Stimulated Raman Scattering (SRS) is a powerful method for quantifying the uptake, flow, and flux of active pharmaceutical ingredients (APIs). While there are numerous APIs that have unique structures and vibrational spectra, the majority do not have strong or unique vibrational bands. We have developed a sparse spectral sampling SRS imaging system to address these needs that can rapidly and specifically tune into specific Raman shifts from the fingerprint to high wavenumber region in < 5 ms per step. These hyperspectral SRS images can be analyzed using computational analytical approaches to visualize and quantify the dynamics of multiple molecular species simultaneously.

Mapping the localization of multiple proteins in their native 3D context would be useful for biomedicine. We harness the narrow spectrum of Raman spectroscopy and introduce Raman Dye Imaging and Tissue Clearing (RADIANT), an optical method for imaging multiple targets in thick samples in one shot. We expanded the range of bioorthogonal Raman dyes and developed a tissue clearing strategy for them. RADIANT allowed us to image >10 targets in millimeter thick brain slices, extending the imaging depth 10-100 fold compared to prior methods. RADIANT will facilitate the exploration of the intricate 3D protein interactions in complex systems.

Saturated fatty acids (sFA) are more toxic to cells than unsaturated fatty acids (uFA). Previously, we developed a chemical imaging strategy to track the spatiotemporal dynamics of specific deuterium-labeled fatty acids in living Caenorhabditis elegans, called isotope-labeling coupled stimulated Raman scattering (iSRS) microscopy, and showed that sFAs are incorporated into lipid droplets (LDs) slower than uFAs. To study how fatty acids are integrated into LDs at different rates, we performed a high-throughput forward genetic screen using iSRS microscopy and identified mutants with enhanced sFA integration into LDs. Currently, we are sequencing these candidates to identify the molecular players in fatty acid metabolism and lipotoxicity.

The onset and progression of dermal inflammation are easy to diagnose, but challenging to quantify, stage, and measure. CARS and SRS imaging are modalities capable of providing insight into the dynamics of structural and drug/fluid concentration changes throughout a time course of tissue imaging. This work displays two main avenues of exploration using dermatitis-induced mouse models. First, we track inflammation development and resolution over a four-day time course, capturing CARS and SRS image data at multiple time points to use in a machine learning (ML) based approach trained to classify the extent of inflammation in the provided images. Second, we treat mice with anti-inflammatory agents to determine whether these agents truly help with inflammation resolution, using our ML-based approach trained on structural and concentration rich images as a proxy for the pharmacodynamic response. We additionally use ML interpretability methods to aid in the justification of our results.

Chronic skin wounds are a tremendous clinical problem, and our understanding of them has been limited by a lack of established tools and biomarkers sensitive to different wound features. Using label-free two-photon excited fluorescence, second harmonic generation, and coherent anti-Stokes Raman scattering microscopy, we demonstrate the ability to monitor skin wounds in vivo over 10 days. Specifically, differences in skin wound epithelial metabolism were identified among aged, diabetic, and young mice. Recent work involving automated wound segmentation through convolutional neural networks indicate an ability to provide rapid quantitative readouts of different aspects of wound structure and function.

In recent studies, stimulated Raman scattering (SRS) and transient absorption microscopy (TAM) have been employed for label-free mapping of biomolecules (e.g., proteins and lipids) in brain tissues and hemoglobin in red blood cells, respectively. In this study, we combined SRS and TAM to simultaneously image cell densities and capillary structure in vivo at the highest reported imaging depth, ~300 µm, for either technique. This multimodal approach resulted in label-free identification of endothelial cells and pericytes in vivo with 90% accuracy using a machine learning classifier. Simultaneous two-photon excited fluorescence microscopy serving as the ground truth.

We developed a Raman scattering microscope using a 0th-order Bessel beam to illuminate the inside of a biological sample. We constructed a Raman spectroscopic system in which the Bessel beam illuminates the sample from the side, and the illuminated area in the sample is imaged on the slit of a spectrophotometer. By using the developed microscope, polymer beads and biological samples were observed to confirm the imaging property and the improvement of the image contrast compared to the conventional line-illumination Raman microscopy.

Spectroscopic stimulated Raman scattering (SRS) imaging has become a useful tool finding a broad range of applications. Due to the limited spectral coverage of current SRS systems, most SRS applications utilized a narrow Raman band (<300 cm-1). Multi-window SRS imaging covering C-H, C-D and fingerprint regions offers richer chemical information. In this work, we present a multi-window SRS imaging system with a rapid widely tunable fiber laser. We implemented auto-balanced detection to enhance the signal-to-noise ratio of stimulated Raman loss by 23 times. We demonstrated SRS metabolic imaging of fungi, cancer cells, and Caenorhabditis elegans across the C-H, silent and fingerprint Raman windows. Our results showcase the potential of the multi-window SRS system for a broad range of applications.

