Stimulated Raman Scattering Microscopy Research Articles

Advances in Super-resolution Stimulated Raman Scattering Microscopy

Advances in Super-resolution Stimulated Raman Scattering Microscopy

Imaging Metabolic Flow of Water in Plants with Isotope-Traced Stimulated Raman Scattering Microscopy.

Water plays a vital role in the life cycle of plants, participating in various critical biochemical reactions during both non-photosynthetic and photosynthetic processes. Direct visualization of the metabolic activities of water in plants with high spatiotemporal resolution is essential to reveal the functional utilization of water. Here, stimulated Raman scattering (SRS) microscopy is applied to monitor the metabolic processes of deuterated water (D2O) in model plant Arabidopsis thaliana (A. thaliana). The work shows that in plants uptaking D2O/water solution, proton-transfer from water to organic metabolites results in the formation of C-D bonds in newly synthesized biomolecules (lipid, protein, and polysaccharides, etc.) that allow high-resolution detection with SRS. Reversible metabolic pathways of oil-starch conversion between seed germination and seed development processes are verified. Spatial heterogeneity of metabolic activities along the vertical axis of plants (root, stem, and tip meristem), as well as the radial distributions of secondary growth on the horizontal cross-sections are quantified. Furthermore, metabolic flow of protons from plants to animals is visualized in aphids feeding on A. thaliana. Collectively, SRS microscopy has potential to trace a broad range of matter flows in plants, such as carbon storage and nutrition metabolism.

High Sensitivity Stimulated Raman Scattering Microscopy with Electronic Resonance Enhancement.

Raman microscopy is an important tool for labelfree microscopy. However, spontaneous Raman microscopy suffers from slow image acquisition rates and susceptibility to fluorescence background. Coherent Raman microsocopy techniques such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy, by contrast, offer fast imaging capability and robustness against sample fluorescence. Yet, their rather low sensitivity impedes their broader application. This review discusses sensitivity enhancement of SRS microscopy to M detection levels by using electronically pre-resonant excitation. We present the foundations of this approach, discuss its technological implementation, and show first successful applications. A special emphasis is given to outlining new experimental developments allowing novel types of investigations.

Assessing Drug Uptake and Response Differences in 2D and 3D Cellular Environments Using Stimulated Raman Scattering Microscopy.

The architecture of cell culture, two-dimensional (2D) versus three-dimensional (3D), significantly impacts various cellular factors, including cell-cell interactions, nutrient and oxygen gradients, metabolic activity, and gene expression profiles. This can result in different cellular responses during cancer drug treatment, with 3D-cultured cells often exhibiting higher resistance to chemotherapeutic drugs. While various genetic and proteomic analyses have been employed to investigate the underlying mechanisms of this increased resistance, complementary techniques that provide experimental evidence of spatial molecular profiling data are limited. Stimulated Raman scattering (SRS) microscopy has demonstrated its capability to measure both intracellular drug uptake and growth inhibition. In this work, we applied three-band (C-D, C-H, and fingerprint regions) SRS imaging to 2D and 3D cell cultures and performed a comparative analysis of drug uptake and response with the goal of understanding whether the difference in drug uptake explains the drug resistance in 3D culture compared to 2D. Our investigations revealed that despite similar intracellular drug levels in 2D and 3D A549 cells during lapatinib treatment, the growth of 3D spheroids was less impacted, supporting an enhanced drug tolerance in the 3D microenvironment. We further elucidated drug penetration patterns and the resulting heterogeneous cellular responses across different spheroid layers. Additionally, we investigated the role of the extracellular matrix in modulating drug delivery and cell response and discovered that limited drug penetration in 3D could also contribute to lower drug response. Our study provides valuable insights into the intricate mechanisms of increased drug resistance in 3D tumor models during cancer drug treatments.

The Duality of Raman Scattering.

