Unveiling the Spatiotemporal and Dose Responses within a Single Live Cancer Cell to Photoswitchable Upconversion Nanoparticle Therapeutics Using Hybrid Hyperspectral Stimulated Raman Scattering and Transient Absorption Microscopy.

Photodynamic therapy (PDT) provides an alternative approach to targeted cancer treatment, but the therapeutic mechanism of advanced nanodrugs applied to live cells and tissue is still not well understood. Herein, we employ the hybrid hyperspectral stimulated Raman scattering (SRS) and transient absorption (TA) microscopy developed for real-time in vivo visualization of the dynamic interplay between the unique photoswichable lanthanide-doped upconversion nanoparticle-conjugated rose bengal and triphenylphosphonium (LD-UCNP@CS-Rb-TPP) probe synthesized and live cancer cells. The Langmuir pharmacokinetic model associated with SRS/TA imaging is built to quantitatively track the uptakes and pharmacokinetics of LD-UCNP@CS-Rb-TPP within cancer cells. Rapid SRS/TA imaging quantifies the endocytic internalization rates of the LD-UCNP@CS-Rb-TPP probe in individual HeLa cells, and the translocation of LD-UCNP@CS-Rb-TPP from mitochondria to cell nuclei monitored during PDT can be associated with mitochondria fragmentations and the increased nuclear membrane permeability, cascading the dual organelle ablations in cancer cells. The real-time SRS spectral changes of cellular components (e.g., proteins, lipids, and DNA) observed reflect the PDT-induced oxidative damage and the dose-dependent death pattern within a single live cancer cell, thereby facilitating the real-time screening of optimal light dose and illumination duration controls in PDT. This study provides new insights into the further understanding of drug delivery and therapeutic mechanisms of photoswitchable LD-UCNP nanomedicine in live cancer cells, which are critical in the optimization of nanodrug formulations and development of precision cancer treatment in PDT.

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

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

Quantitative Real-Time Analysis of Living Materials by Stimulated Raman Scattering Microscopy.

Composite materials built in part from living organisms have the potential to exhibit useful autonomous, adaptive, and self-healing behavior. The physicochemical, biological, and mechanical properties of such materials can be engineered through the genetic manipulation of their living components. Successful development of living materials will require not only new methods for design and preparation but also new analytical tools that are capable of real-time noninvasive mapping of chemical compositions. Here, we establish a strategy based on stimulated Raman scattering microscopy to monitor phosphatase-catalyzed mineralization of engineered bacterial films in situ. Real-time label-free imaging elucidates the mineralization process, quantifies both the organic and inorganic components of the material as functions of time, and reveals spatial heterogeneity at multiple scales. In addition, we correlate the mechanical performance of films with the extent of mineralization. This work introduces a promising strategy for quantitatively analyzing living materials, which should contribute to the accelerated development of such materials in the future.

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

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

Visualization of Modified Bisarylbutadiyne-Tagged Small Molecules in Live-Cell Nuclei by Stimulated Raman Scattering Microscopy.

Visualizing the distribution of small-molecule drugs in living cells is an important strategy for developing specific, effective, and minimally toxic drugs. As an alternative to fluorescence imaging using bulky fluorophores or cell fixation, stimulated Raman scattering (SRS) imaging combined with bisarylbutadiyne (BADY) tagging enables the observation of small molecules closer to their native intracellular state. However, there is evidence that the physicochemical properties of BADY-tagged analogues of small-molecule drugs differ significantly from those of their parent drugs, potentially affecting their intracellular distribution. Herein, we developed a modified BADY to reduce deviations in physicochemical properties (in particular, lipophilicity and membrane permeability) between tagged and parent drugs, while maintaining high Raman activity in live-cell SRS imaging. We highlight the practical application of this approach by revealing the nuclear distribution of a modified BADY-tagged analogue of JQ1, a bromodomain and extra-terminal motif inhibitor with applications in targeted cancer therapy, in living HeLa cells. The modified BADY, methoxypyridazyl pyrimidyl butadiyne (MPDY), revealed intranuclear JQ1, while BADY-tagged JQ1 did not show a clear nuclear signal. We anticipate that the present approach combining MPDY tagging with live-cell SRS imaging provides important insight into the behavior of intracellular drugs and represents a promising avenue for improving drug development.

