Stimulated Raman scattering (SRS) is a fundamental optical process, discovered more than 60 years ago. While the early SRS spectroscopy studies have provided valuable insights to materials systems, the advent of SRS microscopy in 2008 has launched a rapidly growing field in chemical imaging. However, fundamental understanding of the molecular response under SRS spectroscopy and microscopy are 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.

Stimulated Raman scattering (SRS) microscopy using picosecond near-IR pulses has provided a great penetration depth with reduced fluorescence interference when imaging biological samples for bioenergy applications. These tools have provided insight into 1) tracking the degradation of chemical composites in biomass feedstocks to investigate the recalcitrant factors during the deconstruction processes, 2) monitoring the production of chemicals in photosynthetic plants and wood-digesting microorganisms, and 3) probing plant-bacteria interactions. However, the above processes are usually slow and require continuous imaging for an extended period. This is challenging for classic SRS because the laser power needed to achieve enough sensitivity causes photodamage in the samples during such long experiments. Quantum-squeezed light with reduced noise in the intensity quadrature can improve the sensitivity of classic SRS microscopy beyond the shot noise limit. The successful squeezing of one of the picosecond pulses in the above SRS will improve sensitivity and reduce photodamage, greatly expanding the range of studies available to SRS microscopy.

We developed metasurface-enhanced mid-infrared microscopy for living cells, where cells are cultured on a metasurface-bottomed microwell and are probed from the bottom using a confocal laser scanning microscope with a quantum cascade laser (QCL) as the source. The mid-infrared light interacts with the cells through the near-field of the plasmonic nanoantennas in the metasurface, and the reflected light is detected in the far field. Using this setup, we imaged fixed cells at different vibrational bands for the chemical imaging of different biomolecules, including proteins, lipids, and nucleic acids. Additionally, we have monitored the real-time adhesion and spreading of living cells on the metasurface with protein contrast.

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 from receiving 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, especially for clinical applications. We will provide updates on our latest progress in experimental techniques such as polarization pump-probe microscopy that allow for cleaner decomposition of measured signals into fundamental physical interactions. We will also discuss supervised learning algorithms for classification and their application to metastatic melanoma diagnosis, and compare them to conventional data analysis methods such as fitting.

Phototoxicity is a universal problem in optical imaging. Effective and quantitative evaluation of phototoxicity allows for the optimization of imaging conditions and the minimization of functional perturbation to live biological samples. We found that microtubule polymerization dynamics is a sensitive and reliable readout that can quantify phototoxicity in various imaging conditions. We quantified the photosensitizing effect of common organelle stains under the illumination of different laser wavelengths and compared the phototoxicity of different conditions. By controlling oxygen levels during light treatment, we found that the type I photosensitizing effect is involved in the hypoxia condition. We also evaluated the photoperturbation of IR laser pulses at different average and peak power levels in nonlinear optical imaging. Applying a recently developed real-time precision opto-control system, phototoxicity and reactive oxygen species generated at specific organelles can be studied.

Super-resolution imaging has facilitated groundbreaking discoveries by surpassing classical limitations. However, the commonly employed fluorescence-based approach exhibits inherent drawbacks, such as low labeling efficiency and potential toxicity. To address these challenges, we propose photothermal relaxation localization (PEARL) microscopy, a broadly applicable label-free chemical imaging technique that exploits the unique temporal characteristics of sub-diffraction limit objects. These features can be extracted through higher harmonic demodulation, effectively transcending the diffraction limit. Unlike existing methods, PEARL demonstrates a highly linear power dependence, enabling enhanced signal levels without the need for high peak power. Furthermore, PEARL's applicability extends to virtually any absorptive material. Experimental validations encompassing a range of materials, from gold nanoparticles to subcellular droplets, corroborate PEARL's ability to achieve superior resolution. In summary, PEARL introduces a versatile and potent optical super-resolution imaging technique with the potential to catalyze advancements across diverse scientific domains.

