Chapter 14
Raman Cryomicroscopic Imaging and Sample Holder
for Spectroscopic Subzero Temperature Measurements
Guanglin Yu, Rui Li, and Allison Hubel
Abstract
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.
Key words Raman spectroscopy, Hyperspectral imaging, Cryopreservation, Cell, Low-temperature
microscopy
1 Introduction
Current theories of cell freezing damage were developed in the
1960s. Darkening of the cell observed by conventional cryomicroscopy has been associated with formation of ice crystals inside the
cell, which leads to cell death [1]. The fundamental knowledge of
the mechanisms of cell freezing damage has not changed much in
the 50-plus years. However, the tools available to interrogate cell
freezing responses have greatly evolved. A previous study in our lab
has utilized low-temperature Raman spectroscopy to verify the
presence of ice inside a frozen cell and to illustrate the partitioning
of dimethyl sulfoxide (DMSO) across the cell membrane at low
temperatures [2]. The high spatial resolution of Raman microscopy
also allows for investigation of the freezing damage imposed to
subcellular structures such as mitochondria [3], granting a better
understanding of the mechanism of cell damage during freezing.
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_14, © Springer Science+Business Media, LLC, part of Springer Nature 2021
351
Raman Cryomicroscopic Imaging and Sample Holder
for Spectroscopic Subzero Temperature Measurements
Guanglin Yu, Rui Li, and Allison Hubel
Abstract
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.
Key words Raman spectroscopy, Hyperspectral imaging, Cryopreservation, Cell, Low-temperature
microscopy
1 Introduction
Current theories of cell freezing damage were developed in the
1960s. Darkening of the cell observed by conventional cryomicroscopy has been associated with formation of ice crystals inside the
cell, which leads to cell death [1]. The fundamental knowledge of
the mechanisms of cell freezing damage has not changed much in
the 50-plus years. However, the tools available to interrogate cell
freezing responses have greatly evolved. A previous study in our lab
has utilized low-temperature Raman spectroscopy to verify the
presence of ice inside a frozen cell and to illustrate the partitioning
of dimethyl sulfoxide (DMSO) across the cell membrane at low
temperatures [2]. The high spatial resolution of Raman microscopy
also allows for investigation of the freezing damage imposed to
subcellular structures such as mitochondria [3], granting a better
understanding of the mechanism of cell damage during freezing.
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_14, © Springer Science+Business Media, LLC, part of Springer Nature 2021
351