Stimulated Raman Scattering Microscopy Research Articles

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.

Stimulated Raman Scattering Microscopy Reveals Aberrant Triglyceride Accumulation in Lymphatic Metastasis of Papillary Thyroid Carcinoma.

Lipid metabolic alterations are known to play a crucial role in cancer metastasis. As a key hub in lipid metabolism, intracellular neutral lipid accumulation in lipid droplets (LDs) has become a signature of aggressive human cancers. Nevertheless, it remains unclear whether lipid accumulation displays distinctive features in metastatic lesions compared to the primary ones. Here, we integrated multicolor stimulated Raman scattering (SRS) imaging with confocal Raman spectroscopy on the same platform to quantitatively analyze the amount and composition of LDs in intact human thyroid tissues in situ without any processing or labeling. Inspiringly, we found aberrant accumulation of triglycerides (TGs) in lymphatic metastases but not in normal thyroid, primary papillary thyroid carcinoma (PTC), or normal lymph node. In addition, the unsaturation degree of unsaturated TGs was significantly higher in the lymphatic metastases from patients diagnosed with late-stage (T3/T4) PTC compared to those of patients diagnosed with early-stage (T1/T2) PTC. Furthermore, both public sequencing data analysis and our RNA-seq transcriptomic experiment showed significantly higher expression of alcohol dehydrogenase-1B (ADH1B), which is critical to lipid uptake and transport, in lymphatic metastases relative to the primary ones. In summary, these findings unravel the lipid accumulation as a novel marker and therapeutic target for PTC lymphatic metastasis that has a poor response to the regular radioactive iodine therapy.

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.

Discrimination of lipid composition and cellular localization in human liver tissues by stimulated Raman scattering microscopy.

The molecular mechanisms driving the progression from nonalcoholic fatty liver (NAFL) to fibrosing steatohepatitis (NASH) are insufficiently understood. Techniques enabling the characterization of different lipid species with both chemical and spatial information can provide valuable insights into their contributions to the disease progression. We extend the utility of stimulated Raman scattering (SRS) microscopy to characterize and quantify lipid species in liver tissue sections from patients with NAFL and NASH. We applied a dual-band hyperspectral SRS microscopy system for imaging tissue sections in both the C-H stretching and fingerprint regions. The same sections were imaged with polarization microscopy for detecting birefringent liquid crystals in the tissues. Our imaging and analysis pipeline provides accurate classification and quantification of free cholesterol, saturated cholesteryl esters (CEs), unsaturated CE, and triglycerides in liver tissue sections. The subcellular resolution enables investigations of the heterogeneous distribution of saturated CE, which has been under-examined in previous studies. We also discovered that the birefringent crystals, previously found to be associated with NASH development, are predominantly composed of saturated CE. Our method allows for a detailed characterization of lipid composition in human liver tissues and enables further investigation into the potential mechanism of NASH progression.

Rapid single-particle chemical imaging of nanoplastics by SRS microscopy.

Plastics are now omnipresent in our daily lives. The existence of microplastics (1 µm to 5 mm in length) and possibly even nanoplastics (<1 μm) has recently raised health concerns. In particular, nanoplastics are believed to be more toxic since their smaller size renders them much more amenable, compared to microplastics, to enter the human body. However, detecting nanoplastics imposes tremendous analytical challenges on both the nano-level sensitivity and the plastic-identifying specificity, leading to a knowledge gap in this mysterious nanoworld surrounding us. To address these challenges, we developed a hyperspectral stimulated Raman scattering (SRS) imaging platform with an automated plastic identification algorithm that allows micro-nano plastic analysis at the single-particle level with high chemical specificity and throughput. We first validated the sensitivity enhancement of the narrow band of SRS to enable high-speed single nanoplastic detection below 100 nm. We then devised a data-driven spectral matching algorithm to address spectral identification challenges imposed by sensitive narrow-band hyperspectral imaging and achieve robust determination of common plastic polymers. With the established technique, we studied the micro-nano plastics from bottled water as a model system. We successfully detected and identified nanoplastics from major plastic types. Micro-nano plastics concentrations were estimated to be about 2.4 ± 1.3 × 105 particles per liter of bottled water, about 90% of which are nanoplastics. This is orders of magnitude more than the microplastic abundance reported previously in bottled water. High-throughput single-particle counting revealed extraordinary particle heterogeneity and nonorthogonality between plastic composition and morphologies; the resulting multidimensional profiling sheds light on the science of nanoplastics.