Compact simultaneous label-free autofluorescence multi-harmonic microscopy for user-friendly photodamage-monitored imaging.

Significance: Label-free nonlinear optical microscopy has become a powerful tool for biomedical research. However, the possible photodamage risk hinders further clinical applications. Aim: To reduce these adverse effects, we constructed a new platform of simultaneous label-free autofluorescence multi-harmonic (SLAM) microscopy, featuring four-channel multimodal imaging, inline photodamage monitoring, and pulse repetition-rate tuning. Approach: Using a large-core birefringent photonic crystal fiber for spectral broadening and a prism compressor for pulse pre-chirping, this system allows users to independently adjust pulse width, repetition rate, and energy, which is useful for optimizing imaging conditions towards no/minimal photodamage. Results: It demonstrates label-free multichannel imaging at one excitation pulse per image pixel and thus paves the way for improving the imaging speed by a faster optical scanner with a low risk of nonlinear photodamage. Moreover, the system grants users the flexibility to autonomously fine-tune repetition rate, pulse width, and average power, free from interference, ensuring the discovery of optimal imaging conditions with high SNR and minimal phototoxicity across various applications. Conclusions: The combination of a stable laser source, independently tunable ultrashort pulse, photodamage monitoring features, and a compact design makes this new system a robust, powerful, and user-friendly imaging platform.

On-the-fly Raman microscopy guaranteeing the accuracy of discrimination.

Accelerating the measurement for discrimination of samples, such as classification of cell phenotype, is crucial when faced with significant time and cost constraints. Spontaneous Raman microscopy offers label-free, rich chemical information but suffers from long acquisition time due to extremely small scattering cross-sections. One possible approach to accelerate the measurement is by measuring necessary parts with a suitable number of illumination points. However, how to design these points during measurement remains a challenge. To address this, we developed an imaging technique based on a reinforcement learning in machine learning (ML). This ML approach adaptively feeds back "optimal" illumination pattern during the measurement to detect the existence of specific characteristics of interest, allowing faster measurements while guaranteeing discrimination accuracy. Using a set of Raman images of human follicular thyroid and follicular thyroid carcinoma cells, we showed that our technique requires 3,333 to 31,683 times smaller number of illuminations for discriminating the phenotypes than raster scanning. To quantitatively evaluate the number of illuminations depending on the requisite discrimination accuracy, we prepared a set of polymer bead mixture samples to model anomalous and normal tissues. We then applied a home-built programmable-illumination microscope equipped with our algorithm, and confirmed that the system can discriminate the sample conditions with 104 to 4,350 times smaller number of illuminations compared to standard point illumination Raman microscopy. The proposed algorithm can be applied to other types of microscopy that can control measurement condition on the fly, offering an approach for the acceleration of accurate measurements in various applications including medical diagnosis.

Evaluation of chemical disposition in skin by stimulated Raman scattering microscopy.

Tracking drug disposition in the skin in a non-destructive and at least semi-quantitative fashion is a relevant objective for the assessment of local (cutaneous) bioavailability. Confocal Raman spectroscopy has been shown potentially useful in this regard and, importantly, recent advances have enabled the presence of applied chemicals in the viable epidermis below the stratum corneum (SC) to be determined without ambiguity and having addressed the challenges of (a) background signals from endogenous species and noise and (b) signal attenuation due to absorption and scattering. This study aimed to confirm these observations using a different vibrational spectroscopy approach - specifically, stimulated Raman scattering (SRS) microscopy - and the more conventional in vitro skin penetration test (IVPT). SRS is a nonlinear optical imaging technique which enables more precise location of the skin surface and enhanced skin depth resolution relative to confocal Raman microscopy. The method can also probe larger areas of the sample under investigation and identify the localization of the permeating chemical in specific structural components of the skin. Here, SRS was shown capable of tracking the uptake and distribution of 4-cyanophenol (CP), the same model compound used in the recent confocal Raman investigation, at depths beyond the SC following skin treatment with different vehicles and for different times. The SRS results correlated well with those from the confocal Raman experiments, and both were consistent with independent IVPT measurements. Acquired images clearly delineated CP preference for the intercellular lipid layers of the SC relative to the corneocytes. The stage is now set to apply these and other correlative techniques to examine commercial drug products.

Multi-molecular hyperspectral PRM-SRS microscopy.