Time-resolved mid-infrared photothermal imaging with boxcar gating is discussed to study heat diffusion dynamics in various complex environments, including water interfaces and nanoparticle clusters. Thermal transients and heat diffusion interface dynamics can be studied with hyper-temporal imaging stacks.

Membranes are essential components of living cells. Chemical composition and biophysical organization of membrane profoundly impact many cellular processes. Characterizing membrane property in a cellular context is thus critical to our understanding of lipid biology. The dynamic and heterogeneous nature of cellular membranes presents a challenge for studying their biophysical properties and organization in vivo. Stimulated Raman scattering (SRS) microscopy offers a powerful solution. In this presentation, I will highlight how Raman imaging provides unique insights into membrane phase behavior and organization. In particular, SRS imaging reveals aberrant solid phase in intracellular membrane, shedding light on the biology of lipotoxicity.

Stimulated Raman scattering (SRS) microscopy offers label-free, high-speed measurements of small molecules in living cells and tissues, holding promise for potential biomedical applications such as drug screening and therapeutic drug monitoring. Nevertheless, with conventional SRS microscopy, fluctuations in the background Raman signal from cells and tissues limit quantitative measurements at drug concentrations below a few mM. Here we present novel SRS microscopy using temporal filtering combined with a phase-locking technique to break the detection limit due to the background Raman signals. We demonstrate that the new phase-locked system enables drug measurements at sub millimolar concentrations with linear calibration curves.

The low-frequency Raman region below 400 wavenumbers has rich chemical information to identify elements and compounds with low vibrational energies. Such low-frequency vibrational modes are usually probed by spontaneous Raman scattering which has low acquisition speed or femtosecond impulsive Raman scattering that requires ultrashort laser pulse and interferometry. High-speed hyperspectral stimulated Raman scattering (hSRS) microscopy has been developed based on spectral focusing but cannot detect low-frequency vibrational transitions. We extended the frequency range of picosecond hSRS microscopy to measure vibrational transitions below 400 cm-1. Using spectral focusing, we can perform hSRS imaging for a ~200 cm-1 spectral window tunable from 200-4000 cm-1.

We present a novel widefield super-resolution imaging technique based on Fluorescence Enhanced Photothermal IR (FE-PTIR) spectroscopy. The key enabling innovation is the realization that fluorescence emission, is on average, up to 100x more sensitive to temperature changes to than current O-PTIR contrast mechanisms of expansion and refractive index changes up on sample heating my wavelength specific absorption of the pump IR beam. Improved sensitivity allows the defocusing of the IR beam to cover an area of up to 70x70um, with measurement via 2D high sensitivity fluorescence camera, providing for true, rapid widefield chemical imaging with <500nm spatial resolution . FE-PTIR thus allows the IR spectroscopic analysis of specifically labeled regions of biological cells and tissue, for example to study conformational stages of a specifically labeled class of target proteins. FE-PTIR can enable the study protein misfolding associated with neurodegenerative diseases. Various examples from these applications will be provided.

Aging related biological mechanisms are often difficult to probe in situ without exogenous fluorophores. Here, we leverage label-free stimulated Raman scattering (SRS) microscopy to provide new insights into aging in C. elegans. We demonstrate multispectral SRS imaging of whole worms in vivo with quantitative chemical insights across different ages. We show that both lipid and protein synthesis and compartmentalization are associated with aging in worms. Moreover, we observe notable SRS image differences when worms are subjected to calorie restriction, suggesting a promising avenue towards understanding calorie restriction’s enhancing effects on longevity when coupled to proteomic and metabolomic analysis.

We report a z-scan-free stimulated Raman scattering tomography enabled by generation of ultraslow Bessel beams (B2-SRST) for rapid and deeper label-free volumetric chemical imaging. We demonstrate the utility of B2-SRST in a variety of samples (e.g., polymer beads phantoms and biotissues) for rapid label-free deeper tissue 3D chemical imaging.

