Hyperspectral Stimulated Raman Scattering Research Articles

Insights into pharmaceutical co-crystallization using coherent Raman microscopy

Formulating active pharmaceutical ingredients (APIs) as co-crystals requires a thorough understanding of co-crystallization behavior under different process conditions. This study employs two forms of coherent Raman microscopy, narrowband coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) with spectral focusing, to study co-crystallization via liquid-assisted ball milling. Indomethacin and nicotinamide served as the model API and co-former, and the results were compared with established analytical methods. Narrowband CARS, with univariate peak position analysis, was useful to visualize co-crystal formation, but suffered some degree of signal mixing that affected component identification. Hyperspectral SRS imaging, combined with classical least squares multivariate analysis, separated the different components with high confidence and proved to be a robust and rapid tool to qualitatively and quantitatively image co-crystallization. The coherent Raman imaging results explained divergent co-crystallization endpoints obtained with the conventional solid-state analysis methods. CARS and SRS microscopies also revealed the presence of otherwise undetected trace forms. Finally, we also demonstrated the dramatic reversal of partial co-crystal formation during milling, depending on ethanol content. Overall, the study demonstrates the added value coherent Raman microscopy can provide for analysis of co-crystallization processes.

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.

Quantitative assessment and monitoring of microplastics and nanoplastics distributions and lipid metabolism in live zebrafish using hyperspectral stimulated Raman scattering microscopy

Microplastics (MP) and nanoplastics (NP) pollutions pose a rising environmental threat to humans and other living species, given their escalating presence in essential resources that living subjects ingest and/or inhale. Herein, to elucidate the potential health implications of MP/NP, we report for the first time by using label-free hyperspectral stimulated Raman scattering (SRS) imaging technique developed to quantitatively monitor the bioaccumulation and metabolic toxicity of MP/NP within live zebrafish larvae during their early developmental stages. Zebrafish embryos are exposed to environmentally related concentrations (3–60 μg/ml) of polystyrene (PS) beads with two typical sizes (2 μm and 50 nm). Zebrafish are administered isotope-tagged fatty acids through microinjection and dietary intake for in vivo tracking of lipid metabolism dynamics. In vivo 3D quantitative vibrational imaging of PS beads and intrinsic biomolecules across key zebrafish organs reveals that gut and liver are the primary target organs of MP/NP, while only 50 nm PS beads readily aggregate and adhere to the brain and blood vessels. The 50 nm PS beads are also found to induce more pronounced hepatic inflammatory response compared to 2 μm counterparts, characterized by increased biogenesis of lipid droplets and upregulation of arachidonic acid detected in zebrafish liver. Furthermore, Raman-tagged SRS imaging of fatty acids uncovers that MP/NP exposure significantly reduces yolk lipid utilization and promotes dietary lipid storage in zebrafish, possibly associated with developmental delays and more pronounced food dilution effects in zebrafish larvae exposed to 2 μm PS beads. The hyperspectral SRS imaging in this work shows that MP/NP exposure perturbs the development and lipid metabolism in zebrafish larvae, furthering the understanding of MP/NP ingestions and consequent toxicity in different organs in living species.

Transient stimulated Raman scattering spectroscopy and imaging

Stimulated Raman scattering (SRS) has been developed as an essential quantitative contrast for chemical imaging in recent years. However, while spectral lines near the natural linewidth limit can be routinely achieved by state-of-the-art spontaneous Raman microscopes, spectral broadening is inevitable for current mainstream SRS imaging methods. This is because those SRS signals are all measured in the frequency domain. There is a compromise between sensitivity and spectral resolution: as the nonlinear process benefits from pulsed excitations, the fundamental time-energy uncertainty limits the spectral resolution. Besides, the spectral range and acquisition speed are mutually restricted. Here we report transient stimulated Raman scattering (T-SRS), an alternative time-domain strategy that bypasses all these fundamental conjugations. T-SRS is achieved by quantum coherence manipulation: we encode the vibrational oscillations in the stimulated Raman loss (SRL) signal by femtosecond pulse-pair sequence excited vibrational wave packet interference. The Raman spectrum was then achieved by Fourier transform of the time-domain SRL signal. Since all Raman modes are impulsively and simultaneously excited, T-SRS features the natural-linewidth-limit spectral line shapes, laser-bandwidth-determined spectral range, and improved sensitivity. With ~150-fs laser pulses, we boost the sensitivity of typical Raman modes to the sub-mM level. With all-plane-mirror high-speed time-delay scanning, we further demonstrated hyperspectral SRS imaging of live-cell metabolism and high-density multiplexed imaging with the natural-linewidth-limit spectral resolution. T-SRS shall find valuable applications for advanced Raman imaging.

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.

Spectral fingerprinting of cellular lipid droplets using stimulated Raman scattering microscopy and chemometric analysis.