Lipids play crucial roles in many biological processes. Mapping spatial distributions and examining the metabolic dynamics of different lipid subtypes in cells and tissues are critical to better understanding their roles in aging and diseases. Commonly used imaging methods (such as mass spectrometry-based, fluorescence labeling, conventional optical imaging) can disrupt the native environment of cells/tissues, have limited spatial or spectral resolution, or cannot distinguish different lipid subtypes. Here we present a hyperspectral imaging platform that integrates a Penalized Reference Matching algorithm with Stimulated Raman Scattering (PRM-SRS) microscopy. Using this platform, we visualize and identify high density lipoprotein particles in human kidney, a high cholesterol to phosphatidylethanolamine ratio inside granule cells of mouse hippocampus, and subcellular distributions of sphingosine and cardiolipin in human brain. Our PRM-SRS displays unique advantages of enhanced chemical specificity, subcellular resolution, and fast data processing in distinguishing lipid subtypes in different organs and species.

Determining topical product bioequivalence with stimulated Raman scattering microscopy.

Generic drugs are essential for affordable medicine and improving accessibility to treatments. Bioequivalence (BE) is typically demonstrated by assessing a generic product's pharmacokinetics (PK) relative to a reference-listed drug (RLD). Accurately estimating cutaneous PK (cPK) at or near the site of action can be challenging for locally acting topical products. Certain cPK approaches are available for assessing local bioavailability (BA) in the skin. Stimulated Raman scattering (SRS) microscopy has unique capabilities enabling continuous, high spatial and temporal resolution and quantitative imaging of drugs within the skin. In this paper, we developed an approach based on SRS and a polymer-based standard reference for the evaluation of topical product BA and BE in human skin ex vivo. BE assessment of tazarotene-containing formulations was achieved using cPK parameters obtained within different skin microstructures. The establishment of BE between the RLD and an approved generic product was successfully demonstrated. Interestingly, within the constraints of the current study design the results suggest similar BA between the tested gel formulation and the reference cream formulation, despite the differences in the formulation/dosage form. Another formulation containing polyethylene glycol as the vehicle was demonstrated to be not bioequivalent to the RLD. Compared to using the SRS approach without a standard reference, the developed approach enabled more consistent and reproducible results, which is crucial in BE assessment. The abundant information from the developed approach can help to systematically identify key areas of study design that will enable a better comparison of topical products and support an assessment of BE.

Differences in Lipid Order and Dynamics in Plasma Membranes Assessed by Nonlinear Optical Microscopy.

When amphiphilic polar dyes were added to the cells, they intercalated predominantly in the outer leaf of the plasma membrane, making them active for second harmonic generation (SHG). The fluorescence of the dye enabled simultaneous 3D imaging of SHG and two-photon excited fluorescence (TPF). Because SHG intensity is sensitive to the alignment of the dyes, which reflects lipid ordering in the plasma membrane, we assessed the difference in lipid ordering by comparing the SHG intensity normalized to the TPF intensity. Together with an enzyme release assay that detects pore formation in the plasma membrane, our SHG assay revealed how polycations affect lipid ordering at low concentrations, where membrane damage has not yet been examined. By scaling the results of the assays with the charge concentration of the two polycations, polyethylenimine (PEI) and poly-l-lysine (PLL), we found that PEI reduced the lipid order more than PLL, and PLL formed more pores than PEI. A comparison of the SHG and TPF images of the wounded cells revealed that one of the lipid dynamics (flip-flop) was significantly enhanced in the bleb membrane. Moreover, the SHG assay indicated that the biocompatible polymer, poly(N-(2-hydroxypropyl)methacrylamide), did not affect the lipid order. Thus, our technique allows the assessment of the plasma membrane structure at the molecular level.

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

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

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

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

Stimulated Raman Scattering Microscopy Reveals Bioaccumulation of Small Microplastics in Protozoa from Natural Waters.