Timely and accurate identification and antimicrobial susceptibility testing (AST) is essential to slow down the emergence of antimicrobial resistance and consequently reduce deaths due to drug-resistant infections. Here, we present multi-color fluorescence in situ hybridization (FISH) and stimulated Raman scattering (SRS) metabolic imaging to enable rapid identification and AST (iAST) of clinical samples. We have successfully developed FISH probes that utilizes oligonucleotide probes targeting the ribosomal RNA (rRNA) of bacteria strains associated with urinary tract infections (UTIs), which enables rapid and direct single-cell identification of microbes. By fast tuning between C-D and C-H vibration, we are able to use CD/(CD+CH) SRS signal ratio to quantitate the intracellular deuterium oxide (D2O) metabolism at single cell level. High-throughput, high-speed AST through robotic handling of liquid specimens and a multi-well chamber design has been demonstrated. We test the efficacy of our approach on common UTI associated bacterial isolates and clinical specimens, and demonstrate a 2 h diagnostic time, with an identification and categorical susceptible/resistant accuracy over 95%.

Drug resistance is influenced by molecular mechanisms that affect intracellular drug exposure and a drug’s ability to reach its protein target. Visualizing subcellular behavior of small-molecule drugs requires single-cell chemical analysis with high spatial and temporal resolution. In this work, we utilized label-free hyperspectral stimulated Raman scattering (hsSRS) microscopy to directly visualize and quantify tyrosine kinase inhibitor (TKI) drugs inside living cells. We report our live cell imaging acquisition and processing pipeline and demonstrate the capability of this hsSRS imaging method to characterize the roles of organic cation transporters (OCTs) on drug uptake. We observe significant transporter-dependent changes in intracellular drug concentration of multiple TKIs in response to the inhibition of OCTs.

Hyperspectral Stimulated Raman Imaging (SRS) has shown great promise as a label-free chemical imaging technique in biomedical and medical research. We present recent developments in SRS integrating a compact and portable all-fiber laser with balanced detection into an imaging system, aiming to enhance ease-of-use, specificity, and reliability in acquiring high-speed, multicolor chemical images. The system's adaptability is highlighted by its seamless integration with a Nikon Eclipse Ti widefield microscope, providing a compact and robust extension for varied imaging setups. The system incorporates a balanced detector to enable shot-noise-limited measurements, accommodating over 100mW of Stokes power on the detector.

Understanding metabolite dynamics in living systems is crucial for biomedical research. Fluorescence-based imaging has contributed tremendously to revealing mechanisms of organelle trafficking and interactions, however, not all biomolecules or organelles are labeled efficiently simultaneously without substantial perturbation from the labeling molecules. Vibrational spectroscopic imaging techniques are increasingly recognized as unique tools for investigating biomolecules in their natural microenvironment. Here, we developed a fluorescence-guided multimodal imaging approach to study organelle distribution and compositional changes in single cells. These advancements enable quantitative mapping of chemical dynamics, providing insights into subcellular biomolecule dynamics and mechanisms regulating cell physiology.

The indispensability of label-free microscopy in rendering detailed cellular observations of biological processes is well-established. This talk introduces an innovative progression in this field towards a super-resolution, label-free microscope that employs an ambient-air, field detector. In this technique, by employing bright, ultra-broadband, femtosecond laser pulses at near petahertz frequencies, molecular vibrations are driven coherently and efficiently. The same laser provides optical pulses with ultrashort duration to directly access and detects the field oscillations of the emitted molecular response in space and time. This novel metrology allows for simultaneous and broadband detection of the entire molecular fingerprint and beyond, with high detection sensitivity, dynamic range, and spatial resolution.