Hyperspectral stimulated Raman scattering (SRS) microscopy is a powerful method for direct visualisation and compositional analysis of cellular lipid droplets. Here we report the application of spectral phasor analysis as a convenient method for the segmentation of lipid droplets using the hyperspectral SRS spectrum in the high wavenumber and fingerprint region of the spectrum. Spectral phasor analysis was shown to discriminate six fatty acids based on vibrational spectroscopic features in solution. The methodology was then applied to studying fatty acid metabolism and storage in a mammalian cancer cell model and during drug-induced steatosis in a hepatocellular carcinoma cell model. The accumulation of fatty acids into cellular lipid droplets was shown to vary as a function of the degree of unsaturation, whilst in a model of drug-induced steatosis, the detection of increased saturated fatty acid esters was observed. Taking advantage of the fingerprint and high wavenumber regions of the SRS spectrum has yielded a greater insight into lipid droplet composition in a cellular context. This approach will find application in the label-free profiling of intracellular lipids in complex disease models.

Multimodal imaging of a liver-on-a-chip model using labelled and label-free optical microscopy techniques.

A liver-on-a-chip model is an advanced complex in vitro model (CIVM) that incorporates different cell types and extracellular matrix to mimic the microenvironment of the human liver in a laboratory setting. Given the heterogenous and complex nature of liver-on-a-chip models, brightfield and fluorescence-based imaging techniques are widely utilized for assessing the changes occurring in these models with different treatment and environmental conditions. However, the utilization of optical microscopy techniques for structural and functional evaluation of the liver CIVMs have been limited by the reduced light penetration depth and lack of 3D information obtained using these imaging techniques. In this study, the potential of both labelled as well as label-free multimodal optical imaging techniques for visualization and characterization of the cellular and sub-cellular features of a liver-on-a-chip model was investigated. (1) Cellular uptake and distribution of Alexa 488 (A488)-labelled non-targeted and targeted antisense oligonucleotides (ASO and ASO-GalNAc) in the liver-on-a-chip model was determined using multiphoton microscopy. (2) Hyperspectral stimulated Raman scattering (SRS) microscopy of the C-H region was used to determine the heterogeneity of chemical composition of circular and cuboidal hepatocytes in the liver-on-a-chip model in a label-free manner. Additionally, the spatial overlap between the intracellular localization of ASO and lipid droplets was explored using simultaneous hyperspectral SRS and fluorescence microscopy. (3) The capability of light sheet fluorescence microscopy (LSFM) for full-depth 3D visualization of sub-cellular distribution of A488-ASO and cellular phenotypes in the liver-on-a-chip model was demonstrated. In summary, multimodal optical microscopy is a promising platform that can be utilized for visualization and quantification of 3D cellular organization, drug distribution and functional changes occurring in liver-on-a-chip models, and can provide valuable insights into liver biology and drug uptake mechanisms by enabling better characterization of these liver models.

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.

Visualizing drug-induced lipid accumulation in lysosomes of live cancer cells with stimulated Raman imaging.

The low pH of the lysosomal compartment often results in sequestration of chemotherapeutic agents that contain positively charged basic functional groups, leading to anti-cancer drug resistance. To visualize drug localization in lysosomes and its influence on lysosomal functions, we synthesize a group of drug-like compounds that contain both a basic functional group and a bisarylbutadiyne (BADY) group as a Raman probe. With quantitative stimulated Raman scattering (SRS) imaging, we validate that the synthesized lysosomotropic (LT) drug analogs show high lysosomal affinity, which can also serve as a photostable lysosome tracker. We find that long-term retention of the LT compounds in lysosomes leads to the increased amount and colocalization of both lipid droplets (LDs) and lysosomes in SKOV3 cells. With hyperspectral SRS imaging, further studies find that the LDs stuck in lysosomes are more saturated than the LDs staying out of the lysosomes, indicating impaired lysosomal lipid metabolism by the LT compounds. These results demonstrate that SRS imaging of the alkyne-based probes is a promising approach to characterizing the lysosomal sequestration of drugs and its influence on cell functions.

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.

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.

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.

Imaging Low-Temperature Phases of Ice with Polarization-Resolved Hyperspectral Stimulated Raman Scattering Microscopy.

Water freezes into various phases of ice under different cryogenic temperatures and pressure conditions, such as ice Ih and ice XI at normal pressure. Vibrational imaging with high spectral, spatial, and polarization resolutions could provide detailed information on ice, including the phases and crystal orientations at the microscopic level. Here, we report in situ stimulated Raman scattering (SRS) imaging of ice to analyze the vibrational spectral changes of the OH stretching modes associated with the phase transition between ice Ih and ice XI. In addition, polarization-resolved measurements were performed to reveal the microcrystal orientations of the two phases of ice, with the spatial-dependent anisotropy pattern indicating the inhomogeneous distribution of their orientations. Furthermore, the angular patterns were theoretically explained by third-order nonlinear optics with the known crystal symmetries of the ice phases. Our work may provide new opportunities to investigate many intriguing physical chemistry properties of ice under low-temperature conditions.

Simultaneous Dual-Band Hyperspectral Stimulated Raman Scattering Microscopy with Femtosecond Optical Parametric Oscillators.

