the device to the other side when a direct current flows through
the device.
3. Freezing responses of other cell types, such as mesenchymal
stem cells (MSCs) [1] and human-induced pluripotent stem
cells (hiPSCs) [13], have also been studied using
low-temperature Raman spectroscopy. Please refer to these
publications for more information.
4. Culture medium generally contains a small amount phenol red
as a pH indicator. Phenol red auto-fluoresces, and this phenomenon can exceed the magnitude of Raman signals
[23]. Phenol red-free medium is recommended for culturing
cells to be used in Raman imaging. If phenol red-free medium
is not available, cells need to be washed in phenol red-free
buffer solution, such as DPBS, several times to remove traces
of phenol red before they are introduced to the freezing
solution.
5. Circulating bath should always be turned on before switching
on the temperature controller. Otherwise, heat accumulated at
the bottom surface of the Peltier device can significantly decline
the cooling capability of the Peltier. Circulating bath should
also be turned off after shutting down the temperature controller. Otherwise, the Peltier device might overheat and cause
damage.
6. A very thin piece of mica sheet is used to cover the sample. The
mica sheet allows great transmission of photons scattered from
the sample and has little intrinsic Raman scattering to interfere
with spectra acquired of the sample. Thus air objectives instead
of water or oil immersion objectives are used. Water- or
oil-immersed objectives are not recommended for use at low
temperatures due to freezing and disruption of the temperature
field.
7. All samples need to be seeded at a specific ice nucleation
temperature using a liquid nitrogen-chilled needle. Otherwise,
ice nucleation in the sample will occur spontaneously at varying
temperatures. Seeding temperature affects the chemical gradient across the cell membrane, and intracellular ice formation is
more likely when lower seeding temperatures are used due to
decreased water permeability of the cell membrane [22].
8. A high flow rate of nitrogen gas can disturb the temperature
field of the sample, so low flow rates are recommended. Purging can only be performed after ice nucleation but before
further cooling of the sample to prevent deviation of the sample
temperature from the preset cooling profile.
9. If more than one cryoprotectant is used in the freezing solution, such as sucrose in combination with glycerol, it is likely
that Raman signals of different cryoprotectants and cells
Raman Cryomicroscopic Imaging
359
the device.
3. Freezing responses of other cell types, such as mesenchymal
stem cells (MSCs) [1] and human-induced pluripotent stem
cells (hiPSCs) [13], have also been studied using
low-temperature Raman spectroscopy. Please refer to these
publications for more information.
4. Culture medium generally contains a small amount phenol red
as a pH indicator. Phenol red auto-fluoresces, and this phenomenon can exceed the magnitude of Raman signals
[23]. Phenol red-free medium is recommended for culturing
cells to be used in Raman imaging. If phenol red-free medium
is not available, cells need to be washed in phenol red-free
buffer solution, such as DPBS, several times to remove traces
of phenol red before they are introduced to the freezing
solution.
5. Circulating bath should always be turned on before switching
on the temperature controller. Otherwise, heat accumulated at
the bottom surface of the Peltier device can significantly decline
the cooling capability of the Peltier. Circulating bath should
also be turned off after shutting down the temperature controller. Otherwise, the Peltier device might overheat and cause
damage.
6. A very thin piece of mica sheet is used to cover the sample. The
mica sheet allows great transmission of photons scattered from
the sample and has little intrinsic Raman scattering to interfere
with spectra acquired of the sample. Thus air objectives instead
of water or oil immersion objectives are used. Water- or
oil-immersed objectives are not recommended for use at low
temperatures due to freezing and disruption of the temperature
field.
7. All samples need to be seeded at a specific ice nucleation
temperature using a liquid nitrogen-chilled needle. Otherwise,
ice nucleation in the sample will occur spontaneously at varying
temperatures. Seeding temperature affects the chemical gradient across the cell membrane, and intracellular ice formation is
more likely when lower seeding temperatures are used due to
decreased water permeability of the cell membrane [22].
8. A high flow rate of nitrogen gas can disturb the temperature
field of the sample, so low flow rates are recommended. Purging can only be performed after ice nucleation but before
further cooling of the sample to prevent deviation of the sample
temperature from the preset cooling profile.
9. If more than one cryoprotectant is used in the freezing solution, such as sucrose in combination with glycerol, it is likely
that Raman signals of different cryoprotectants and cells
Raman Cryomicroscopic Imaging
359