ConspectusFirst predicted more than 100 years ago, Raman scattering is a cornerstone of photonics, spectroscopy, and imaging. The conventional framework of understanding Raman scattering was built on Raman cross section σRaman. Carrying a dimension of area, σRaman characterizes the interaction strength between light and molecules during inelastic scattering. The numerical values of σRaman turn out to be many orders of magnitude smaller in comparison to the linear absorption cross sections σAbsorption of similar molecular systems. Such an enormous gap has been the reason for researchers to believe the extremely feeble Raman scattering ever since its discovery. However, this prevailing picture is conceptually problematic or at least incomplete due to the fact that Raman scattering and linear absorption belong to different orders of light-matter interaction.In this Account, we will summarize an alternate way to think about Raman scattering, which we term stimulated response formulation. To capture the third-order interaction nature of Raman scattering, we introduced stimulated Raman cross section, σSRS, defined as the intrinsic molecular property in response to the external photon fluxes. Foremost, experimental measurement of σSRS turns out to be not weak at all or even larger when fairly compared with electronic counterparts of the same order. The analytical expression for σSRS derived from quantum electrodynamics also supports the measurement and proves that σSRS is intrinsically strong. Hence, σRaman and σSRS can be extremely small and large, respectively, for the same molecule at the same time. Our subsequent theoretical studies show that stimulated response formulation can unify spontaneous emission, stimulated emission, spontaneous Raman, and stimulated Raman via eq 10, in a coherent and symmetric way. In particular, an Einstein-coefficient-like equation, eq 12a, was derived, showing that σRaman can be explicitly expressed as σSRS multiplied by an effective photon flux arising from zero-point fluctuation of the vacuum. The feeble vacuum fluctuation hence explains how σSRS can be intrinsically strong while, at the same time, σRaman ends up being many orders of magnitude smaller when both compared to the electronic counterparts. These two sides of the same coin prompted us to propose "the duality of Raman scattering" (Table 1). Finally, this formulation naturally leads to a quantitative treatment of stimulated Raman scattering (SRS) microscopy, providing an intuitive, molecule-centric explanation as to how SRS microscopy can outperform regular Raman microscopy. Hence, as unveiled by the new formulation, a duality of Raman scattering has emerged, with implications for both fundamental science and practical technology.

Label-Free Screening of Drug-Induced Liver Injury Using Stimulated Raman Scattering Microscopy and Spectral Phasor Analysis.

Hepatic toxicity is a leading cause of the termination of clinical trials and the withdrawal of therapeutics following regulatory approval. The detection of drug-induced liver injury (DILI) is therefore of importance to ensure patient safety and the effectiveness of novel small molecules and drugs. DILI encompasses drug-induced steatosis (DIS) and drug-induced phospholipidosis (DIPL) which involve the accumulation of excess intracellular lipids. Here, we develop hyperspectral stimulated Raman scattering (SRS) microscopy as a label-free methodology for discriminating DIS and DIPL in mammalian cell culture. We demonstrate that hyperspectral SRS imaging in tandem with spectral phasor analysis is capable of discriminating DIS and DIPL based on the nature and distribution of intracellular lipids resulting from each process. To demonstrate the practical application of this methodology, we develop a panel of alkyne-tagged propranolol analogues that display varying DILI effects. Using hyperspectral SRS imaging together with spectral phasor analysis, our label-free methodology corroborated the standard fluorescence-based assay for DILI. As a label-free screening method, it offers a convenient and expedient methodology for visualizing hepatotoxicity in cell cultures which could be integrated into the early stages of the drug development process for screening new chemical entities for DILI.

Frequency-Domain Low-Wavenumber Hyperspectral Stimulated Raman Scattering Microscopy.

In recent years, stimulated Raman scattering (SRS) microscopy has experienced rapid technological advancements and has found widespread applications in chemical analysis. Hyperspectral SRS (hSRS) microscopy further enhances the chemical selectivity in imaging by providing a Raman spectrum for each pixel. Time-domain hSRS techniques often require interferometry and ultrashort femtosecond laser pulses. They are especially suited to measuring low-wavenumber Raman transitions but are susceptible to scattering-induced distortions. Frequency-domain hSRS microscopy, on the other hand, offers a simpler optical configuration and demonstrates high tolerance to sample scattering but typically operates within the spectral range of 400-4000 cm-1. Conventional frequency-domain hSRS microscopy is widely employed in biological applications but falls short in detecting chemical bonds with a weaker vibrational energy. In this work, we extend the spectral coverage of picosecond spectral-focusing hSRS microscopy to below 100 cm-1. This frequency-domain low-wavenumber hSRS approach can measure the weaker vibrational energy from the sample and has a strong tolerance to sample scattering. By expanding spectral coverage to 100-4000 cm-1, this development enhances the capability of spectral-domain SRS microscopy for chemical imaging.