Microplastics (MPs) are pollutants of global concern, and bioaccumulation determines their biological effects. Although microorganisms form a large fraction of our ecosystem's biomass and are important in biogeochemical cycling, their accumulation of MPs has never been confirmed in natural waters because current tools for field biological samples can detect only MPs > 10 µm. Here, we show that stimulated Raman scattering microscopy (SRS) can image and quantify the bioaccumulation of small MPs (<10 µm) in protozoa. Our label-free method, which differentiates MPs by their SRS spectra, detects individual and mixtures of different MPs (e.g., polyethylene, polypropylene, polyvinyl chloride, polyethylene terephthalate, polystyrene, and poly(methyl methacrylate)) in protozoa. The ability of SRS to quantify cellular MP accumulation is similar to that of flow cytometry, a fluorescence-based method commonly used to determine cellular MP accumulation. Moreover, we discovered that protozoa in water samples from Yangtze River, Xianlin Wastewater Treatment Plant, Lake Taihu and the Pearl River Estuary accumulated MPs < 10 µm, but the proportion of MP-containing cells was low (∼2-5%). Our findings suggest that small MPs could potentially enter the food chain and transfer to organisms at higher trophic levels, posing environmental and health risks that deserve closer scrutiny.

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

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

Quantitative analysis of drug tablet aging by fast hyper-spectral stimulated Raman scattering microscopy.

Pharmaceutical development of solid-state formulations requires testing active pharmaceutical ingredients (API) and excipients for uniformity and stability. Solid-state properties such as component distribution and grain size are crucial factors that influence the 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 microscopy and confocal Raman spectroscopy are capable of high chemical and spatial resolution but cannot achieve the measurement speeds necessary for integration into the pharmaceutical production and quality assurance processes. To fill this gap, we demonstrate fast chemical imaging by epi-detected sparse spectral sampling stimulated Raman scattering to quantify API and excipient degradation and distribution.

Combination of deep learning and 2D CARS figures for identification of amyloid-ß plaques.

In vivo imaging and accurate identification of amyloid-ß (Aß) plaque are crucial in Alzheimer's disease (AD) research. In this work, we propose to combine the coherent anti-Stokes Raman scattering (CARS) microscopy, a powerful detection technology for providing Raman spectra and label-free imaging, with deep learning to distinguish Aß from non-Aß regions in AD mice brains in vivo. The 1D CARS spectra is firstly converted to 2D CARS figures by using two different methods: spectral recurrence plot (SRP) and spectral Gramian angular field (SGAF). This can provide more learnable information to the network, improving the classification precision. We then devise a cross-stage attention network (CSAN) that automatically learns the features of Aß plaques and non-Aß regions by taking advantage of the computational advances in deep learning. Our algorithm yields higher accuracy, precision, sensitivity and specificity than the results of conventional multivariate statistical analysis method and 1D CARS spectra combined with deep learning, demonstrating its competence in identifying Aß plaques. Last but not least, the CSAN framework requires no prior information on the imaging modality and may be applicable to other spectroscopy analytical fields.

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

Multiplex optical detection in live cells is challenging due to overlapping signals and poor signal-to-noise associated with some chemical reporters. To address this, the application of spectral phasor analysis to stimulated Raman scattering (SRS) microscopy for unmixing three bioorthogonal Raman probes within cells is reported. Triplex detection of a metallacarborane using the B-H stretch at 2480-2650 cm-1 , together with a bis-alkyne and deuterated fatty acid can be achieved within the cell-silent region of the Raman spectrum. When coupled to imaging in the high-wavenumber region of the cellular Raman spectrum, nine discrete regions of interest can be spectrally unmixed from the hyperspectral SRS dataset, demonstrating a new capability in the toolkit of multiplexed Raman imaging of live cells.

Stimulated Raman scattering microscopy reveals a unique and steady nature of brain water dynamics.

The biological activities of substances in the brain are shaped by their spatiotemporal dynamics in brain tissues, all of which are regulated by water dynamics. In contrast to solute dynamics, water dynamics have been poorly characterized, owing to the lack of appropriate analytical tools. To overcome this limitation, we apply stimulated Raman scattering multimodal multiphoton microscopy to live brain tissues. The microscopy system allows for the visualization of deuterated water, fluorescence-labeled solutes, and cellular structures at high spatiotemporal resolution, revealing that water moves faster than fluorescent molecules in brain tissues. Detailed analyses demonstrate that water, unlike solutes, diffuses homogeneously in brain tissues without differences between the intra- and the extracellular routes. Furthermore, we find that the water dynamics are steady during development and ischemia, when diffusions of solutes are severely affected. Thus, our approach reveals routes and uniquely robust properties of water diffusion in brain tissues.

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

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