Effect of glycerol on photobleaching of cytochrome Raman lines in frozen yeast cells

Abstract

We applied a Raman spectroscopy approach to investigate the effect of a cryoprotectant on the redox state of cytochromes on freezing yeast cells. The redox activity of cytochromes was studied using time-resolved photobleaching of the resonance Raman lines. It is found that ice formation causes a drastic change in the redox state of cytochromes in cells frozen without cryoprotectant, whereas in the presence of glycerol the effects of ice formation are more gradual. The photobleaching rate of cells frozen in glycerol solution shows a gradual slowing with temperature decrease and an abrupt slowdown below - 48 °C. This abrupt decrease was interpreted as originating from changes in protein conformational dynamics. Our findings provide important new insights into the transition from active to inactive cytochrome states as cells undergo freezing in the presence and absence of cryoprotectant.

Raman microscopy of cryofixed biological specimens for high-resolution and high-sensitivity chemical imaging

“…The combination of cryofixation and subsequent low-temperature measurement of biological specimens has overcome significant limitations in Raman microscopy, realizing a high SNR and a large FOV as well as high spatial and spectral resolution. The technique significantly reduces photodamage and motion artifacts, allowing the visualization of intracellular molecules and subcellular structures invisible in the measurement without freezing, while also enhancing quantification capabilities of Raman imaging [46], [47]. The cryofixation of chemical states, such as redox states, can further extend the advantage of Raman imaging in its capability of sensing molecular states and their environments.…”