Controlling behaviors of biomolecules in living cells is a challenging task. Conventional methods such as treating cells with compounds or using genetic methods do not have spatial precision. Optical tweezers can only manual manipulation of a few targets. Laser ablation methods require pre-scanning and manual target selection. Optogenetics methods also require pre-imaging and only have precision at the single-cell level. We develop a real-time precision opto-control (RPOC) platform that can selectively and precisely control biomolecules using lasers. RPOC can automatically probe and control biomolecules in dynamic living samples with high spatial accuracy, a fast response time, and high chemical selectivity.

We developed a mid-infrared photothermal imaging (MIPI) - fluorescence in-situ hybridization (FISH) microscope that enables simultaneous identity probing and metabolic activity imaging for bacteria in a complex environment. We added the 13C-glucose in the bacteria culture medium and monitored the newly synthesized protein composition by MIPI at amide I band. Bacterial taxa were targeted with fluorescently-labelled FISH probes and imaged with the widefield epi-fluorescence imaging that built on to the MIP microscope. The sub-micrometer spatial resolution of MIPI enables the characterization at single bacterium level. The proposed platform provides the link between genotype and phenotype and would be a powerful tool to deepen our understanding of microbiome.

Volumetric chemical imaging is highly desired for investigating biochemical processes at the sub-cellular level. Here, we report bond-selective intensity diffraction tomography (BS-IDT) based on 3D quantitative phase detection of the mid-infrared photothermal effect. BS-IDT demonstrates volumetric chemical imaging with incoherent diffraction-limited resolution and a high speed up to ~6 Hz per volume. The mid-IR spectrum extracted from BS-IDT shows high fidelity compared with ground truth measured by an FTIR spectrometer. The 3D chemical imaging results from cancer cells and Caenorhabditis elegans validate BS-IDT’s superior performance.

Fluorescence-detected photothermal mid-infrared (F-PTIR) microscopy is demonstrated experimentally and applied to characterize the chemical composition within micrometer-size phase-separated domains of ritonavir/copovidone amorphous solid dispersions formed upon water sorption. In F-PTIR, temperature-sensitive changes in fluorescence quantum efficiency report on highly localized absorption of mid-infrared radiation. Two-photon excited ultraviolet autofluorescence supported label-free F-PTIR microscopy of tryptophan microcrystals and lyophilized lysozyme particles. F-PTIR provides two degrees of chemical specificity, informing on infrared absorption selectively in the local environments immediately adjacent to fluorescent regions of interest.

The absorption of mid-infrared light has the specificity to enable biochemical identification and characterization of tissue. Mid-infrared spectroscopic imaging (MIRSI) combines the molecular specificity of vibrational spectroscopy with the spatial detail provided by microscopy. The combination of machine learning and MIRSI has facilitated the identification of tissue sub-type and cancer grade in a label-free and quantitative manner. We will present results comparing various MIRSI technologies and discuss the advantages of each technology. We also present imaging data and results of high-resolution MIRSI of ovarian, cervical, and bone samples.

Vibrational imaging based on mid-infrared photothermal (MIP) microscopy with the sub-micrometer spatial resolution is an indispensable analytical tool for a wide range of fields, such as polymer and medical science. In this talk, we will report multimodal imaging of polymer blends and biomolecules combining a MIP imaging with other optical microscopic techniques such as Raman or fluorescence imaging. Since Raman scattering exhibits complementary information of molecular vibrations and fluorescence imaging helps to understand biological dynamics by visualizing the distribution of biomarkers, our demonstration of multimodal imaging offers accurate and detailed characterization of molecular structures and functions of various samples.

Periodically patterned photobleaching followed by spatial Fourier transform analysis of the recovery is shown to enable mapping of molecular diffusivity within spatially heterogeneous media. Both pixel by pixel analysis method and segmented analysis method of Fourier transform fluorescence recovery after photobleaching (FT-FRAP) measurements and implemented for quantitatively evaluating diffusion of macromolecules in spatially and chemically complex media. With the development of FT-FRAP, the time-dependent recovery in fluorescence due to diffusion is measured in the spatial Fourier domain, with substantial improvements in the signal-to-noise ratio, the mathematical simplicity, and the compatibility with multi-photon excitation.

