While the use of fluorescent probes the study the spatial distribution of a target molecule within a cell to determine cellular dynamics at the molecular level, the presence of the probe itself may alter the physical and chemical conditions within the cells. This latest research, published in the journal Analytical Chemistry by researchers from the University of Tokyo in Japan, uses a novel coherent anti-stokes Raman scattering (CARS) microspectroscopy technique to enable unstained imaging of live cells. The coherent anti-stokes Raman scattering (CARS) phenomenon was originally observed in the 1965 at the Ford Motor company during a "three wave mixing experiment" to investigate the third order vibrational signatures of molecules much like in Raman vibrational spectroscopy. CARS uses light in the visible and near-infrared regions to probe molecular resonances that would normally be in the mid- to far-infrared region which are readily absorbed by water making the analysis of biological samples difficult. The technique uses two overlapped laser beams whose frequencies are separated by the resonance frequency of interest. When a 'resonant' molecule is hit by these intense beams it will emit an 'anti-Stokes' photon. By determining the number of photons released, the amount of resonant molecules in the illuminated area can be calculated. One of the major limitations to the use of the technique has been the spectral bandwidth of the lasers available to excite the bonds, however the researchers employed a supercontinuum light source that enabled imaging over a range of 3500cm-1. This light source makes it possible not only to image the fingerprint region but also the C-H, N-H and O-H stretching regions. In addition, to this multiplex CARS imaging, two-photon excited fluorescence (TPEF) can also be measured to enable multiple nonlinear spectral imaging if a fluorescent label is added to the system. This nonlinear spectral imaging has been used to study not only the distribution of molecular species within a cell, but also the cell activity of growing and dying cells. They found that they could they could gain three dimensional images of cells at high speed and could visualise septum and membranous organelles by following their C-H stretches. In addition, by incorporating a fluorescence label such as green fluorescent protein to the mitochondria the TPEF image could be used to differentiate the known, labelled mitochondria from other organelles. The researchers discovered a strong Raman band in the mitochondria of the yeast cells that sharply reflects the metabolic activity of the mitochondria, which the researchers have dubbed the "Raman spectroscopic signature of life". This method could be particularly useful for studying cancer cells in vitro and their response to drugs as not only could the cell life cycle be followed, but also for monitoring the metabolic activity of the mitochondria which is suppressed in cancer cells. Research published in the journal Cancer Cell earlier this year by researchers from the University of Alberta (UA) in Canada, showed that mitochondrial metabolism changes in cancer cells could be targeted and promote apoptosis and inhibit cancer growth. "Cancer cells actively suppress their mitochondria, which alters their metabolism, and this appears to offer cancer cells a significant advantage in growth compared to normal cells, as well as protection from many standard chemotherapies," said Professor Evangelos Michelakis of UA and lead author of the report.
Japanese researchers have used an advanced microspectroscopy technique to study the cell cycle of yeast without the need for fluorescent imaging agents that can potentially alter the cellular environment.