This conference presentation was prepared for SPIE BiOS, 2023.

To uncover the pathological mechanism of neurodegenerative diseases, simple and cost-effective analytical methods are highly desirable to obtain biochemistry and morphology information on Tau protein aggregation. Here, we demonstrate the fluorescence-guided computational mid-infrared photothermal microscope, termed Fluorescence-guided Bond-selective Intensity Diffraction Tomography (FBS-IDT). FBS-IDT enables hyperspectral 3D bond-selective refractive index map recovery on Tau fibrils in cells. Depth-resolved mid-infrared fingerprint spectra extraction and related secondary protein structure analysis are demonstrated. 3D reconstruction of β sheet in Tau fibril is realized mainly based on the mid-infrared photothermal effects.

The imaging speed of the current mid-infrared photothermal (MIP) microscope is limited to tens of seconds per frame due to the long pixel dwell time and slow sample scanning process, which is insufficient for capturing dynamics inside living systems. In this work, we developed a video-rate MIP microscope by employing a lock-in free demodulation scheme to resolve single IR pulse induced contrast. We further developed a synchronous pump-probe Galvo scanning for reaching a line rate over 2.5 kHz. The system is capable of resolving chemical dynamics in living cells in a uniform imaging field of view over 300 μm.

Label-free spectroscopic detection of single viruses provides component analysis of virus strains. Current methods suffer from low throughput and weak signal contrast of individual virions. Here, we report a wide-field interferometric defocus-enhanced mid-infrared photothermal (WIDE-MIP) microscopy for high-throughput fingerprinting of single viruses. Comprehensive theoretical frameworks for WIDE-MIP signal is developed and experimentally validated by acquiring defocused interferometric and photothermal images. WIDE-MIP not only provides the amide I and amide II vibrations in viral proteins, but also reveals the unique IR signature of thymine and uracil residues vibration in DNA vaccinia viruses and RNA vesicular stomatitis viruses, respectively.

Raman probes based on alkyne or nitrile tags hold promise for highly multiplexed imaging. In order to functionalize the Raman probes to trace biological phenomena in live cells, we have established a general strategy to control Raman signal intensity based on the resonant Raman effect, and prepared activatable Raman probes that show enhanced Raman signals upon reaction with enzymes under physiological conditions. Recently, we expanded our molecular design strategy by developing aggregation-based Raman probes for detecting enzyme activities in tissues, photo-activatable Raman probes, and photo-switchable Raman probes for super-resolution Raman imaging.

Innovations in optical spectroscopy and microscopy have revolutionized our understanding in live biological systems at the sub-cellular levels. In this talk, I will present our recent advancements in developing and applying stimulated Raman scattering (SRS) imaging coupled with novel vibrational tags for specific and highly sensitive investigations of complex biological (i.e. cancer- and neuronal-) systems.

Alkyne-tagged Raman probes have shown high promise for noninvasive and sensitive visualization of small biomolecules in live cells. Here, we report a general strategy for Raman imaging-based local environment sensing by hydrogen-deuterium exchange (HDX) of terminal alkynes. We first demonstrate, in multiple Raman probes, that deuterations of the alkynyl hydrogens lead to remarkable shifts of alkyne Raman peaks, providing resolvable signals suited for imaging-based analysis with high specificity. Both our analytical derivation and experimental characterizations subsequently establish that HDX kinetics are linearly proportional to both alkyne pKas and environmental pDs. We establish that alkyne-HDX exhibits high sensitivity to various DNA structures and subtle pD variations in live cells. After validating the quantitative nature of this strategy, our work lays the foundation for utilizing alkyne-HDX strategy to quantitatively sense the local chemical and cellular environments.

Recent developments have shown the utility of vibrational tags in both Raman-based microscopy and infrared-based microspectroscopy. In this context, the availability of probes that feature a strong response is highly desirable. Here we develop vibrational tags that have strong and narrow vibrational lines in the cell silent region of the spectrum. We screened numerous chemical motifs with density functional theory (DFT). Design criteria included a distinct resonance frequency, a long vibrational lifetime, and overall strength of the vibrational response. We discuss the utility of these optimized probes for cellular imaging studies with stimulated Raman scattering and Fourier transform infrared microspectroscopy.

