Methods and Applications of Raman Microspectroscopy to Single-Cell Analysis

Abstract

Raman spectroscopy is a powerful biochemical analysis technique that allows for the dynamic characterization and imaging of living biological cells in the absence of fluorescent stains. In this review, we summarize some of the most recent developments in the noninvasive biochemical characterization of single cells by spontaneous Raman scattering. Different instrumentation strategies utilizing confocal detection optics, multispot, and line illumination have been developed to improve the speed and sensitivity of the analysis of single cells by Raman spectroscopy. To analyze and visualize the large data sets obtained during such experiments, sophisticated multivariate statistical analysis tools are necessary to reduce the data and extract components of interest. We highlight the most recent applications of single cell analysis by Raman spectroscopy and their biomedical implications that have enabled the noninvasive characterization of specific metabolic states of eukaryotic cells, the identification and characterization of stem cells, and the rapid identification of bacterial cells. We conclude the article with a brief look into the future of this rapidly evolving research area.

Label‐free live cell imaging by Confocal Raman Microscopy identifies CHO host and producer cell lines

“…In order to picture different parts of single cells and identify protein accumulation in producer cell lines, we performed a k‐means cluster analysis 5, 34. Thereby, areas in the cells were visualized by false colors attributed to different characteristic bands that correspond to protein‐rich (putative endoplasmatic reticulum (ER) in red) or protein‐poorer (”rest of the cell“, RC in blue) regions.…”

“…bond stretching). This energy shift is measured and the resulting Raman spectrum thus provides a ”molecular fingerprint“ with information about specific molecular bonds and their corresponding micro‐environment 4, 5. It has been widely used in the identification of chemical compounds in areas ranging from basic research (e.g.…”

“…The strength of Raman microscopy lies in the combination of high lateral resolution of about 300 nm (NA 1.4, λ ex 532 nm) gained from the optical microscope and the sensitive chemical information derived from the Raman spectra 5, 16. The resulting Raman image is composed of thousands of spectra where each pixel represents the chemical profile for all Raman‐active components simultaneously.…”

Design and first applications of a flexible Raman micro-spectroscopic system for biological imaging

“…This focus is optically conjugated with the spectrometer slit, so that each line of the CCD camera sensor acquires an independent spectrum from an individual sample spot. In this way fast hyperspectral Raman imaging is performed by scanning the sample in one lateral direction only [43]. With the CCD camera used in our system it is possible to record up to 400 spectra simultaneously.…”

Rapid 3D fluorescence imaging of individual optically trapped living immune cells

“…Because OTs require only focused light, they can be implemented through sealed chambers, and, for near-infrared wavelengths, they have also been shown to be non-damaging to numerous cell types [9,10]. In addition to immobilizing small objects, OTs have been used to measure biomechanical and chemical properties, to perform highly sensitive force spectroscopy on molecules and cells, and to initiate biological interactions such as those between host and pathogens [11][12][13][14][15][16][17].…”