoprotein particles in human kidney, a high cholesterol to phosphatidylethanolamine ratio inside granule cells of mouse hippocampus, and subcellular distributions of sphingosine and cardiolipin in human brain. Our PRM-SRS displays unique advantages of enhanced chemical specificity, subcellular resolution, and fast data processing in distinguishing lipid subtypes in different organs and species.

Expanding the Range of Bioorthogonal Tags for Multiplex Stimulated Raman Scattering Microscopy.

Multiplex optical detection in live cells is challenging due to overlapping signals and poor signal-to-noise associated with some chemical reporters. To address this, the application of spectral phasor analysis to stimulated Raman scattering (SRS) microscopy for unmixing three bioorthogonal Raman probes within cells is reported. Triplex detection of a metallacarborane using the B-H stretch at 2480-2650 cm-1 , together with a bis-alkyne and deuterated fatty acid can be achieved within the cell-silent region of the Raman spectrum. When coupled to imaging in the high-wavenumber region of the cellular Raman spectrum, nine discrete regions of interest can be spectrally unmixed from the hyperspectral SRS dataset, demonstrating a new capability in the toolkit of multiplexed Raman imaging of live cells.

Label-Free Cytometric Evaluation of Mitosis via Stimulated Raman Scattering Microscopy and Spectral Phasor Analysis.

Hyperspectral stimulated Raman scattering (SRS) microscopy is a robust imaging tool for the analysis of biological systems. Here, we present a unique perspective, a label-free spatiotemporal map of mitosis, by integrating hyperspectral SRS microscopy with advanced chemometrics to assess the intrinsic biomolecular properties of an essential process of mammalian life. The application of spectral phasor analysis to multiwavelength SRS images in the high-wavenumber (HWN) region of the Raman spectrum enabled the segmentation of subcellular organelles based on innate SRS spectra. Traditional imaging of DNA is primarily reliant on using fluorescent probes or stains which can affect the biophysical properties of the cell. Here, we demonstrate the label-free visualization of nuclear dynamics during mitosis coupled with an evaluation of its spectral profile in a rapid and reproducible manner. These results provide a snapshot of the cell division cycle and chemical variability between intracellular compartments in single-cell models, which is central to understanding the molecular foundations of these fundamental biological processes. The evaluation of HWN images by phasor analysis also facilitated the differentiation between cells in separate phases of the cell cycle based solely on their nuclear SRS spectral signal, which offers an interesting label-free approach in combination with flow cytometry. Therefore, this study demonstrates that SRS microscopy combined with spectral phasor analysis is a valuable method for detailed optical fingerprinting at the subcellular level.

Measurement of chemical penetration in skin using Stimulated Raman scattering microscopy and multivariate curve resolution - alternating least squares.

The mechanistic understanding of skin penetration underpins the design, efficacy and risk assessment of many high-value products including functional personal care products, topical and transdermal drugs. Stimulated Raman scattering (SRS) microscopy, a label free chemical imaging tool, combines molecular spectroscopy with submicron spatial information to map the distribution of chemicals as they penetrate the skin. However, the quantification of penetration is hampered by significant interference from Raman signals of skin constituents. This study reports a method for disentangling exogeneous contributions and measuring their permeation profile through human skin combining SRS measurements with chemometrics. We investigated the spectral decomposition capability of multivariate curve resolution - alternating least squares (MCR-ALS) using hyperspectral SRS images of skin dosed with 4-cyanophenol. By performing MCR-ALS on the fingerprint region spectral data, the distribution of 4-cyanophenol in skin was estimated in an attempt to quantify the amount permeated at different depths. The reconstructed distribution was compared with the experimental mapping of CN, a strong vibrational peak in 4-cyanophenol where the skin is spectroscopically silent. The similarity between MCR-ALS resolved and experimental distribution in skin dosed for 4 h was 0.79 which improved to 0.91 for skin dosed for 1 h. The correlation was observed to be lower for deeper layers of skin where SRS signal intensity is low which is an indication of low sensitivity of SRS. This work is the first demonstration, to the best of our knowledge, of combining SRS imaging technique with spectral unmixing methods for direct observation and mapping of the chemical penetration and distribution in biological tissues.

Direct Counting and Imaging Chain Lengths of Lipids by Stimulated Raman Scattering Microscopy.

