Simultaneous observation of cytotoxicity and drug distribution at the effector site will bring new insights into understanding mechanism of action of drug. Here, we demonstrated the application of Raman microscopy to visualize drug-induced cytotoxicity and drug distribution as well as its mechanism of action at a real-time and single-cell level. The drug concentration and treatment-dependent decrease of reduced cyt c was found as an early marker of cell apoptosis which is more sensitive than the conventional cell viability assay. This method allows for us to screen the drug cytotoxicity and approach the mechanism at the same time, thereby reducing the cost and time of drug screening.
One major obstacle hampering the utilization of human induced pluripotent stem cells (hiPS) derived cells from bench to bedside is the safety concern of residual undifferentiated hiPS cells. To ensure the clinical use of differentiated cells, a method which can monitor the regenerative processes and assess the cell population without harming and modifying those cells is very critical in this field. Raman microscopy has emerged as a powerful tool in label-free observation and discrimination of cell types without external labels. Cell proliferation, differentiation, and maturation all trigger molecular changes that can be detected via Raman, enabling non-destructive characterization of cell and tissue constructs. By using Raman microscopy, we are aiming at establishing a non-invasive and quantitative evaluation method to monitor hepatic differentiation. In the future, this technique would be an invaluable tool in the quality control and safety assessment of hiPS-derived cell products.
KEYWORDS: Raman spectroscopy, Raman scattering, Live cell imaging, Microscopy, Molecules, Signal detection, Resonance enhancement, Molecular spectroscopy, Microscopes, Signal to noise ratio
Resonance Raman scattering is useful for improving a signal-to-noise ratio and a data acquisition speed in Raman imaging. However, the detection of non-resonance Raman scattering is often hindered by resonance signals and fluorescent background. To aid this dilemma in using resonance Raman scattering, we have developed a confocal Raman microscope with dual-wavelength excitation. Living HeLa cells were measured simultaneously at two different excitation wavelengths. At 532 nm excitation, cytochromes were detected by the resonance effect. At 660 nm excitation, non-resonance signals from proteins and lipids were obtained without any clear influence from cytochromes and fluorescent background.
Amino acids and peptides are basic components of proteins and have vital importance in various biological functions and diseases. In this research, we have attempted to detect and distinguish 20 kinds of amino acids and 39 kinds of peptides without any labeling. By using Raman microscopy, more than two thousand Raman spectra were obtained within five minutes from each analyte, at femtomolecular levels. Furthermore, deep learning analyses of the spectra yielded accuracies greater than 96 percent in discriminating between the amino acids and the peptides.
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