We use D2O probed stimulated Raman scattering (DO-SRS) and multiphoton fluorescence (MPF) microscopy to visualize metabolic changes in cells and tissues during aging processes and diseases. We further developed deconvolution algorithm to enhance the spatial resolution to generate super resolution metabolic imaging. The cellular spatial distribution of de novo lipogenesis, protein synthesis, NADH, Flavin, unsaturated lipids, and cholesterol were all imaged and quantified with hyperspectral detection of various macromolecules as multiplex imaging. Our study shows that the integrated metabolic imaging and spectroscopy platform can be applied to study nutrition regulated metabolic dynamics in cancer and during aging processes.

A suite of novel chemical imaging methods has been developed to collectively inform on the design and optimization of storage and delivery platforms for therapeutic molecules. Brought into dramatic relief by cold-storage constraints in COVID-19 vaccines, the shelf-lives and storage conditions of pharmaceutical materials can profoundly impact global access to life-saving medications. Our group has developed a suite of complementary chemical imaging methods that collectively provide unique insights into critical factors affecting stability and bioavailability of amorphous solid dispersions (ASDs), used routinely for pharmaceutical formulations. Methods and applications of these novel approaches will be summarized.

Simultaneous observation of cytotoxicity and drug distribution at the effector site will bring new insights into understanding mechanism of action of drug. Here, we demonstrated the application of Raman microscopy to visualize drug-induced cytotoxicity and drug distribution as well as its mechanism of action at a real-time and single-cell level. The drug concentration and treatment-dependent decrease of reduced cyt c was found as an early marker of cell apoptosis which is more sensitive than the conventional cell viability assay. This method allows for us to screen the drug cytotoxicity and approach the mechanism at the same time, thereby reducing the cost and time of drug screening.

Raman spectroscopy (RS) has been widely used in a variety of biomedical applications. Nevertheless, there have been multiple technological and regulatory hurdles to move RS from the research lab to the bedside. From the regulatory point of view, especially in Europe, the new EU regulation on medical devices (Medical Device Regulation, MDR2017/745) complicates the translation. From the technical point of view the lack of imaging capabilities for fiber-optic Raman probes, the lack of depth information and the low imaging speed create challenges. Here, we present some of our recent developments to address current challenges.

Breast cancer is one of the most common cancers among women worldwide. The process of diagnosis is completed with conventional histology (Hematoxylin and eosin staining followed by immunohistochemistry staining), which is known to be labor-intensive, time-consuming, and subject to inter- as well as intra-laboratory variations. We present here a multi-color Stimulated Raman Histology (SRH) technique to overcome these limitations. The multi-color SRH can map lipid, cellular protein, and collagen in a label-free manner to differentiate cancerous breast tissue from healthy tissue. Our technique can be a promising candidate for labor-efficient, fast, and highly sensitive breast cancer histology.

More patients diagnosed with stage I (local) melanoma die than any other metastatic stage, because there exists no biomarker to reliably diagnose metastatic melanoma, preventing many patients to receive appropriate treatment. We pursue an approach based on femtosecond pump-probe microscopy of melanin; a natural pigment found in most melanoma. The measured pump-probe signals of melanin are complex superpositions of multiple nonlinear processes, making interpretation challenging. We demonstrate how polarization control and data fitting are used to decompose melanin signals into their underlying nonlinear interactions. False colored images of a small set of melanoma tumors, based on specific nonlinear interactions, correlate with clinical concern. This approach of decomposing pump-probe signals is applicable to a multitude of different samples.

The capability to precisely control biomolecules in live cells would give a better understanding of site-specific biofunctions. We developed a real-time precision opto-control (RPOC) platform which uses chemical-specific optical signals to trigger another laser beam only at desired locations to control biomolecules with submicron precision, nanosecond response time, and high chemical specificity. Stimulated Raman scattering and fluorescence signals are used for chemical detection. Using RPOC, we demonstrate real-time and selective control of chemical states of photochromic molecules at different subcellular locations. When used with photoswitchable inhibitors, RPOC can control intercellular microtubule polymerization and lipid droplet dynamics site-specifically based on chemical preference.

We revisit wide field CARS imaging using speckle field illuminations. In the proposed scheme we break the CARS coherence using fast varying pump speckle illuminations while keeping static the Stokes speckle. Acquiring a large number of Stokes images enables dynamic speckle illumination (DSI) and random illumination microscopy (RIM). We show that the first one enables quasi-confocal axial sectioning (1/z) while the second one, through post-processing, can retrieve a superior image contrast, noise level and spatial resolution as an important step towards robust nonlinear super-resolution CARS microscopy.