Direct counting and mapping the chain lengths of fatty acids on a microscopic scale are of particular importance but remain an unsolvable challenge. Although the current hyperspectral stimulated Raman scattering (SRS) microscopy has gained exceptional capability in chemical imaging of the degree of desaturation, the complete lipid characterization, including the carbon chain length quantification, is awaiting a major breakthrough. Here, we pushed the spectral resolution limit of hyperspectral SRS microscopy to 5.4 cm-1 by employing a highly efficient spectral compressor, which achieved spectral narrowing of the fs laser without much energy loss. The SRS imaging with such high spectral resolution enabled us to differ eight types of saturated lipids with carbon chain lengths from C8:0 to C22:0 by interrogating their subtly red-shifting Raman bands of alkyl C-C gauche stretches between 1070 and 1110 cm-1. The SRS microscopy with superior spectral resolution will pave the way for comprehensive lipid characterization and contribute to uncovering the abnormal pathways of lipid metabolism in cancer.

Phototoxic effects of nonlinear optical microscopy on cell cycle, oxidative states, and gene expression.

Nonlinear optical imaging modalities, such as stimulated Raman scattering (SRS) microscopy, use pulsed-laser excitation with high peak intensity that can perturb the native state of cells. In this study, we used bulk RNA sequencing, quantitative measurement of cell proliferation, and fluorescent measurement of the generation of reactive oxygen species to assess phototoxic effects of near-IR pulsed laser radiation, at different time scales, for laser excitation settings relevant to SRS imaging. We define a range of laser excitation settings for which there was no significant change in mouse Neuro2A cells after laser exposure. This study provides guidance for imaging parameters that minimize photo-induced perturbations in SRS microscopy to ensure accurate interpretation of experiments with time-lapse imaging or with paired measurements of imaging and sequencing on the same cells.

Quantitative Imaging of Intracellular Density with Ratiometric Stimulated Raman Scattering Microscopy.

Cell size and density are tightly controlled in mammalian cells. They impact a wide range of physiological functions, including osmoregulation, tissue homeostasis, and growth regulation. Compared to size, density variation for a given cell type is typically much smaller, implying that cell-type-specific density plays an important role in cell function. However, little is known about how cell density affects cell function or how it is regulated. Current tools for intracellular cell density measurements are limited to either suspended cells or cells grown on 2D substrates, neither of which recapitulate the physiology of single cells in intact tissue. While optical measurements have the potential to noninvasively measure cell density in situ, light scattering in multicellular systems prevents direct quantification. Here, we introduce an intracellular density imaging technique based on ratiometric stimulated Raman scattering microscopy (rSRS). It uses intrinsic vibrational information from intracellular macromolecules to quantify dry mass density. Moreover, water is used as an internal standard to correct for aberration and light scattering effects. We demonstrate real-time measurement of intracellular density and show that density is tightly regulated across different cell types and can be used to differentiate cell types as well as cell states. We further demonstrate dynamic imaging of density change in response to osmotic challenge as well as intracellular density imaging of a 3D tumor spheroid. Our technique has the potential for imaging intracellular density in intact tissue and understanding density regulation and its role in tissue homeostasis.

Multiple Parallel Fusion Network for Predicting Protein Subcellular Localization from Stimulated Raman Scattering (SRS) Microscopy Images in Living Cells.

Stimulated Raman Scattering Microscopy (SRS) is a powerful tool for label-free detailed recognition and investigation of the cellular and subcellular structures of living cells. Determining subcellular protein localization from the cell level of SRS images is one of the basic goals of cell biology, which can not only provide useful clues for their functions and biological processes but also help to determine the priority and select the appropriate target for drug development. However, the bottleneck in predicting subcellular protein locations of SRS cell imaging lies in modeling complicated relationships concealed beneath the original cell imaging data owing to the spectral overlap information from different protein molecules. In this work, a multiple parallel fusion network, MPFnetwork, is proposed to study the subcellular locations from SRS images. This model used a multiple parallel fusion model to construct feature representations and combined multiple nonlinear decomposing algorithms as the automated subcellular detection method. Our experimental results showed that the MPFnetwork could achieve over 0.93 dice correlation between estimated and true fractions on SRS lung cancer cell datasets. In addition, we applied the MPFnetwork method to cell images for label-free prediction of several different subcellular components simultaneously, rather than using several fluorescent labels. These results open up a new method for the time-resolved study of subcellular components in different cells, especially cancer cells.

A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy.