We present Raman image-activated cell sorting (RIACS), a technology that performs real-time coherent Raman image-based sorting of single live cells with a throughput of up to ~100 cells per second. Specifically, the fast image acquisition of the RIACS is enabled by multicolor stimulated Raman scattering (SRS) imaging based on a pulse-pair-resolved wavelength-switchable laser. To show the broad utility of the RIACS, we show its applicability to diverse cell types and sizes. The technology is highly versatile and holds promise for numerous applications that are previously difficult or undesirable with fluorescence-based technologies.

We present label-free transient absorption microscopy (TAM) of mitochondrial redox in live muscle fibers. Image stacks consisting of TAM images at different pump-probe delays, processed with principal component analysis (PCA) reveal picosecond timescale differences between reduced and oxidized mitochondria, in addition to heterogeneity among mitochondria within individual muscle fibers. We will discuss these findings in light of the photoinduced transient conformational and vibronic states of mitochondrial respiratory chain hemeproteins and relaxation pathways.

We present a novel version of mid-infrared photothermal microscopy in which thermosensitive fluorescent probes are harnessed to sense the mid-infrared photothermal effect. The fluorescence intensity can be modulated at the level of 1% per Kelvin, which is 100 times larger than the modulation of scattering intensity. In addition, fluorescence emission is free of interference, thus much improving the image quality. Moreover, fluorophores can target specific organelles or biomolecules, thus augmenting the specificity of photothermal imaging. Spectral fidelity is confirmed by fingerprinting a single bacterium. A wide-field fluorescence-detected mid-infrared photothermal microscope developed allows video-rate bond-selective imaging of biological specimens.

Coherent Raman Scattering microscopy (CRS) is a powerful approach for label-free, chemically specific imaging, based on the characteristic vibrational contrast of the sample molecules. Here, we introduce the all-new Leica STELLARIS 8 CRS microscope, a multi-modal optical discovery instrument that offers a unique combination of biochemical, biophysical and molecular contrasts. This is achieved by a seamless integration of Stimulated Raman Scattering (SRS), CARS, two-photon fluorescence and second-harmonic generation, with the STELLARIS confocal microscope. We will present applications in neurodegenerative disease, cancer, 3D biology, stem cell and developmental biology, and pharmacology.

Optical Photothermal Infrared (O-PTIR) spectroscopy has established itself as a cutting edge vibrational microspectroscopy tool, offering significant advantages over traditional FTIR/QCL and Raman spectroscopic tools, providing submicron simultaneous IR+Raman microscopy, in non-contact mode with high sensitivity. A new modality, “counter-propagating” has been engineered to provide for enhanced IR (and Raman) spatial resolution and sensitivity, with the use of high NA refractive objectives. This improves spatial resolution to ~300nm for both IR and Raman, whilst also improving sensitivity, image quality and facilitating immersion objective studies in fluid. Further, we coupled a widefield epifluorescence to facilitate a novel concept – fluorescence guided (or fluorescence co-located) O-PTIR microspectroscopy. Several life sciences examples from bacteria, cells and tissues will be provided to demonstrate these capabilities and how they can enable new experiments and research findings.

We present recent developments on our portable light source for multicolor coherent Raman imaging. The source combines a rapid and wide tunability for accessing Raman bands between 700 and 3200 cm-1 with high stability for long term measurements utilizing software PID-stabilization by means of an integrated optical miniature spectrometer. We present a long-term power stability with a standard deviation of less than 0.3% and a wavelength stability of better than 5pm over the course of 100h. This development constitutes an important step for advancing CRI microscopy in terms of portability and stability for applications in medical diagnostics or environmental sensing.

We have developed a new multimodal microscope enabling “Fluorescence-Guided” Optical Photothermal Infrared (O-PTIR) spectroscopy. O-PTIR has proven itself a breakthrough in IR spectroscopy, providing IR spectroscopic analysis with spatial resolution ~20x better than traditional IR in a non-contact optical configuration. We have now coupled O-PTIR with fluorescence microscopy, a long established, cornerstone technique in life science research. The new fluorescence guided O-PTIR instrument allows: (1) a sample to be analyzed via fluorescence microscopy to map sample regions that are labeled one or more fluorophores; and (2) use fluorescence images to direct infrared spectroscopic analysis in the vicinity of the fluorescently labeled regions. We have used fluorescence guided O-PTIR to perform spectroscopic analysis of the protein secondary structure of fluorescently labeled amyloid plaques in mouse brain tissue, with the goal of providing insights in neurodegenerative disease research.