Super-Resolution Stimulated Raman Scattering Microscopy with Graphical User Interface-Supported A-PoD.

Raman microscopy is a vibrational imaging technology that can detect molecular chemical bond vibrational signals. Since this signal is originated from almost every vibrational mode of molecules with different vibrational energy levels, it provides spatiotemporal distribution of various molecules in living organisms without the need for any labeling. The limitations of low signal strength in Raman microscopy have been effectively addressed by incorporating a stimulated emission process, leading to the development of stimulated Raman scattering (SRS) microscopy. Furthermore, the issue of low spatial resolution has been resolved through the application of computational techniques, specifically image deconvolution. In this article, we present a comprehensive guide to super-resolution SRS microscopy using an Adam-based pointillism deconvolution (A-PoD) algorithm, complemented by a user-friendly graphical user interface (GUI). We delve into the crucial parameters and conditions necessary for achieving super-resolved images through SRS imaging. Additionally, we provide a step-by-step walkthrough of the preprocessing steps and the use of GUI-supported A-PoD. This complete package offers a user-friendly platform for super-resolution SRS microscopy, enhancing the versatility and applicability of this advanced microscopy technique to reveal nanoscopic multimolecular nature. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Super-resolution stimulated Raman scattering microscopy with graphical user interface-supported A-PoD Support Protocol: Deuterium labeling on cells with heavy water for metabolic imaging.

Super-resolution stimulated Raman scattering microscopy enhanced by quantum light and deconvolution

Stimulated Raman scattering (SRS) microscopy is a powerful tool for label-free chemical contrast bio-imaging. However, its spatial resolution is limited by diffraction; its noise level is also fundamentally limited by the shot noise due to the quantum nature of light. In this work, we apply the squeezed light technique associated with the deconvolution method to achieve quantum-enhanced super-resolution SRS microscopy. To generate squeezed pump light, we design a unique cascaded scheme by using two nonlinear crystals, in which the second-harmonic generation (SHG) from the first crystal is used to boost the SHG of the second crystal sequentially. Such a cascaded light squeezed scheme suppresses the shot noise down to 89.7% (1 dB), which can be readily applied to the existing conventional SRS microscopy. We combine the squeezed light-controlled SRS with the Richardson–Lucy deconvolution method to break the diffraction limit by improving the spatial resolution of ∼2.2-fold compared to conventional SRS imaging. We realize the quantum-enhanced super-resolution SRS imaging in a variety of samples (e.g., oleic acid, porcine muscle tissue), suggesting the potential of squeezed light SRS with deconvolution for label-free super-resolution chemical imaging in biological and biomedical systems.

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

AbstractMultiplex 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.

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.

Electronically preresonant stimulated Raman scattering microscopy in the visible

We report an experimental scheme for stimulated Raman scattering (SRS) microscopy with excitation in the visible spectral region. This allows electronically preresonant (epr) SRS microscopy of a broad range of chromophores with sensitivities as low as 1 μM. Our experiment is based on two synchronously near-infrared pumped optical parametric oscillators (OPO). One of the outputs is modulated at a fourth of the repetition rate with a novel broadband electro-optical modulator. Using a combination of spectral focusing and tuning of the OPO, we show the recording of epr-SRS spectra over the whole range of molecular vibrations at a speed up to 20 times faster than classical wavelength tuning. The imaging capabilities of this setup are demonstrated with material scientific and cellular samples.

Investigating ionizing radiation-induced changes in breast cancer cells using stimulated Raman scattering microscopy.

