2. Turn on the excitation laser and adjust it to the desired power
(10 mW) using the digital power meter. Use a piece of silicon
wafer to check the alignment of the optics of Raman microscopy, as the silicon wafer should always yield consistent Raman
signal intensity at the same laser power. Fine-tune the alignment of the optics if Raman signal intensity of the silicon wafer
is lower than usual.
3. Turn on the circulating bath to cool down the propylene glycol
solution to its desired temperature in advance. Connect the
circulating bath to the heat sink to circulate the solution and
cool down the heat sink (see Note 5).
4. After incubation of cells in the freezing solution has been
completed, place a drop of experimental cell suspension
(1–3 μL) on the top surface of the cooling stage using a pipette
or syringe. Gently cover the suspension with a piece of mica (see
Note 6) in order to prevent evaporation/sublimation of the
sample during each experiment.
5. Cool the sample to the seeding temperature at a desired cooling rate using the temperature controller; after which, use a
liquid nitrogen-chilled needle to induce ice nucleation of the
sample by touching the edge of the sample (see Note 7). Please
refer to the instruction manual of temperature controller on
temperature ramp settings.
6. Wrap Parafilm around the objective and cooling stage to build
an enclosed space. Use Scotch tape to seal off all the edges of
Parafilm.
7. Use a length of tubing to feed nitrogen gas from the pressurized cylinder into the enclosed space. Open the valve of nitrogen gas cylinder and purge sufficient amount of nitrogen gas
into the enclosed space to prevent water condensation and
crystallization on the sample surface and objective during further cooling of the sample (see Note 8).
8. After purging is finished, close the valve of nitrogen gas cylinder. Continue cooling the sample from the seeding temperature to a final temperature of À50
C at a preset cooling rate
using the temperature controller.
9. Initiate Raman imaging of cells after the sample is in equilibrium with the surroundings. The sample may need 5–10 min to
be physically stable after cooling process is accomplished.
3.2 Raman Imaging
of a Single Cell
1. Raman images are generated by integrating Raman spectra at
each pixel based on characteristic wave numbers of common
intracellular and extracellular materials. Raman signal of amide
I is used to illustrate the distribution of protein and lipid to
delineate the size and shape of frozen cells. Images of ice are
generated from the Raman OH stretching band with
Raman Cryomicroscopic Imaging
355
(10 mW) using the digital power meter. Use a piece of silicon
wafer to check the alignment of the optics of Raman microscopy, as the silicon wafer should always yield consistent Raman
signal intensity at the same laser power. Fine-tune the alignment of the optics if Raman signal intensity of the silicon wafer
is lower than usual.
3. Turn on the circulating bath to cool down the propylene glycol
solution to its desired temperature in advance. Connect the
circulating bath to the heat sink to circulate the solution and
cool down the heat sink (see Note 5).
4. After incubation of cells in the freezing solution has been
completed, place a drop of experimental cell suspension
(1–3 μL) on the top surface of the cooling stage using a pipette
or syringe. Gently cover the suspension with a piece of mica (see
Note 6) in order to prevent evaporation/sublimation of the
sample during each experiment.
5. Cool the sample to the seeding temperature at a desired cooling rate using the temperature controller; after which, use a
liquid nitrogen-chilled needle to induce ice nucleation of the
sample by touching the edge of the sample (see Note 7). Please
refer to the instruction manual of temperature controller on
temperature ramp settings.
6. Wrap Parafilm around the objective and cooling stage to build
an enclosed space. Use Scotch tape to seal off all the edges of
Parafilm.
7. Use a length of tubing to feed nitrogen gas from the pressurized cylinder into the enclosed space. Open the valve of nitrogen gas cylinder and purge sufficient amount of nitrogen gas
into the enclosed space to prevent water condensation and
crystallization on the sample surface and objective during further cooling of the sample (see Note 8).
8. After purging is finished, close the valve of nitrogen gas cylinder. Continue cooling the sample from the seeding temperature to a final temperature of À50
C at a preset cooling rate
using the temperature controller.
9. Initiate Raman imaging of cells after the sample is in equilibrium with the surroundings. The sample may need 5–10 min to
be physically stable after cooling process is accomplished.
3.2 Raman Imaging
of a Single Cell
1. Raman images are generated by integrating Raman spectra at
each pixel based on characteristic wave numbers of common
intracellular and extracellular materials. Raman signal of amide
I is used to illustrate the distribution of protein and lipid to
delineate the size and shape of frozen cells. Images of ice are
generated from the Raman OH stretching band with
Raman Cryomicroscopic Imaging
355