Raman spectroscopy provides a comprehensive molecular information of a biological sample without any invasive and destructive treatment prior to a measurement. However, the slow measurement speed limits imaging applications of Raman spectroscopy. I present acceleration of Raman spectroscopy and imaging by multiplexed spatial sampling. Multiline illumination Raman microscopy, scanning a sample with multiple line-shaped laser foci, improved the imaging speed by 4 orders of magnitude in comparison to conventional confocal Raman microscopy. I also present a selective-sampling approach for accelerating Raman spectroscopy and imaging. I discuss the potential new applications of accelerated Raman spectroscopy and imaging in life sciences.

We demonstrate a novel approach to super-resolution in stimulated Raman scattering based on single-pixel detection scheme. We exploit speckle structured illumination, and enable fast super-resolution microscopy using Raman contrasts. The methodology is straightforward to implement and provides label-free super-resolution imaging of optically thick specimens, therefore paving the way for probing complex biological systems when exogenous labelling is challenging.

The detection sensitivity of SRS microscopy is fundamentally limited by shot noise. To address this barrier, we revisit SRS from the perspective of energy deposition. The SRS process pumps molecules to their vibrational excited states. The thereafter relaxation heats up the surrounding and induces a change in refractive index, as suggested by simulation and fluorescence thermometer measurement. By introducing a CW laser beam to probe the refractive index change, we demonstrate stimulated Raman photothermal (SRPT) imaging of particles, cells, and tissues with high signal to noise ratio at the fingerprint region. SRPT microscopy opens a new way to vibrational spectroscopic imaging with ultrahigh sensitivity.

Stimulated Raman scattering (SRS) microscopy is a powerful chemical imaging tool for visualizing biomolecule distribution in biological cells and tissues. Recent research has focused on SRS imaging in the C-H region due to the strong signals from lipids and proteins. However, these signals are regarded as non-specific. To improve the specificity of C-H imaging, we sought to use advanced machine learning to extract hidden information from C-H SRS imaging. This is possible because cells in tissue often have distinct sizes, shapes, and compositions. In this talk, I will present our recent efforts on machine learning/deep learning augmented cell imaging and classification with SRS. This approach potentially enables label-free mapping and tracking of different cells in various tissue.

We present several approaches to high-speed broadband stimulated Raman scattering (SRS) and broadband coherent anti-Stokes Raman scattering (CARS) spectroscopy and microscopy for label-free chemical identification of molecules through their intrinsic vibrational spectrum. (1) A 32-channels lock-in amplifier for parallel detection of broadband SRS spectra. (2) A broadband CARS microscope covering the entire fingerprint with down to 1-ms pixel dwell time, based on a low-repetition-rate amplified laser allowing white-light supercontinuum generation in bulk media. (3) Artificial-intelligence-based methods to enhance the signal-to-noise ratio and remove the non-resonant background signal from CARS spectra. (4) Video-rate wide-field CARS microscopy over the fingerprint region.

Broadband coherent anti-Stokes Raman scattering (BCARS) micro-/spectroscopy is a powerful method of chemical imaging that is self-referenced to a co-generated background signal; thus, enabling direct, unnormalized comparison between microscopy platforms and samples. The workflow required to extract the self-referenced Raman spectra, however, has typically required milliseconds per spectrum; thus, hindering real-time analysis, visualization, and user interactivity. In this work, we demonstrate a new workflow using linear machine learning methods that is intrinsically interpretable, informed by the physics of the problem, and yet enables real-time processing with improved quantitation. Additionally, we will demonstrate how this can translate into automated region-of-interest selection.

We demonstrated the potential of applying infrared spectroscopy for neuroscience by increasing both the spatial resolution and the specificity of chemical structure analysis in tissues and cells, thus overcoming the current limitations of conventional fluorescence microscopy and infrared microspectroscopy. Specifically, we used a novel instrumental setup that combines optical photothermal infrared spectroscopy (OPTIR) with an epifluorescent imaging module (EF) to allow IR spectroscopic analysis of fluorescently microglia cells surrounding amyloid plaques in comparison to microglial cells in amyloid-free tissue. We have demonstrated the capability of EF-OPTIR microscopy to locate specific cells and amyloid proteins and assess amyloid structures with sub-micron spatial resolution.

We developed an optical infrared photothermal (OPTIR) metabolic imaging platform that enables in situ lipid metabolism characterization at sub-cellular resolution. The cell-type specific lipid metabolic imaging was achieved through the fluorescence module integrated into the OPTIR system. We successfully imaged newly-synthesized lipids after azide-palmitic acid treatment in cells across different model systems including neuroglioma, human-induced pluripotent stem cells (iPSCs), iPSC-derived microglia cells, and iPSC-derived brain organoids. The metabolic heterogeneity in different cell types in brain organoids was observed. Collectivity, the proposed metabolic imaging platform opens a vast potential for in situ metabolism characterization at the single-cell level.