Stimulated Raman Scattering Microscopy: A Review

Stimulated Raman Scattering Microscopy: A Review

Spontaneous-stimulated Raman co-localization dual-modal analysis approach for efficient identification of tumor cells

The study of highly heterogeneous tumor cells, especially acute myeloid leukemia (AML) cells, usually relies on invasive analytical methods such as morphology, immunology, cytogenetics, and molecular biology classification, which are complex and time-consuming to perform. Mortality is high if patients are not diagnosed in a timely manner, so rapid label-free analysis of gene expression and metabolites within single-cell substructures is extremely important for clinical diagnosis and treatment. As a label-free and non-destructive vibrational detection technique, spontaneous Raman scattering provides molecular information across the full spectrum of the cell but lacks rapid imaging localization capabilities. In contrast, stimulated Raman scattering (SRS) provides a high-speed, high-resolution imaging view that can offer real-time subcellular localization assistance for spontaneous Raman spectroscopic detection. In this paper, we combined multi-color SRS microscopy with spontaneous Raman to develop a co-localized Raman imaging and spectral detection system (CRIS) for high-speed chemical imaging and quantitative spectral analysis of subcellular structures. Combined with multivariate statistical analysis methods, CRIS efficiently differentiated AML from normal leukocytes with an accuracy of 98.1 % and revealed the differences in the composition of nuclei and cytoplasm of AML relative to normal leukocytes. Compared to conventional Raman spectroscopy blind sampling without imaging localization, CRIS increased the efficiency of single-cell detection by at least three times. In addition, using the same approach for further identification of AML subtypes M2 and M3, we demonstrated that intracytoplasmic differential expression of proteins is a marker for their rapid and accurate classifying. CRIS analysis methods are expected to pave the way for clinical translation of rapid tumor cell identification.

Stain-Free Histopathology with Stimulated Raman Scattering Microscopy.

Stain-Free Histopathology with Stimulated Raman Scattering Microscopy.

Widely tunable fiber optical parametric oscillator synchronized with a Ti:sapphire laser for stimulated Raman scattering microscopy

Stimulated Raman scattering (SRS) microscopy is a powerful vibrational imaging technique with high chemical specificity. However, the insufficient tuning range or speed of light sources limits the spectral range of SRS imaging and, hence, the ability to identify molecular species. Here, we present a widely tunable fiber optical parametric oscillator with a tuning range of 1470 cm−1, which can be synchronized with a Ti:sapphire laser. By using the synchronized light sources, we develop an SRS imaging system that covers the fingerprint and C–H stretching regions, without balanced detection. We validate its broadband imaging capability by visualizing a mixed polymer sample in multiple vibrational modes. We also demonstrate SRS imaging of HeLa cells, showing the applicability of our SRS microscope to biological samples.

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.

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.

Spatiotemporal Observation of Monosodium Urate Crystals Deposition in Synovial Organoids Using Label-Free Stimulated Raman Scattering.

Gout, a common form of arthritis, is characterized by the deposition of monosodium urate (MSU) crystals in joints. MSU deposition in synovial tissues would initiate arthritis flares and recurrence, causing irreversible joint damage. However, the dynamic deposition of MSU crystals in tissues lacks experimental observation. In this study, we used chemical-specific, label-free stimulated Raman scattering (SRS) microscopy to investigate the spatiotemporal deposition and morphological characteristics of MSU crystals in human synovial organoids. Our findings revealed a critical 12-h window for MSU deposition in the lining layer of gouty synovium. Moreover, distinctive inflammatory reactions of the lining and sublining synovial layers in gout using SRS microscopy were further verified by immunofluorescence. Importantly, we identified a crucial proinflammatory role of sublining fibroblast-like synoviocytes, indicating a need for targeted medication treatment on these cells. Our work contributes to the fundamental understanding of MSU-based diseases and offers valuable insights for the future development of targeted gout therapies.

Photoswitchable polyynes for multiplexed stimulated Raman scattering microscopy with reversible light control

Optical imaging with photo-controllable probes has greatly advanced biological research. With superb chemical specificity of vibrational spectroscopy, stimulated Raman scattering (SRS) microscopy is particularly promising for super-multiplexed optical imaging with rich chemical information. Functional SRS imaging in response to light has been recently demonstrated, but multiplexed SRS imaging with reversible photocontrol remains unaccomplished. Here, we create a multiplexing palette of photoswitchable polyynes with 16 Raman frequencies by coupling asymmetric diarylethene with super-multiplexed Carbow (Carbow-switch). Through optimization of both electronic and vibrational spectroscopy, Carbow-switch displays excellent photoswitching properties under visible light control and SRS response with large frequency change and signal enhancement. Reversible and spatial-selective multiplexed SRS imaging of different organelles are demonstrated in living cells. We further achieve photo-selective time-lapse imaging of organelle dynamics during oxidative stress and protein phase separation. The development of Carbow-switch for photoswitchable SRS microscopy will open up new avenues to study complex interactions and dynamics in living cells with high spatiotemporal precision and multiplexing capability.

