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Abstract Measurements and imaging of the mechanical response of biological cells are critical for understanding the mechanisms of many diseases, and for fundamental studies of energy, signal and force transduction. The recent emergence of Brillouin microscopy as a powerful non-contact, label-free way to non-invasively and non-destructively assess local viscoelastic properties provides an opportunity to expand the scope of biomechanical research to the sub-cellular level. Brillouin spectroscopy has recently been validated through static measurements of cell viscoelastic properties, however, fast (sub-second) measurements of sub-cellular cytomechanical changes have yet to be reported. In this report, we utilize a custom multimodal spectroscopy system to monitor for the very first time the rapid viscoelastic response of cells and subcellular structures to a short-duration electrical impulse. The cytomechanical response of three subcellular structures - cytoplasm, nucleoplasm, and nucleoli - were monitored, showing distinct mechanical changes despite an identical stimulus. Through this pioneering transformative study, we demonstrate the capability of Brillouin spectroscopy to measure rapid, real-time biomechanical changes within distinct subcellular compartments. Our results support the promising future of Brillouin spectroscopy within the broad scope of cellular biomechanics. 

Development of a simple, label-free screening technique capable of precisely and directly sensing interaction-in-solution over a size range from small molecules to large proteins such as antibodies could offer an important tool for researchers and pharmaceutical companies in the field of drug development. In this work, we present a thermostable Raman interaction profiling (TRIP) technique that facilitates low-concentration and low-dose screening of binding between protein and ligand in physiologically relevant conditions. TRIP was applied to eight protein–ligand systems, and produced reproducible high-resolution Raman measurements, which were analyzed by principal component analysis. TRIP was able to resolve time-depending binding between 2,4-dinitrophenol and transthyretin, and analyze biologically relevant SARS-CoV-2 spike-antibody interactions. Mixtures of the spike receptor–binding domain with neutralizing, nonbinding, or binding but nonneutralizing antibodies revealed distinct and reproducible Raman signals. TRIP holds promise for the future developments of high-throughput drug screening and real-time binding measurements between protein and drug. 

Molecular, morphological, and physiological heterogeneity is the inherent property of cells which governs differences in their response to external influence. Tumor cell metabolic heterogeneity is of a special interest due to its clinical relevance to tumor progression and therapeutic outcomes. Rapid, sensitive, and noninvasive assessment of metabolic heterogeneity of cells is a great demand for biomedical sciences. Fluorescence lifetime imaging (FLIM), which is an all-optical technique, is an emerging tool for sensing and quantifying cellular metabolism by measuring fluorescence decay parameters of endogenous fluorophores, such as NAD(P)H. To achieve accurate discrimination between metabolically diverse cellular subpopulations, appropriate approaches to FLIM data collection and analysis are needed. In this paper, the unique capability of FLIM to attain the overarching goal of discriminating metabolic heterogeneity is demonstrated. This has been achieved using an approach to data analysis based on the nonparametric analysis, which revealed a much better sensitivity to the presence of metabolically distinct subpopulations compared to more traditional approaches of FLIM measurements and analysis. The approach was further validated for imaging cultured cancer cells treated with chemotherapy. These results pave the way for accurate detection and quantification of cellular metabolic heterogeneity using FLIM, which will be valuable for assessing therapeutic vulnerabilities and predicting clinical outcomes. 

Scanning confocal Raman spectroscopy was applied for detecting and identifying topically applied ocular pharmaceuticals on rabbit corneal tissue. Raman spectra for Cyclosporin A, Difluprednate, and Dorzolamide were acquired together with Raman spectra from rabbit corneas with an unknown amount of applied drug. Kernel principle component analysis (KPCA) was then used to explore a transform that can describe the acquired set of Raman spectra. Using this transform, we observe some spectral similarity between cornea spectra and Cyclosporin A, with little similarity to Dorzolamide and Difluprednate. Further investigation is needed to identify why these differences occur. 

The optical activity of Raman scattering provides insight into the absolute configuration and conformation of chiral molecules. Applications of Raman optical activity (ROA) are limited by long integration times due to a relatively low sensitivity of the scattered light to chirality (typically 10^-3 to 10^-5). We apply ROA techniques to hyper-Raman scattering using incident circularly polarized light and a right-angle scattering geometry. We explore the sensitivity of hyper- Raman scattering to chirality as compared to spontaneous Raman optical activity. Using the excitation wavelength at around 532 nm, the photobleaching is minimized, while the hyper-Raman scattering benefits from the electronic resonant enhancement. For S/R-2-butanol and L/D-tartaric acid, we were unable to detect the hyper-Raman optical activity at the sensitivity level of 1%. We also explored parasitic thermal effects which can be mitigating by varying the repetition rate of the laser source used for excitation of hyper-Raman scattering.