Infrared microspectroscopy has been shown to be a powerful tool for biomedical applications, e.g. to assess bacteria, cells and tissues. Quantum cascade laser-based optical photothermal infrared (O-PTIR) spectroscopy and imaging have been introduced which offers submicron spatial resolution, non-contact measurements, and measurement of samples in aqueous media. O-PTIR spectra and single wavenumber images of cells were recorded with subcellular resolution, and the spectral data were used to develop a six-cell classification model for blood cells and their changes related to infections. Tissue sections of a murine model for infection disease have also been studied be O-PTIR spectroscopy and imaging.

Mid-infrared spectroscopic imaging (MIRSI) combines the molecular specificity of vibrational spectroscopy with the spatial detail provided by microscopy. Combining machine learning and MIRSI has facilitated the identification of tissue subtypes and cancer grades in a label-free and quantitative manner. We will present results from tissue subtyping and segmentation in ovarian, cervical, and endometrial cancer tissue with a new, high-resolution photothermal MIRSI technology. We will also show imaging data and analysis results from clinical bone samples and characterize fibrosis.

Enzymes are vital in most physiological and biochemical processes and may function as critical biomarkers of disease. Notably, many biological events and signaling networks involve multiplex enzyme species. Thus, mapping multiple enzyme activities is significant for elucidating enzymatic functions in life and disease. Here, we report a novel technology to map the catalytic efficacy of phosphatase and caspase in live cancer cells and in C. elegans by mid-infrared photothermal (MIP) imaging of nitrile chameleons. This technology will certainly bring new insight into the roles of enzyme in biochemical activities and explore new knowledge of enzymatic activity in health and disease.

Infrared (IR) absorption microscopy is a sensitive label-free chemical imaging tool for biomolecules in cells and tissues. However, quantitative measurements in hydrated biological samples remain a challenge due to the strong water absorption in the fingerprint region, in particular near 1650 cm-1, which critically overlaps with the protein amide I peak. Here, we present a new approach to overcome the challenge by using a Solvent Absorption Compensation (SAC) unit, which dynamically adjusts the intensity of incident light to compensate for the strong water absorption. We demonstrate the capability to perform quantitative absorption imaging of proteins (amide I) in live fibroblast cells in a 25 µm thick layer of water. This work enables future label-free, quantitative chemical imaging of biomolecules in the living cell.

Imaging techniques that utilize mid-infrared (MIR) light obtain their contrast from the molecular resonances of the sample. When each pixel in the image contains spectral information, the resulting data cube [xyw] yields an information rich visualization of chemical content. However, detector technologies designed for the MIR struggle to produce high definition spectral images in real-time. This can be circumvented by detecting the MIR image on a camera designed for visible light. By using non-degenerate two-photon absorption, we demonstrate the acquisition of high-definition spectral data cubes in under a second.

The detection scheme of conventional mid-infrared (mid-IR) spectroscopy and imaging as well as the strong absorption of water restricts the application of mid-IR spectroscopy/imaging. We applied optoacoustic sensing for the non-invasive retrieval of mid-IR signals in biological tissues and developed Mid-IR Optoacoustic Microscopy (MiROM). This results in a label-free bond-selective microscopy that is able to monitor lipids, proteins, and carbohydrates down to a single-cell level with a lateral resolution of ~5 µm. We will discuss MiROM’s most recent developments for spatiotemporal metabolic monitoring in living cells and tissues as well as perspectives and recent advances for its application in vivo.

AF-PTIR microscopy is shown to enable chemically-specific imaging of an active pharmaceutical ingredient (API) and discrimination between two crystal forms of the API with parts per million (ppm) detection limit. AF-PTIR is based on detecting highly-localized temperature-induced variations in native autofluorescence intensity following the absorption of mid-IR radiation generated by an array of 32 independent narrowband quantum cascade lasers (QCLs). AF-PTIR was demonstrated to improve the detection limit in trace crystal form impurities analysis by two orders of magnitude as compared to commercial powder X-ray diffraction instrumentation, suggesting potential applications in pharmaceutical formulations analysis.

