The architecture of cell culture, two-dimensional (2D) versus three-dimensional (3D), significantly impacts various cellular factors, including cell-cell interactions, nutrient and oxygen gradients, metabolic activity, and gene expression profiles. This can result in different cellular responses during cancer drug treatment, with 3D-cultured cells often exhibiting higher resistance to chemotherapeutic drugs. While various genetic and proteomic analyses have been employed to investigate the underlying mechanisms of this increased resistance, complementary techniques that provide experimental evidence of spatial molecular profiling data are limited. Stimulated Raman scattering (SRS) microscopy has demonstrated its capability to measure both intracellular drug uptake and growth inhibition. In this work, we applied three-band (C-D, C-H, and fingerprint regions) SRS imaging to 2D and 3D cell cultures and performed a comparative analysis of drug uptake and response with the goal of understanding whether the difference in drug uptake explains the drug resistance in 3D culture compared to 2D. Our investigations revealed that despite similar intracellular drug levels in 2D and 3D A549 cells during lapatinib treatment, the growth of 3D spheroids was less impacted, supporting an enhanced drug tolerance in the 3D microenvironment. We further elucidated drug penetration patterns and the resulting heterogeneous cellular responses across different spheroid layers. Additionally, we investigated the role of the extracellular matrix in modulating drug delivery and cell response and discovered that limited drug penetration in 3D could also contribute to lower drug response. Our study provides valuable insights into the intricate mechanisms of increased drug resistance in 3D tumor models during cancer drug treatments.

Hepatic toxicity is a leading cause of the termination of clinical trials and the withdrawal of therapeutics following regulatory approval. The detection of drug-induced liver injury (DILI) is therefore of importance to ensure patient safety and the effectiveness of novel small molecules and drugs. DILI encompasses drug-induced steatosis (DIS) and drug-induced phospholipidosis (DIPL) which involve the accumulation of excess intracellular lipids. Here, we develop hyperspectral stimulated Raman scattering (SRS) microscopy as a label-free methodology for discriminating DIS and DIPL in mammalian cell culture. We demonstrate that hyperspectral SRS imaging in tandem with spectral phasor analysis is capable of discriminating DIS and DIPL based on the nature and distribution of intracellular lipids resulting from each process. To demonstrate the practical application of this methodology, we develop a panel of alkyne-tagged propranolol analogues that display varying DILI effects. Using hyperspectral SRS imaging together with spectral phasor analysis, our label-free methodology corroborated the standard fluorescence-based assay for DILI. As a label-free screening method, it offers a convenient and expedient methodology for visualizing hepatotoxicity in cell cultures which could be integrated into the early stages of the drug development process for screening new chemical entities for DILI.

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.

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.

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.

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.

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.

UNASSIGNED: Label-free nonlinear optical microscopy has become a powerful tool for biomedical research. However, the possible photodamage risk hinders further clinical applications.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.

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.

Nonlinear microscopy (NM) enables us to investigate the morphology or monitor the physiological processes of the skin through the use of ultrafast lasers. Fiber (or fiber-coupled) lasers are of great interest because they can easily be combined with a handheld, scanning nonlinear microscope. This latter feature greatly increases the utility of NM for pre-clinical applications and in vivo tissue imaging. Here, we present a fiber-coupled, sub-ps Ti-sapphire laser system being optimized for in vivo, stain-free, 3D imaging of skin alterations with a low thermal load of the skin. The laser is pumped by a low-cost, 2.1 W, 532 nm pump laser and delivers 0.5-1 ps, high-peak-power pulses at a ~20 MHz repetition rate. The spectral bandwidth of the laser is below 2 nm, which results in a low sensitivity for dispersion during fiber delivery. The reduction in the peak intensity due to the increased pulse duration is compensated by the lower repetition rate of our laser. In our proof-of-concept imaging experiments, a ~1.8 m long, commercial hollow-core photonic bandgap fiber was used for fiber delivery. Fresh and frozen skin biopsies of different skin alterations (e.g., adult hemangioma, basal cell cancer) and an unaffected control were used for high-quality, two-photon excitation fluorescence microscopy (2PEF) and second-harmonic generation (SHG) z-stack (3D) imaging.