High-throughput vibrational cytometry based on nonlinear Raman microspectroscopy

摘要

Flow cytometry is a technology that allows a single cell or particle to be measured for a variety of characteristics, determined by looking at their properties while they flow in a liquid stream. High speed of flow and huge number of objects to be analyzed imposed some strict criteria on which methods can be used for analysis. All the known commercial instruments are currently using light scattering for particle sizing and fluorescence detection for chemical recognition. However, vibrational spectroscopy is the only non-invasive optical spectroscopy tool, which has proven to provide chemically-specific information about the interrogated sample. It is proposed that vibrational spectroscopy, based on nonlinear Raman scattering can be used to serve as an analytical tool for cytometry by providing rapid and accurate chemical recognition of flowing materials. To achieve a desired speed (> 10,000 cell/particles per second), we have substantially upgraded our previous system for nonlinear Raman microspectroscopy. By increasing the size of the excitation volume to the size of a cell and by keeping the incident intensity at the same level, a dramatic increase of the nonlinear Raman signal is achieved. This allows high-quality vibrational spectra to be acquired within 10-100 microsecond from a single yeast cell without any observable damage to the irradiated cell. This is four orders of magnitude better than any previous attempts involving Raman microspectroscopy.