Label-free and nondestructive mid-infrared (MIR) vibrational hyperspectral imaging has gained significance as a valuable analytical tool for analyzing biomedical tissue samples. However, the complex chemical composition and heterogeneous nature of tissue specimens along with the weak interaction of infrared light with biomolecules, pose limitations on the analytical performance of traditional MIR chemical imaging. In this talk, I will introduce an advanced MIR spectrochemical tissue imaging technique that utilizes plasmonic metasurfaces to enable the capture of quantitative molecular maps of large-area murine brain tissue sections. The proposed surface-enhanced chemical imaging method using plasmonic metasurfaces has great potential for translational biomedical research and diagnostic clinical histopathology.

The mid-infrared photothermal microscope enables super-resolution imaging of living systems in water. However, the slow tuning of the laser limits spectroscopic imaging output to one color per second. To solve this, we developed a microsecond-scale spectroscopic detection scheme with a fast-tuning quantum cascade laser array. Each laser in the array emits at a slightly different wavelength, operating independently. A sequential emission from 32 lasers is completed within 4 microseconds. The absorption spectrum is acquired by capturing the temperature rise curve and calculating the first derivative. This improved spectroscopic imaging covers 150 cm-1 wavenumbers at 5 cm-1 resolution in one second, enabling real-time multiplexed molecular analysis.

We utilize spectral focusing hyperspectral stimulated Raman scattering (SRS) imaging for longitudinally assessment of the interactions between live zebrafish and microplastics (MP) throughout the development process. Our study delves into the pathways of MP uptake and the size-dependent accumulation of MP within different zebrafish organs during various developmental phases. The altered lipid metabolic dynamics uncovered by Raman-tagged hyperspectral SRS imaging further reveal the hepatic inflammation and disruptions in energy metabolism within MP-exposed zebrafish, providing fresh insights on the potential impacts and fate of MP on diverse living organisms.

Rapid diagnosis of biopsies, including core-needle biopsy and gastroscopic biopsy, is crucial for clinical decision makings. We applied stimulated Raman scattering (SRS) microscopy on fresh biopsy specimens without fixation, sectioning or staining. We further combined SRS with various deep neural networks for fast histological imaging and automated diagnosis. These include the integration of U-Net for femtosecond-SRS histology, as well as the use of convolutional neural network for histological classifications and gradings. Our results indicated that SRS histology integrated with deep learning algorithm provides potentials for delivering rapid diagnosis that could aid the surgical management of cancers.

Label-free chemical imaging by stimulated Raman scattering (SRS) has seen rapid developments and widespread biomedical applications. Nevertheless, the cross-section of the Raman process sets up a physical limit that impedes further evolution of SRS. Computational imaging is an appealing approach to break the physical barrier through the synergistic integration of advanced instrumentation and data science. In this talk, I will introduce a series of computational SRS work to push the physical limits in the aspects of imaging speed, signal fidelity, chemical specificity, and spatial resolution.

FT-FRAP with structured illumination is demonstrated for recovery of 2D molecular diffusion maps within heterogeneous systems. Distribution of the bleach power in comb illumination patterns maintained high integrated power, but avoids “hot spots” from local heating typical in point-bleach multi-photon excitation. Spatial Fourier transform approaches support recovery of diffusion coefficient imaging, in which 2D maps of spatially varying diffusion coefficients are recovered within a given field of view. Proof-of-concept simulations with known ground-truth behaviors enable algorithm assessment. Experiments are described to characterize molecular and macromolecular mobility within biological tissues and tissue-mimetic materials. Additional applications include mapping of small molecule diffusion within model heterogeneous pharmaceutical formulations to understand bioavailability and stability.

Recent oncology research highlights that senescence, once deemed beneficial in cancer treatments, can contribute to cancer relapse. Detecting therapy-induced senescent cells is challenging due to their complexity and lack of specific markers. Nonlinear optical (NLO) microscopy provides a fast, non-invasive, label-free detection solution. To distinguish between senescent and proliferating cells, here we present the development of a deep learning architecture based on multimodal NLO microscopy images coming from Stimulated Raman Scattering, Two Photon Excited Fluorescence and Optical Transmission. Despite limited labeled data, Transfer Learning, Data Augmentation, and Ensemble Learning techniques allowed us to achieve an accuracy over 90%. Ultimately, the predictions of the neural network are evaluated using the Grad-CAM visualization approach, which allows highlighting the most important features in the input images responsible for the labels assigned by the network. This work reveals the effectiveness of deep learning in senescence classification, potentially advancing treatment strategies.