Stimulated Raman scattering (SRS) microscopy is a label-free quantitative optical technique for imaging molecular distributions in cells and tissues by probing their intrinsic vibrational frequencies. Despite its usefulness, existing SRS imaging techniques have limited spectral coverage due to either a wavelength tuning constraint or narrow spectral bandwidth. High-wavenumber SRS imaging is commonly used to map lipid and protein distribution in biological cells and visualize cell morphology. However, to detect small molecules or Raman tags, imaging in the fingerprint region or "silent" region, respectively, is often required. For many applications, it is desirable to collect SRS images in two Raman spectral regions simultaneously for visualizing the distribution of specific molecules in cellular compartments or providing accurate ratiometric analysis. In this work, we present an SRS microscopy system using three beams generated by a femtosecond oscillator to acquire hyperspectral SRS image stacks in two arbitrary vibrational frequency bands, between 650-3280 cm-1, simultaneously. We demonstrate potential biomedical applications of the system in investigating fatty acid metabolism, cellular drug uptake and accumulation, and lipid unsaturation level in tissues. We also show that the dual-band hyperspectral SRS imaging system can be adapted for the broadband fingerprint region hyperspectral imaging (1100-1800 cm-1) by simply adding a modulator.

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging.

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging technologies. Hyperspectral SRS and CARS imaging offer Raman spectral information at every pixel, which enables better separation of different chemical compositions. Although both techniques require two excitation lasers, their signal detection schemes and spectral properties are quite different. The goal of this protocol is to perform both hyperspectral SRS and CARS imaging on a single platform and compare the two microscopy techniques for imaging different biological samples. The spectral focusing method is employed to acquire spectral information using femtosecond lasers. By using standard chemical samples, the sensitivity, spatial resolution, and spectral resolution of SRS and CARS in the same excitation conditions (i.e., power at the sample, pixel dwell time, objective lens, pulse energy) are compared. The imaging contrasts of CARS and SRS for biological samples are juxtaposed and compared. The direct comparison of CARS and SRS performances would allow for optimal selection of the modality for chemical imaging.

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

Hyperspectral stimulated Raman scattering microscopy facilitates differentiation of low-grade and high-grade human prostate cancer

Histopathology currently acts as a gold standard for human prostate cancer (PCa) diagnosis. However, the subjective nature of histopathology leads to inevitable discordance among pathologists. Specifically, the inter-observer discordance could be up to 40% for the differentiation between Gleason score 6 (low-grade) and 7 (high-grade) of PCa. According to clinical guidelines, patients with high-grade PCa need to be actively treated, while patients with low-grade PCa could undergo active surveillance due to its slowly growing feature. Therefore, differentiating high-grade and low-grade PCa is an urgent clinical need. By integrating stimulated Raman scattering microscopy and confocal Raman spectroscopy, our previous study found the aberrant cholesteryl ester (CE) accumulation in human PCa tissues. However, no significant difference in CE accumulation between the low-grade and high-grade PCa was found, primarily because the previous study only analyzed the composition of manually selected lipid droplets (LDs) without quantitative analysis of the whole field of view. Here, we employed hyperspectral stimulated Raman scattering (HSRS) microscopy to test the hypothesis of CE as a marker for differentiation of low-grade and high-grade human PCa. First, lipid, lipofuscin, and protein were quantitatively mapped in human prostate tissues based on HSRS imaging of C–H vibrational region and multivariate curve resolution analysis. Then, within the lipid channel, CE percentage and unsaturation level of LDs were quantitatively mapped according to the height ratio between Raman bands at 2870 and 2850 cm−1, and between Raman bands at 3006 and 2850 cm−1, respectively. In total of 6 normal prostate tissues, 9 low-grade and 9 high-grade PCa tissues from patients, we found lipofuscin accumulation in all the normal prostate but none in PCa. While all the high-grade PCa tissues had LD accumulation, only 3 low-grade PCa tissues had LD accumulation. Notably, among all the PCa tissues with LD accumulation, no significant difference in LD amount was found between low-grade and high-grade PCa. Fortunately, both CE percentage and unsaturation level of the LDs were significantly different between low-grade and high-grade PCa. Furthermore, it was shown that CE percentage could differentiate low-grade and high-grade PCa with high sensitivity and specificity. Taken together, our study may provide a new opportunity towards more accurate PCa diagnosis.

Multivariate analysis of hyperspectral stimulated Raman scattering microscopy images

AbstractCoherent Raman scattering microscopy is an attractive new technology for label‐free imaging. Both coherent anti‐Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy offer the possibility to record hyperspectral imaging data. While for the analysis of spontaneous Raman microscopy data multivariate methods are nowadays routinely employed, until to date most of the coherent Raman imaging data are interpreted using univariate data analysis. In this work, we report a quantitative comparison of the performance of different multivariate methods used for the analysis of hyperspectral SRS data from different model samples. Our data show for all samples that multivariate methods outperform univariate analysis. Using metrics to quantify method performance, we find that of the methods tested, multivariate curve resolution (MCR) gives the best results. We show that the combination with a selection of essential components based on first‐order autocorrelation, gives a simple workflow for the MCR‐based analysis of hyperspectral coherent Raman imaging data.