Measuring Drug Response with Single-Cell Growth Rate Quantification.

Intratumoral heterogeneity is a substantial cause of drug resistance development during chemotherapy or other drug treatments for cancer. Therefore, monitoring and measuring cell exposure and response to drugs at the single-cell level are crucial. Previous research suggested that the single-cell growth rate can be used to investigate drug-cell interactions. However, currently established methods for quantifying single-cell growth are limited to isolated or monolayer cells. Here, we introduce a technique that accurately measures both 2D and 3D cell growth rates using label-free ratiometric stimulated Raman scattering (SRS) microscopy. We use deuterated amino acids, leucine, isoleucine, and valine, as tracers and measure the C-D SRS signal from deuterium-labeled proteins and the C-H SRS signal from unlabeled proteins simultaneously to determine the cell growth rate at the single-cell level. The technique offers single-cell level drug sensitivity measurement with a shorter turnaround time (within 12 h) than most traditional assays. The submicrometer resolution of the imaging technique allows us to examine the effects of chemotherapeutic drugs, including kinase inhibitors, mitotic inhibitors, and topoisomerase II inhibitors, on both the cell growth rate and morphology. The capability of quantifying 3D cell growth rates provides insight into a deeper understanding of the cell-drug interaction in the actual tumor environment.

Assessing drug uptake and response differences in 2D and 3D cellular environments using stimulated Raman scattering microscopy.

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 SRS imaging to 2D and 3D cell cultures and provided 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 is 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 we 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.

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.

Simultaneous Dual-Band Hyperspectral Stimulated Raman Scattering Microscopy with Femtosecond Optical Parametric Oscillators.

Stimulated Raman scattering (SRS) microscopy is a label-free quantitative optical technique for imaging molecular distributions in cells and tissues by probing their intrinsic vibrational frequencies. Despite its usefulness, existing SRS imaging techniques have limited spectral coverage due to either a wavelength tuning constraint or narrow spectral bandwidth. High-wavenumber SRS imaging is commonly used to map lipid and protein distribution in biological cells and visualize cell morphology. However, to detect small molecules or Raman tags, imaging in the fingerprint region or "silent" region, respectively, is often required. For many applications, it is desirable to collect SRS images in two Raman spectral regions simultaneously for visualizing the distribution of specific molecules in cellular compartments or providing accurate ratiometric analysis. In this work, we present an SRS microscopy system using three beams generated by a femtosecond oscillator to acquire hyperspectral SRS image stacks in two arbitrary vibrational frequency bands, between 650-3280 cm-1, simultaneously. We demonstrate potential biomedical applications of the system in investigating fatty acid metabolism, cellular drug uptake and accumulation, and lipid unsaturation level in tissues. We also show that the dual-band hyperspectral SRS imaging system can be adapted for the broadband fingerprint region hyperspectral imaging (1100-1800 cm-1) by simply adding a modulator.

Quantitative Imaging of Intracellular Density with Ratiometric Stimulated Raman Scattering Microscopy.

Cell size and density are tightly controlled in mammalian cells. They impact a wide range of physiological functions, including osmoregulation, tissue homeostasis, and growth regulation. Compared to size, density variation for a given cell type is typically much smaller, implying that cell-type-specific density plays an important role in cell function. However, little is known about how cell density affects cell function or how it is regulated. Current tools for intracellular cell density measurements are limited to either suspended cells or cells grown on 2D substrates, neither of which recapitulate the physiology of single cells in intact tissue. While optical measurements have the potential to noninvasively measure cell density in situ, light scattering in multicellular systems prevents direct quantification. Here, we introduce an intracellular density imaging technique based on ratiometric stimulated Raman scattering microscopy (rSRS). It uses intrinsic vibrational information from intracellular macromolecules to quantify dry mass density. Moreover, water is used as an internal standard to correct for aberration and light scattering effects. We demonstrate real-time measurement of intracellular density and show that density is tightly regulated across different cell types and can be used to differentiate cell types as well as cell states. We further demonstrate dynamic imaging of density change in response to osmotic challenge as well as intracellular density imaging of a 3D tumor spheroid. Our technique has the potential for imaging intracellular density in intact tissue and understanding density regulation and its role in tissue homeostasis.