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many biological and biomedical applications. However, long-term time-lapse SRS imaging of live cells has not been widely adopted. SRS microscopy often uses a high numerical aperture (NA) water-immersion objective and a high NA oil-immersion condenser to achieve high-resolution imaging. In this case, the gap between the objective and the condenser is only a few millimeters. Therefore, most commercial stage-top environmental chambers cannot be used for SRS imaging because of their large thickness with a rigid glass cover. This paper describes the design and fabrication of a flexible chamber that can be used for time-lapse live-cell imaging with transmitted SRS signal detection on an upright microscope frame. The flexibility of the chamber is achieved by using a soft material - a thin natural rubber film. The new enclosure and chamber design can be easily added to an existing SRS imaging setup. The testing and preliminary results demonstrate that the flexible chamber system enables stable, long-term, time-lapse SRS imaging of live cells, which can be used for various bioimaging applications in the future.

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy.

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps at high speed, and high spatial resolution of thin samples by directly interrogating their molecular vibrations. In its standard implementation, SRS microscopy is narrowband and forms images with only a single vibrational frequency at a time. However, this approach not only hinders the chemical specificity of SRS but also neglects the wealth of information encoded within vibrational spectra. These limitations can be overcome by broadband SRS, an implementation capable of extracting a vibrational spectrum per pixel of the image in parallel. This delivers hyperspectral data that, when coupled with chemometric analysis, maximizes the amount of information retrieved from the specimen. Thus, broadband SRS improves the chemical specificity of the system, allowing the quantitative determination of the concentration of the different constituents of a sample. Here, we report a protocol for chemical imaging with broadband SRS microscopy, based on a home-built SRS microscope operating with a custom differential multichannel-lock-in amplifier detection. It discusses the sample preparation, alignment of the SRS apparatus, and chemometric analysis. By acquiring vibrational Raman spectra, the protocol illustrates how to identify different chemical species within a mixture, determining their relative concentrations.

Practical considerations for quantitative and reproducible measurements with stimulated Raman scattering microscopy.

Stimulated Raman scattering (SRS) microscopy provides rapid label-free 3D chemical imaging with wide-ranging applications including histology, pharmacokinetic studies, and materials characterisation. SRS microscopy has seen a steady increase in utilisation since the early 2000s and has become more accessible due to the increase in availability of facilities, and the development of user-friendly instrumentation. Although some complete SRS systems are now commercially available, many instruments are home-built with highly varied laser sources, optics, and detection mechanisms. Signal intensity is also dependent on the effective spatiotemporal overlap of two (or more) laser beams, thus any drift in alignment can result in variable performance. Currently there is a lack of standard procedures or reference materials for SRS, which has important implications on reproducibility and consistency. These concerns are particularly relevant to the comparison of data from the same instrument at different times and instrument settings as well as between different instruments and laboratories. This tutorial-style review presents the most important practical considerations for sample preparation, instrument set-up, image acquisition and data analysis to obtain reproducible SRS measurements.

Label-Free Imaging of Lipid Droplets in Prostate Cells Using Stimulated Raman Scattering Microscopy and Multivariate Analysis.

Hyperspectral stimulated Raman scattering (SRS) microscopy is a powerful imaging modality for the analysis of biological systems. Here, we report the application of k-means cluster analysis (KMCA) of multi-wavelength SRS images in the high-wavenumber region of the Raman spectrum as a robust and reliable method for the segmentation of cellular organelles based on the intrinsic SRS spectrum. KMCA has been applied to the study of the endogenous lipid biochemistry of prostate cancer and prostate healthy cell models, while the corresponding SRS spectrum of the lipid droplet (LD) cluster enabled direct comparison of their composition. The application of KMCA in visualizing the LD content of prostate cell models following the inhibition of de novo lipid synthesis (DNL) using the acetyl-coA carboxylase inhibitor, 5-(tetradecyloxy)-2-furoic acid (TOFA), is demonstrated. This method identified a reliance of prostate cancer cell models upon DNL for metabolic requirements, with a significant reduction in the cellular LD content after treatment with TOFA, which was not observed in normal prostate cell models. SRS imaging combined with KMCA is a robust method for investigating drug-cell interactions in a label-free manner.

Super-Resolution Vibrational Imaging Using Expansion Stimulated Raman Scattering Microscopy.

