Raman spectroscopy has been gaining in popularity for noninvasive analysis of single cells. Raman spectra and images deliver meaningful information regarding the biochemical, biophysical, and structural properties of cells in various states. Low-temperature Raman spectroscopy has been applied to verify the presence of ice inside a frozen cell and to illustrate the distribution of both penetrating and non-penetrating cryoprotectants. This chapter delineates Raman cryomicroscopic imaging of single cells as well as sample handling for spectroscopic measurements at subzero temperature. The experimental setup is depicted with a special emphasis on a custom-built temperature-controlled cooling stage. The use of Raman cryomicroscopic imaging is demonstrated using Jurkat cells cryopreserved in a sucrose solution. Moreover, strategies for determining intracellular ice formation (IIF) and analysis of sucrose partitioning across the cell membrane are presented.
|Original language||English (US)|
|Title of host publication||Methods in Molecular Biology|
|Publisher||Humana Press Inc.|
|Number of pages||11|
|State||Published - 2021|
|Name||Methods in Molecular Biology|
Bibliographical noteFunding Information:
This work was partially supported by funding from the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under award number R01EB023880. Raman spectroscopy in this demonstration was carried out in the Characterization Facility, University of Minnesota, which receives partial support from the National Science Foundation through the MRSEC program.
- Hyperspectral imaging
- Low-temperature microscopy
- Raman spectroscopy