Water is essential in maintaining cellular activity, from mediating reactions to promoting the shuttling of macromolecules. Spectroscopic studies have indicated that the H-bonding network of water is disrupted by the hydrophobicity of solutes, molecular crowding, and pressure, leading to distinct water structures with the hydration shells of biomacromolecules. Intracellular water is within a very crowded and confined environment and, thus, cannot be adequately described as bulk water; instead, it is expected to exhibit more order or structure due to the confined and crowded environment. Raman spectral imaging is a powerful tool to identify and characterize water throughout a cell, generating subcellular hydration maps. This label-free approach relies on intrinsic molecular vibrational signatures enabling us to obtain spatial information on water and other biomacromolecules in a single experiment. We provide direct evidence that intracellular water does not exhibit the same vibrational properties as bulk water, supporting the importance of considering the effects of confinement and crowding when studying biomolecular systems.

Bioimaging harnessing optical contrasts and chemical specificity is of vital importance in probing complex biology. Vibrational spectroscopy based on mid-infrared excitation can reveal rich chemical information about molecular distributions. However, its full potential for bioimaging is hindered by the achievable sensitivity. Here we report bond-selective fluorescence-detected infrared-excited (BonFIRE) spectro-microscopy. BonFIRE employs two-photon excitation in the mid- and near-infrared to upconvert vibrational excitations to electronic states for fluorescence detection, thus encoding vibrational information into fluorescence. We demonstrate BonFIRE spectral imaging in large spectroscopic windows on samples ranging from single molecules to biological specimens. We then extend BonFIRE imaging from point-scanning to wide-field configuration, enabling ultrafast imaging with a large field of view. We expect BonFIRE to expand the bioimaging toolbox by providing a new level of bond-specific vibrational information and facilitate biological investigations.

Introducing MD-WISE microscopy, a groundbreaking technique that extends the applications of Photoluminescence (PL) imaging. While PL imaging is widely used for visualizing biological activities, detecting chemical species, and characterizing materials, its effectiveness is limited due to overlapping emission spectra of chromophores. However, MD-WISE overcomes this challenge by utilizing nonlinear interactions between mid-infrared and visible excitations, enabling the differentiation of chromophores with similar spectra in a multi-dimensional space. This is achieved through two mechanisms: modulating the optical absorption cross sections of molecular dyes by exciting specific vibrational groups and reducing the Photoluminescence quantum yield of semiconductor nanocrystals. Unlike most nonlinear optical microscopies that require tightly focused beams, MD-WISE operates under widefield imaging conditions, offering a field of view of 30 by 30 microns. By capturing multi-dimensional information in PL images, MD-WISE microscopy has the potential to revolutionize high-speed widefield imaging by simultaneously tracking numerous species and processes.

I will describe the use of plasmon-enhanced fluorescence to boost single-molecule fluorescence signals by more than two orders of magnitude. I will discuss two applications of this approach that directly exploit these ultrabright signals. Firstly, they enable highly sensitive single-molecule DNA diagnostics directly in serum on an affordable and miniaturized microscopy platform. By using a reversible sandwich assay I will describe a continuous monitoring biosensor that is suitable for point-of-care diagnostics. Secondly, the ultrabright signals pave the way to real-time probing of single-molecule dynamics on so-far inaccessible microsecond timescales. I will show recent results where we study the dynamics of monovalent and multivalent DNA constructs in real time with microsecond integration times.