Stimulated Raman scattering (SRS) microscopy is an emerging technology that provides high chemical specificity for endogenous biomolecules and can circumvent common constraints of fluorescence microscopy including limited capabilities to probe small biomolecules and difficulty resolving many colors simultaneously. However, the resolution of SRS microscopy remains governed by the diffraction limit. To overcome this, a new technique called molecule anchorable gel-enabled nanoscale Imaging of Fluorescence and stimulated Raman scattering microscopy (MAGNIFIERS) that integrates SRS microscopy with expansion microscopy (ExM) is described. MAGNIFIERS offers chemical-specific nanoscale imaging with sub-50 nm resolution and has scalable multiplexity when combined with multiplex Raman probes and fluorescent labels. MAGNIFIERS is used to visualize nanoscale features in a label-free manner with CH vibration of proteins, lipids, and DNA in a broad range of biological specimens, from mouse brain, liver, and kidney to human lung organoid. In addition, MAGNIFIERS is applied to track nanoscale features of protein synthesis in protein aggregates using metabolic labeling of small metabolites. Finally, MAGNIFIERS is used to demonstrate 8-color nanoscale imaging in an expanded mouse brain section. Overall, MAGNIFIERS is a valuable platform for super-resolution label-free chemical imaging, high-resolution metabolic imaging, and highly multiplexed nanoscale imaging, thus bringing SRS to nanoscopy.

Quantitative Stimulated Raman Scattering Microscopy: Promises and Pitfalls.

Since its first demonstration, stimulated Raman scattering (SRS) microscopy has become a powerful chemical imaging tool that shows promise in numerous biological and biomedical applications. The spectroscopic capability of SRS enables identification and tracking of specific molecules or classes of molecules, often without labeling. SRS microscopy also has the hallmark advantage of signal strength that is directly proportional to molecular concentration, allowing for in situ quantitative analysis of chemical composition of heterogeneous samples with submicron spatial resolution and subminute temporal resolution. However, it is important to recognize that quantification through SRS microscopy requires assumptions regarding both system and sample. Such assumptions are often taken axiomatically, which may lead to erroneous conclusions without proper validation. In this review, we focus on the tacitly accepted, yet complex, quantitative aspect of SRS microscopy. We discuss the various approaches to quantitative analysis, examples of such approaches, challenges in different systems, and potential solutions. Through our examination of published literature, we conclude that a scrupulous approach to experimental design can further expand the powerful and incisive quantitative capabilities of SRS microscopy.

Nonlinear Optical Microscopy with Ultralow Quantum Light.

Nonlinear optical (NLO) microscopy relies on multiple light-matter interactions to provide unique contrast mechanisms and imaging capabilities that are inaccessible to traditional linear optical imaging approaches, making them versatile tools to understand a wide range of complex systems. However, the strong excitation fields that are necessary to drive higher-order optical processes efficiently are often responsible for photobleaching, photodegradation, and interruption in many systems of interest. This is especially true for imaging living biological samples over prolonged periods of time or in accessing intrinsic dynamics of electronic excited-state processes in spatially heterogeneous materials. This perspective outlines some of the key limitations of two NLO imaging modalities implemented in our lab and highlights the unique potential afforded by the quantum properties of light, especially entangled two-photon absorption based NLO spectroscopy and microscopy. We further review some of the recent exciting advances in this emerging filed and highlight some major challenges facing the realization of quantum-light-enabled NLO imaging modalities.

9-Cyanopyronin probe palette for super-multiplexed vibrational imaging.

Multiplexed optical imaging provides holistic visualization on a vast number of molecular targets, which has become increasingly essential for understanding complex biological processes and interactions. Vibrational microscopy has great potential owing to the sharp linewidth of vibrational spectra. In 2017, we demonstrated the coupling between electronic pre-resonant stimulated Raman scattering (epr-SRS) microscopy with a proposed library of 9-cyanopyronin-based dyes, named Manhattan Raman Scattering (MARS). Herein, we develop robust synthetic methodology to build MARS probes with different core atoms, expansion ring numbers, and stable isotope substitutions. We discover a predictive model to correlate their vibrational frequencies with structures, which guides rational design of MARS dyes with desirable Raman shifts. An expanded library of MARS probes with diverse functionalities is constructed. When coupled with epr-SRS microscopy, these MARS probes allow us to demonstrate not only many versatile labeling modalities but also increased multiplexing capacity. Hence, this work opens up next-generation vibrational imaging with greater utilities.

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.