Altered lipid metabolism of cancer cells has been implicated in increased radiation resistance. A better understanding of this phenomenon may lead to improved radiation treatment planning. Stimulated Raman scattering (SRS) microscopy enables label-free and quantitative imaging of cellular lipids but has never been applied in this domain. We sought to investigate the radiobiological response in human breast cancer MCF7 cells using SRS microscopy, focusing on how radiation affects lipid droplet (LD) distribution and cellular morphology. MCF7 breast cancer cells were exposed to either 0 or 30Gy (X-ray) ionizing radiation and imaged using a spectrally focused SRS microscope every 24hrs over a 72-hr time period. Images were analyzed to quantify changes in LD area per cell, lipid and protein content per cell, and cellular morphology. Cell viability and confluency were measured using a live cell imaging system while radiation-induced lipid peroxidation was assessed using BODIPY C11 staining and flow cytometry. The LD area per cell and total lipid and protein intensities per cell were found to increase significantly for irradiated cells compared to control cells from 48 to 72hrs post irradiation. Increased cell size, vacuole formation, and multinucleation were observed as well. No significant cell death was observed due to irradiation, but lipid peroxidation was found to be greater in the irradiated cells than control cells at 72hrs. This pilot study demonstrates the potential of SRS imaging for investigating ionizing radiation-induced changes in cancer cells without the use of fluorescent labels.

Electronically Preresonant Stimulated Raman Scattering Microscopy of Weakly Fluorescing Chromophores.

Stimulated Raman Scattering microscopy is an important imaging technique. Its broader application, however, is hampered by its comparatively low sensitivity. Using organic fluorophores, it has recently been demonstrated that, similar to spontaneous Raman microscopy, the sensitivity of stimulated Raman microscopy is increased by orders of magnitudes if electronic preresonances are exploited. In this Article, we show that this approach also works with low quantum yield chromophores. We investigate the relevant photophysics and discuss the background arising from preresonant excitation conditions. Applications of preresonant stimulated Raman scattering microscopy for the imaging of weakly fluorescing labels in fixed and live cells are demonstrated.

Absolute Stimulated Raman Cross Sections of Molecules

Stimulated Raman scattering (SRS) is a fundamental optical process that was discovered more than 60 years ago. While the early SRS spectroscopy studies have provided valuable insights into materials systems, the advent of SRS microscopy has launched a rapidly growing field in biological imaging. However, a fundamental understanding of the molecular response under SRS is still lacking. Herein we present a new framework to introduce molecule-intrinsic stimulated Raman scattering cross sections, σSRS, in the unit of Göppert-Mayer (GM). The absolute SRS cross sections determined for real molecular systems challenge the conventional wisdom that Raman spectroscopy is always a weak process. The enormous rate acceleration of SRS, captured by an apparent SRS cross section, stems from a synergistic effect between the field and the molecule. Our new framework goes beyond the conventional optics-centric view and presents a molecule-inclusive perspective, thus offering a comprehensive foundation for the future growth of SRS spectroscopy and microscopy.

High-Speed Stimulated Raman Scattering Microscopy Using Inertia-Free AOD Scanning

High-throughput stimulated Raman scattering (SRS) microscopy is highly desired for large tissue imaging with chemical specificity. However, the mapping speed remains as the major short board of conventional SRS, primarily owing to the mechanical inertia existing in galvanometers or other laser scanning alternatives. Here, we developed inertia-free acousto-optic deflector (AOD)-based high-speed large-field stimulated Raman scattering microscopy, in which both the speed and integration time are ensured by immune of the mechanical response time. To avoid laser beam distortion induced by the intrinsic spatial dispersion of AODs, two spectral compression systems are implemented to compress the broad-band femtosecond pulse to picosecond laser. We achieved an SRS imaging of a 12 × 8 mm2 mouse brain slice in only 8 min at an image resolution of approximately 1 μm and 32 slices from a whole brain in 12 h. The AOD-based inertia-free SRS mapping can be much faster after further upgrading and allow broad-spectrum applications of chemical imaging in the future.

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.