Stimulated Raman Histology (SRH) can be used for label-free histological tissue analysis. However the current approaches are limited to two Raman peaks, thus restricting chemically-specific insights. We present a broadband coherent Raman platform (CRP), which overcomes these limitations combining an all-fiber dual output laser and a multichannel lock-in amplifier. This enables the simultaneous detection of 38 channels across the entire CH spectrum (2800-3100 cm-1) in parallel, facilitating chemometric and multimodal tissue analysis. By incorporating AI-driven virtual H&E staining and tissue segmentation for diagnostic purposes, it advances SRH towards clinical-diagnostic.

Mid-Infrared Spectroscopic Imaging (MIRSI) integrates the molecular precision of vibrational spectroscopy with the comprehensive spatial resolution offered by microscopy. The merger of machine learning with MIRSI has empowered the process of discerning tissue subtypes and grading cancers in a quantitative, label-free way. We plan to showcase findings derived from subtyping and segmenting tissues from ovarian, cervical, and endometrial cancers using an advanced high-resolution photothermal MIRSI technology. Moreover, we will exhibit imaging data along with analysis results from clinical bone samples and outline the characteristics of fibrosis.

Broadband photothermal imaging using synchrotron radiation of the Lawrence Berkeley National Laboratory Advanced Light Source is demonstrated. Synchrotron-based photothermal spectroscopy simultaneously provides a significantly improved bandwidth over commercial laser-based approaches and enables sub-micron chemical imaging with at least a tenfold resolution improvement over FTIR microscopy. Synchrotron PTIR is shown to enable studying low-frequency aromatic bending modes of polymer samples. Furthermore, fluorescence detection of the photothermal effect was employed to demonstrate cell-specific broadband infrared imaging in brain tissues using nucleus-specific fluorescence dyes.

Protein aggregation is associated with many neurodegenerative diseases. We applied mid-infrared photothermal (MIP) to dissect the secondary structure of protein aggregates in living yeast cells modeling Huntington’s disease. We validated that MIP spectroscopy could reveal β-sheet enrichment by significant spectral changes and confirmed the β-sheet enrichment of aggregates in high throughput by wide-field fluorescence-detected MIP. By label-free identification of protein aggregates, we observed a further red shift in spectra for aggregates without GFP tag. Finally, by performing MIP spectroscopy with fine spatial steps, we discovered a partition of secondary structures between β-rich core and ɑ-rich shell of the aggregates.

An approach is described and evaluated for spectrally parallel hyperspectral mid-infrared imaging with optical spatial resolution. Dual-comb mid-infrared spectroscopy using a commercial QCL DCS system (IRis-core, IRsweep) enables acquisition of infrared spectra at high speed (<1 millisecond) through generation of optical beat patterns and radio-frequency detection. The high-speed nature of the spectral acquisition is shown to support spectral mapping in microscopy measurements. Direct detection of the transmitted infrared beam yields excellent spectral information, but the long infrared wavelength imposes low diffraction-limited spatial resolution. Use of fluorescence-detected photothermal infrared (FPTIR) imaging provides high spatial resolution tied directly to the integrated IR absorption. Computational imaging using a multi-agent consensus equilibrium (MACE) approach combines the high spatial resolution of FPTIR and the high spectral information of dual-comb infrared transmission in a single optimized equilibrium hyperspectral data cube.

Deep-tissue chemical imaging is essential for many biomedical applications. Here, we present a shortwave infrared photothermal (SWIP) microscope for millimeter-deep vibrational imaging with micron-resolution and nanoparticle detection sensitivity. By using short-wave infrared light for overtone excitation of CH bonds and photothermal probing, our modality can reach millimeter deep into scattering phantom and tissue. With a fast digitization method, we showed that in our setting the photothermal signal amplitude is 100 times larger than photoacoustic thus enabling higher sensitivity. SWIP can resolve intracellular lipids in tumor spheroid and layered structure in the liver, skin, and brain tissue.