Raman microscopy is a powerful mode of label free nonlinear optical microscopy that is hampered by weak cross-sections, leading to slow imaging. We will discuss two advances in coherent Raman microscopy: 1) high speed coherent anti-Stokes Raman scattering microscopy imaging based on spatial frequency modulation imagining (SPIFI) where a structured line focus is used to image is used to image specimens with a single pixel detector. 2) Doppler Raman microscopy that exploits the extremely low timing jitter of modelocked lasers for ultrasensitive Raman spectroscopy and microscopy.

Stimulated Raman Scattering (SRS) is a label-free technique utilized to analyze the chemical nature of various substances with high-speed. However, SRS can be affected by parasitic effects that can distort the signal. In order to eliminate these spurious effects, we propose a frequency-modulation SRS scheme realized for the first time with an acousto-optic tunable filter, which can perform measurements from the fingerprint to the CH-stretch region of the spectrum without any modification of the optical setup. We demonstrate the efficient cancellation of the parasitic effects with our developed configuration on several samples: human hairs, polymer beads and human cells.

Endothelial dysfunction contributes to the pathophysiology of multiple civilization diseases. Biochemical changes of the dysfunctional endothelium are linked to cellular organelles, including mitochondria, nucleus or lysosomes, so organelle-specific insight is needed for better understanding of endothelial pathobiology. Raman imaging, combining chemical specificity with microscopic resolution, is able to detect biochemical changes at subcellular level. However, the detection of spectroscopic markers associated with specific cell organelles, while desirable, cannot easily be achieved by Raman imaging without labeling. Hence, our work focused on development of Raman reporters that opens a new possibilities in subcellular investigation using labeled Raman microscopy with improved specificity.

Hyperspectral stimulated Raman scattering (hsSRS) microscopy provides rich chemical and spatial information not regularly available to traditional microscopy methods. However, analysis of hsSRS images is often confounded by convolved and overlapping spectral features requiring use of machine learning methods to extract information. Here, we demonstrate the use of our recently published deep learning architecture (the U-within-U-Net) designed for hyperspectral images on hsSRS images. We demonstrate segmentation, classification, and prediction of orthogonal imaging modalities. We also show the architecture is applicable to other hyperspectral imaging modalities with implications for remote sensing and mass spectrometry imaging.

We present a reversible reaction-based Raman probe for live-cell imaging of Glutathione (GSH) by the hyperspectral stimulated Raman scattering microscopy. By introducing a cyano group as the Raman reporter and an alkyne group as the internal standard, we have designed, synthesized, and characterized a series of Raman-based probes for ratiometric imaging and quantification of GSH levels in living cells and organisms.

Chemical neurotransmission is central to brain activity, where aberrations thereof lead to psychiatric or neurodegenerative disorders. The time and length scales over which neurochemical signaling occurs can differ between neurotransmitters, and is not always correlated with neuron electrical activity. Herein, we demonstrate a new, non-perturbative approach based on vibrational microscopy to image neurotransmitter isotopologues for dopamine and GABA, and demonstrate that these neurotransmitter isotopologues enable direct imaging of neurotransmitters in neuronal cells. This method is generalizable to other neurotransmitters, neuromodulators, and neuropeptides, providing a generic toolkit for imaging neurochemistry.

The integrity of mitochondrial function is fundamental to cell life, therefore, the detection and understanding of changes taking place in the organelle may allow faster diagnosis. It turns out that Raman spectroscopy is able to detect changes related to mitochondrial dysfunction much faster by tracking changes in redox state of cytochrome. Our research was focused on selecting the appropriate conditions for the use of MitoBADY (2220 cm-1), both in living and fixed endothelial cells. Interestingly, in our research we were able to observe, Raman signature of life band (1604 cm-1) which also characterizes the functioning of mitochondria. The relationship between the 1604 cm-1 and 2220 cm-1 bands also suggests that the reporter can also be an indicator of mitochondrial activity. Acknowledgement: This work was supported by the National Science Centre, Poland, OPUS grant (UMO-2018/29/B/ST4/00335 to MB).

Photothermal microscopy has enabled highly sensitive label-free imaging of absorbers, from metallic nanoparticles to chemical bonds. Photothermal signals are conventionally detected via modulation of excitation beam and demodulation of probe beam using lock-in amplifier. While convenient, the wealth of thermal dynamics is not revealed. Here, we present a lock-in free, mid-infrared photothermal dynamic imaging (PDI) system by MHz digitization and match filtering at harmonics of modulation frequency. Thermal-dynamic information is acquired at nanosecond resolution within single pulse excitation. Our method not only increases the imaging speed by two orders of magnitude but also obtains four-fold enhancement of signal-to-noise ratio over lock-in counterpart, enabling high-throughput metabolism analysis at single-cell level. Moreover, by harnessing the thermal decay difference between water and biomolecules, water background is effectively separated in mid-infrared PDI of living cells. This ability to nondestructively probe chemically specific photothermal dynamics offers a valuable tool to characterize biological and material specimens.