Measurement of the permeation and distribution of topical drugs remains challenging, in particular within the outmost layer of the skin known as the epidermis. As the epidermis serves as the skin’s major barrier, this makes it difficult, in both preclinical and clinical settings, to quantify the flux and flow of active pharmaceutical compounds into skin. We have developed coherent Raman imaging methods for the three-dimensional mapping and quantification of drug permeation within the epidermis over time, allowing for the visualization of drug permeation and localization alongside microscale measurements of pharmacokinetic parameters including Tmax, Cmax, and AUC. Coherent Raman imaging can additionally be used for the active measurement of skin response to drug intervention, with machine learning methods providing new and unique insight to pharmacodynamics. This information is critical for drug discovery, the development of new topical drugs, and generic topical products.

Fast-acquisition Raman methodologies such as stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy, which generate image contrast using the Raman active vibrational frequency of a given chemical, provide information on the biochemical composition of tissues. This has enabled detailed imaging of the interaction between tumor cells and the surrounding tumor microenvironment (e.g. tumor vasculature, stromal cells) without the need for additional labelling. In addition, we have developed novel, highly Raman active spectroscopically bioorthogonal labels, for the sensitive and specific intracellular visualisation of small-molecules and cells, allowing monitoring of drug uptake and tracking of individual cell populations.

High-speed Raman spectroscopy has enabled high-dimensional phenotyping of cells. Among these, time-domain Raman spectroscopy, such as Fourier-transform coherent anti-Stokes Raman scattering (FT-CARS), has unique capabilities such as high spectral acquisition rate, broadband spectral sensitivity in the fingerprint region, and nonresonant-background-free spectral acquisition. In this talk, we will present our recent achievements in FT-CARS spectroscopy for life science, including super-multiplex flow cytometry, high-speed dual-band spectroscopy, and broadband Raman-activated cell sorting.

Cancer cells in the hypoxic environment alter their metabolism to address the low oxygen stress. Such metabolic changes contribute to cancer resistance to chemotherapy and radiotherapy treatment. Understanding the hypoxia-induced metabolic changes allows for finding new targets to treat hypoxic cancer cells. We applied stimulated Raman scattering spectroscopic imaging to explore the metabolic changes of cancer cells exposed to hypoxia. We found spatial redistribution and content changes of lipid droplets (LDs) in various cancer types. We also studied the sources contributing to the formation of LDs and discovered the relationship between LD formation and endoplasmic reticulum stress.

We propose a new broadband stimulated Raman scattering microscopy scheme. A high-efficient spectral compression method is used to frequency double a solid-state femtosecond laser oscillator and pump a picosecond optical parametric oscillator. The broadband femtosecond beam, used as Stokes beam, is intensity modulated by an electro-optical modulator, and spectrally filtered by a multichannel acousto-optic tunable filter. We implement Hadamard transform acquisitions by applying spectral masks on the broadband Stokes beam to perform compressive sensing for sparse wavenumber analysis to classify environmental micro-fibers, for fast discrimination of natural and synthetic origin materials.

Stimulated Raman scattering (SRS) imaging of fresh tissue is an emerging approach to render label-free pathology and diagnosis based on the chemical contrasts of native biomolecules. However, the contrast of cell nuclei is often too weak to perform reliable segmentation and quantification. To advance the development of label-free digital pathology with SRS, we explored three deep learning-based techniques with multicolor and hyperspectral SRS imaging data, including U-Net, Mask R-CNN, and Autoencoder. Our results show that a combination of machine learning and SRS imaging is a promising pathway to transforming the methodology of SRS label-free pathology for real clinical use.

Raman spectroscopy is a label-free, non-invasive spectroscopic technique, which can be utilized for many biomedical and diagnostic investigations. To do so, chemometric modelling strategies are used, but they lead to a low generalizability of the models. To tackle this issue we investigated transfer learning (TL) approaches for deep learning (DL) based modelling of Raman spectra for classification of three bacterial spore species. In initial test we found that TL can facilitate the usage of DL for time-consuming measurement modalities, because it can help to deal with low dataset sizes.

Periodically patterned photobleaching followed by spatial Fourier transform analysis of the recovery is shown to enable mapping of molecular diffusivity within spatially heterogeneous media. Image segmentation prior to Fourier transform fluorescence recovery after photobleaching (FT-FRAP) enabled quantitatively evaluating diffusion of macromolecules in spatially and chemically complex media. Notably,multi-harmonic analysis by FT-FRAP was able to definitively discriminate and quantify the roles of internal diffusion and exchange to higher mobility interfacial layers in modeling the recovery kinetics within thin amorphous/amorphous phase separated domains, with interfacial diffusion playing a critical role in recovery.