Coherent Raman spectroscopy: Quo vadis?

Although the potential of Coherent Raman Spectroscopy (CRS) in the area of biomedicine, life sciences and material sciences has been well demonstrated, its wide-spread practical application is still rather limited. The two main CRS techniques are Coherent Anti-Stokes Raman Scattering (CARS) and Stimulated Raman Scattering (SRS) spectroscopy or microscopy. Here we present the current state of the art and challenges facing CRS. Although many technological challenges have been addressed to date, showing how to improve resolution, sensitivity and selectivity of CRS, significant efforts are still needed to increase the awareness of these techniques in the academic community, develop reliable protocols, and extend them to practical applications. For this purpose it is also necessary to initiate national and international research networks that can significantly contribute to the development of CRS approaches in areas that have so far made little use of CRS alongside other Raman spectroscopic methods. The purpose of this perspective paper is to present the current state-of-the-art of CRS with a historical background, assess the challenges and present some future development visions.

Fundamental detectability of Raman scattering: A unified diagrammatic approach.

Both spontaneous Raman scattering and stimulated Raman scattering (SRS) are cornerstones of modern photonics, spectroscopy, and imaging. However, a unified understanding of the ultimate detectability of Raman scattering is lacking, due to both historical and technical reasons. Starting from quantum electrodynamics, we formulate the fundamental detectability for both spontaneous Raman scattering and SRS. The key concept is recognizing spontaneous Raman scattering as stimulated Raman process driven by vacuum field fluctuation. A simple and unified expression, Eq. (17), is derived, which can be depicted on a two-dimensional phase-diagram-like graph with inherent symmetry. It turns out that the particle nature of light dictates the ultimate detectability of spontaneous Raman scattering, which can be represented by a line on this detectability diagram. Importantly, if provided with a reasonably strong Stokes photon flux, SRS can breach this fundamental limit and open uncharted territory of drastically accelerated measurement speed and much lower detection concentration relevant to biological imaging. Such new territory in the detectability diagram is otherwise forbidden by the spontaneous counterpart. Diagrammatical analysis explains the empirical observations, provides quantitative insights, and makes new predictions. Notably, recent experimental applications of SRS microscopy can almost entirely be captured by this diagram, further supporting the explanatory power of the theory. Thus, this unified diagrammatic approach outlines a framework to understand all Raman-based measurement and provides a theoretical explanation for the remarkable utility of the emerging SRS microscopy.

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.

Limits of the detection of microplastics in fish tissue using stimulated Raman scattering microscopy.

We demonstrate the detection sensitivity of microplastic beads within fish tissue using stimulated Raman scattering (SRS) microscopy. The intrinsically provided chemical contrast distinguishes different types of plastic compounds within fish tissue. We study the size-dependent signal-to-noise ratio of the microplastic beads and determine a lower boundary for the detectable size. Our findings demonstrate how SRS microscopy can serve as a complementary modality to conventional Raman scattering imaging in order to detect and identify microplastic particles in fish tissue.

Stimulated Raman Scattering Microscopy Reveals Bioaccumulation of Small Microplastics in Protozoa from Natural Waters.

Microplastics (MPs) are pollutants of global concern, and bioaccumulation determines their biological effects. Although microorganisms form a large fraction of our ecosystem's biomass and are important in biogeochemical cycling, their accumulation of MPs has never been confirmed in natural waters because current tools for field biological samples can detect only MPs > 10 μm. Here, we show that stimulated Raman scattering microscopy (SRS) can image and quantify the bioaccumulation of small MPs (<10 μm) in protozoa. Our label-free method, which differentiates MPs by their SRS spectra, detects individual and mixtures of different MPs (e.g., polyethylene, polypropylene, polyvinyl chloride, polyethylene terephthalate, polystyrene, and poly(methyl methacrylate)) in protozoa. The ability of SRS to quantify cellular MP accumulation is similar to that of flow cytometry, a fluorescence-based method commonly used to determine cellular MP accumulation. Moreover, we discovered that protozoa in water samples from Yangtze River, Xianlin Wastewater Treatment Plant, Lake Taihu and the Pearl River Estuary accumulated MPs < 10 μm, but the proportion of MP-containing cells was low (∼2-5%). Our findings suggest that small MPs could potentially enter the food chain and transfer to organisms at higher trophic levels, posing environmental and health risks that deserve closer scrutiny.