Understanding metabolism is of great significance to decipher various physiological and pathogenic processes. While great progress has been made to profile gene expression, how to capture organ-, tissue-, and cell-type-specific metabolic profile (i.e. metabolic tissue atlas) in complex mammalian systems is lagging behind, largely owing to the lack of metabolic imaging tools with high resolution and high throughput. Here, we applied mid-infrared imaging coupled with heavy water (D2O) metabolic labeling to a scope of mouse organs and tissues. Our premise is that, as D2O participates in the biosynthesis of various macromolecules, the resulting broad C-D vibrational spectrum should interrogate a wide range of metabolic pathways. Applying multivariate analysis to the C-D spectrum, we successfully identified both inter-organ and intra-tissue metabolic signatures of mice. A large-scale metabolic atlas map between different organs from the same mice was thus generated. Moreover, leveraging the power of unsupervised clustering methods, spatially-resolved metabolic signatures of brain tissues were discovered, revealing tissue and cell-type specific metabolic profile in situ. As a demonstration of this technique, we characterized metabolic changes during brain development and captured intratumoral metabolic heterogeneity of glioblastoma. Altogether, the integrated platform paves a way to map the metabolic tissue atlas for complex mammalian systems.

Engineered microbes can produce valuable chemicals but often require extensive optimization to reach sufficient production yields. Current quantitation methods such as gas chromatography-mass spectrometry are based on ensemble measurements that ignore the genetic or phenotypic variation among cells. Here, we use hyperspectral stimulated Raman scattering (hsSRS) to directly image free fatty acid synthesis in metabolically engineered Escherichia coli and uncover substantial heterogeneity among and within colonies. Using optimized laser exposures, we perform longitudinal SRS imaging to link the dynamics of biofuel synthesis with cell growth. Further, we develop an analytical protocol to enable mass spectrometry-like chemical specificity estimates of fatty acids.

The gut microbiome has a considerable impact on human health. Furthermore, it is very sensitive to the in vivo environment changes. Entacapone (ENT) is one important human-targeted drug for the treatment of Parkinson’s disease shown to largely impact the growth of individual organisms in the gut microbiome. To resolve ENT incorporation and impact on gut microbiome activity in situ, we employed stimulated Raman scattering - fluorescence in situ hybridization (SRS-FISH) along with ultrafast delay line tuning. The hyperspectral chemical images acquired with this method could better preserve and separate the chemical information, thus enabled high-throughput identity-specific single cell metabolism analysis and mapping of drug incorporation by individual microbiome cells.

The assessment of man-made or natural origin of microscopic debris in the environment requires spectroscopical analysis. However, it is difficult to use commercial FT-IR and spontaneous Raman microspectrometers to analyze micro-fibers due to their small size and strong autofluorescence in some specimens. Using a custom stimulated Raman scattering (SRS) microscope, we assessed the origin of microfibers from different environments by comparing their SRS spectra to a home-built library. Our analysis shows that the majority of the analysed micro-fibers are of natural origin. We show that SRS microscopy overcomes the limitations of FT-IR and spontaneous Raman microscopy for classification of micro-fibers.

Two breast cancer subtypes with contrasting aggressiveness and prognoses - triple negative and normal-like - had their lipid droplets interrogated under methionine and insulin manipulation. Raman scattering results suggest that excess methionine increases droplet volume and de novo lipogenesis but decreases droplet number and alters distribution in both subtypes. These effects are significantly augmented in the triple-negative subtype with the presence of insulin. Flavin autofluorescence increases with insulin but decreases with excess methionine, in which an abundance of unsaturated lipids was also observed. This suggests interplay between methionine and insulin related lipid homeostasis and demonstrates the utility of optical techniques.

Mapping the distribution of chemical molecules throughout a brain is helpful for neuroscience research. We have applied an ultra-broadband multiplex CARS spectroscopic imaging system to construct a whole-brain label-free molecular map in macro and micro scales. We could precisely visualize lipids distributed to white matter, rich in neuronal fibers. Our microscale measurements figured out that cells within the hippocampus and cerebral cortex could be divided into lipid-rich and water-rich cells. Moreover, we applied multivariate curve decomposition analysis for our spectrum and recapitulated the results. Our imaging and analysis techniques will lead to the molecular brain atlas with single-cell resolution.