High-resolution optical imaging techniques allowed the visualization of nanoscale biological structures and molecules. Current fluorescence-based super-resolution techniques are limited in throughput and quantification. We developed a high-resolution versatile imaging modality, called Vibrational Imaging of Swelled Tissues and Analysis (VISTA), based on a sample-expansion vibrational imaging strategy. We achieve label-free super-resolution imaging, and successfully used a U-net convolutional network to predict specific cellular features. We further applied VISTA to investigate protein aggregations in biological samples, including aggregates formed in Huntington’s disease and Alzheimer’s disease. This precise and multiplex high-resolution imaging modality opens new avenues for versatile biomedical studies.

Tissue engineering (TE) constructs are an attractive alternative therapy for patients with e.g., degenerated cartilage. The positive patient outcome relies on the quality of the TE constructs as they should mimic the native tissue in its molecular, chemical, and physical properties. Most quality assessment techniques of TE constructs are destructive to the sample. Therefore, there is an urgent need for a novel non-destructive method to control the in vitro cultivating process. Here, we present Raman Projection Tomography (RPT) which enables live label-free 3D molecular imaging. RPT was employed for the non-destructive quality control of cartilage TE constructs.

Surface-enhanced analogues of coherent Raman scattering (CRS) methods are promising candidates to probe molecular vibrations at much faster rates than what can be achieved with surface-enhanced Raman scattering (SERS). Thus far, experimental challenges have prevented surface-enhanced versions of CRS from growing into a practical tool. One of the major hurdles is the fragility of metallic antenna systems that support surface plasmons when illuminated with ultrafast pulses, causing photo-induced damage to the enhancement structures and lowering the repeatability of surface-enhanced nonlinear Raman measurements. In this contribution we discuss several implementations of surface-enhanced coherent anti-Stokes Raman scattering (SE-CARS) that avoid the use of metallic structures and use dielectric antenna systems instead.

Raman microscopy is a valuable approach to label-free chemical imaging. Recent advances have enabled the development of rapid methods for hyperspectral microscopy using impulsive stimulated Raman scattering (ISRS) that focus on low-frequency Raman modes of materials. The persistent challenge with Raman spectroscopic methods is low signal due to the relatively weak Raman scattering. ISRS microscopy allows for high quality hyperspectral imaging of low and fingerprint Raman vibrational frequencies but is normally subject to poor performance in the presence of optical scattering. We will present methods that solve this issue and allow for high-quality imaging when strong optical scattering is present.

Fluorescence-encoded time-domain coherent Raman spectroscopy (FLETCHERS) is a fluorescence-encoded vibrational spectroscopy technique that boasts both high sensitivity and specificity in the probing of molecular vibrations in the lower fingerprint region. However, to date, all presented data presented so far has been from flowing samples which does not sufficiently demonstrate the method’s applicability for monitoring biological and chemical processes. To amend this, we demonstrate here FLETCHERS imaging of low-concentration samples with an expanded spectral window achieved by utilizing spatial filtering and the confocal collection of fluorescence-encoded light.

We present our recent developments in utilizing a compact and portable light source for high-speed multicolor stimulated Raman scattering imaging (SRS) in biomedical and medical environments. The source combines a rapid and wide tunability for accessing Raman bands between 700 and 3300 cm-1 with high stability in terms of power (deviation < 0.3 %) and wavelength (deviation < 0.5 pm) over more than 100 h. We were able to shorten the Stokes pulse duration to below 3ps, resulting in a twofold increase in SRS signal intensity compared to our previous version (7ps).

We present a ready to use label free coherent Raman microscope (CARS/SRS) to perform instantaneous virtual histology and chemical imaging for scientific applications. The system is based on a picosecond laser source that can address two vibrational wavenumbers simultaneously across the entire vibrational spectrum (400cm-1 – 4000cm-1). The laser source is coupled to a ready to use beam scanning microscope enabling SRS, CARS and multiphoton microscopy. Targeting the CH2 and CH3 chemical bonds, we demonstrate instantaneous stimulated Raman histology in the brain and the GI. This new, ready to use, SRS/CARS and multiphoton microscope enables SRS detection with exquisite sensitivity (shot noise limited) and ratio-metric measurement by targeting simultaneously two vibrational frequencies that can be tuned anywhere across the vibrational spectrum.

This conference presentation was prepared for the Advanced Chemical Microscopy for Life Science and Translational Medicine 2023 conference at SPIE BiOS, 2023.