Fluorescent thermometry has reported noteworthy intracellular temperature gradients, suggesting the possibility of thermal regulation of cellular functions. However, heat conduction calculations assuming an aqueous intracellular environment contradict these findings. To resolve this contradiction, we visualized intracellular heat conduction using label-free mid-infrared photothermal optical diffraction tomography and obtained thermal diffusivity similar to that of water. Furthermore, we found a discrepancy in heat-induced temperature changes measured with label-free and fluorescent thermometry. This suggests that fluorescent thermometers may sense other factors, including the system’s internal energy, in addition to the temperature at a local equilibrium state.

Real-time tracking of intracellular carbohydrates remains a grand challenge. While click chemistry enables bio-orthogonal tagging of small molecules with azido groups, subsequent fluorescence visualization is hindered by larger fluorophores, potentially altering intrinsic metabolism. Here, we introduce a click-free, dye-free method for dynamic tracking of trehalose within single mycobacteria via mid-infrared photothermal (MIP) imaging of azido-trehalose. Through spectroscopic MIP imaging, the metabolic pathways of trehalose and its interactions with intracellular proteins is demonstrated without click reaction and photobleaching, offering valuable insights into mycobacterial physiology. Our approach paves the way for further advancements in studying metabolic dynamics within living cells.

Stimulated Raman photothermal (SRP) is a newly developed chemical imaging scheme that probes the SRS induced thermal effect with a third laser beam. SRP shows high sensitivity, where a >500-fold boost of modulation depth comparing to SRS has been achieved. To further improve the sensitivity of SRP, we introduce spSRP with a ~1 MHz repetition rate, high peak power OPA laser serving as the SRP excitation source. The high peak power increases the energy deposition of SRS process, enables SRP measurement with a single pair of pump and Stokes laser pulses. spSRP is a firm step towards vibrational spectroscopic imaging with ultrahigh sensitivity.

SWIR-based microscopy has opened up windows to cellular and extracellular dynamics in deep tissues and living biological systems. New generation of laser sources, with high pulse energy, wide continuous tunable range, and a compact form, are in high demand to advance nonlinear microscopy and SWIR-based imaging to its full potential for deep-tissue imaging. This talk will discuss our recently developed approach that exploits the spatial and temporal degrees of control of nonlinear effects in step-index MMFs using a 3D-printed programmable fiber piano. By leveraging the rich spatiotemporal degrees of freedom and the high spectral brilliance in SI MMF, We have achieved broadband high-peak-power spanning 560–2200 nm, resulting from combined spectral energy reallocation (up to 166-fold) and temporal shortening (up to 4-fold) uniquely enabled by the fiber shaper. Its potential as a nonlinear imaging source is further demonstrated by applying the MMF source to multiphoton microscopy, where multi-fold signal enhancement is achieved for label-free tissue imaging with adaptive optimization.

Pharmaceutical development of solid-state formulations requires testing for uniformity and stability of active pharmaceutical ingredients and excipients. Solid-state properties such as component distribution and grain size are crucial factors of dissolution profile, which greatly affect drug efficacy and toxicity, and can only be analyzed spatially by chemical imaging (CI) techniques. Current CI techniques such as near infrared and confocal Raman spectroscopy do not offer high chemical and spatial resolution at speeds feasible for integration into the pharmaceutical quality control and quality assurance processes. We demonstrate fast chemical imaging by epi-detected sparse spectral sampling stimulated Raman scattering to quantify API and excipient degradation and distribution.

Raman microscopy enables us to obtain molecular information in biological samples but has suffered from low signal-to-noise ratio (SNR) due to low of Raman scattering cross-section. Here we developed a cryo-stat equipped Raman microscope for low temperature measurement, allowing long time accumulation of Raman signals. We confirmed the SNR improvement in Raman imaging of cryofixed HeLa cells without photodamage under long time observation at low temperature. The reduction of photobleaching in resonant Raman scattering of carotenoid and cytochrome significantly increases the SNR, demonstrated by 7-color high SNR Raman imaging with multiple Raman tags, including EdU, MitoBADY, and alkyne-tagged Coenzyme Q (AltQ2). AltQ2 is a mobile small molecule that cannot be fixed by chemical fixation.

Chemical imaging, especially mid-infrared spectroscopic microscopy, enables label-free biomedical analyses while achieving expansive molecular sensitivity. However, its slow speed and poor image quality impede widespread adoption. We present a microscope that provides high-throughput recording, low noise, and high spatial resolution where the bottom-up design of its optical train facilitates dual-axis galvo laser scanning of a diffraction-limited focal point over large areas using custom infinity-corrected objectives. The data quality enables applications of modern machine learning and capabilities not previously feasible. Distinct from conventional approaches that focus on morphological investigations or immunostaining, this development makes label-free imaging of minimally processed tissue practical.

We have developed a new shot noise limited light source for Coherent Raman microscopy and other nonlinear microscopy modalities such as SHG and multiphoton fluorescence. Compared to the market leading SRS light source it achieves 100x faster tuning for random wavelength access including power control and dispersion compensation. Further switching from 80 to 40MHz repetition rate with subharmonic modulation frequency of 20MHz reduces the pixel dwell time in theory by a factor of 8 for the same signal to noise ratio. We will present a comparison to the prevailing SRS light source along with fingerprint images of relevant biological samples.

Stimulated Raman scattering (SRS) microscopy has emerged as a valuable tool with manifold biomedical applications. Due to the physical limits of Raman cross section, enhancing chemical information bandwidths comes at the price of decreasing the signal-to-noise ratio (SNR). One appealing approach to combat the physical limits is denoising. In this work, we propose a self-supervised Noise2Noise 3D Unet denoiser for three-dimensional SRS imaging. To demonstrate the limit-breaking capability, we denoised three types of three-dimensional SRS datasets, including hyperspectral, volumetric, and longitudinal, under extreme experimental conditions with low SNR. Our results highlight the potential for boosting the physical limits by integration of instrumentation and computation.

We present a fully integrated, clinical-compatible SRS imaging device giving access to the complete Raman spectrum during tumor surgeries. Leveraging the advantages of a compact and robust fiber laser, we have integrated the entire microscopy system into a clinical cart, facilitating deployment in diverse clinical environments. The laser provides rapid tunability within milliseconds across a broad spectral range of 700 to 3300 cm^-1, covering biomedically relevant resonances in the fingerprint region. For detailed examination of larger tissue samples, we have designed a high-speed, low-resolution imaging mode to quickly identify cancerous hot-spots, followed by a high-resolution imaging mode.

Vibrational tags in infrared (IR)-based micro-spectroscopy constitute powerful tools for studies on cellular metabolism. Whereas Raman tags have seen substantial developments, IR tags have not similarly benefitted from systematic design optimization. To improve the utility of IR-based vibrational tags, we modified a series of alkyne-based probes for use in the cell silent region of the IR spectrum. Using density functional theory (DFT) simulations for initial design motifs, the tags were analyzed using linear spectroscopy, and subsequently screened for their utility in cell and tissue imaging. The resulting chemical motifs form a palette of strong vibrational tags for IR-based biological imaging.

Second harmonic generation (SHG) imaging microscopy is a nonlinear optical imaging technique that uses SHG as a contrast mechanism to produce high-resolution images. SHG occurs in materials with non-centrosymmetric crystal structures. Therefore, SHG imaging has been applied for characterization of 2D semiconductors, transition-metal dichalcogenides (TMDs) such as WS₂ and MoSe₂, lithium niobate crystals, PZT thin films, graphene, lanthanides, and even biological tissues. It provides information on the crystal lattice, assesses crystal quality and maps grain boundaries, defects, and mechanical strain. Furthermore, SHG imaging reveals the number of stacked layers as well as their orientation with respect to each other.