2. A 100Â air objective with numerical aperture of 0.9 (Nikon
Instruments, NY, USA).
3. A 532 nm Nd:YAG laser (WITec, Ulm, Germany) as the excitation source (see Note 1).
4. A handheld digital power meter (Thorlabs, New Jersey, USA)
for measurements of laser power.
5. A personal computer installed with Raman spectra and image
acquisition software (e.g., “WITec Control” from WITec).
6. Software for Raman spectra and image analysis (e.g., “Project
FOUR” from WITec).
2.2 TemperatureControlled Cooling
Stage and Sample
Handling
1. A temperature-controlled cooling stage consists of a four-stage
Peltier (Thermonamic Electronics Corp., Jiangxi, China) and a
series 800 temperature controller (Alpha Omega Instruments
Corp., Rhode Island, USA) (see Note 2).
2. A type T thermocouple (OMEGA Engineering, Connecticut,
USA) is used for measuring the temperature on the top surface
of the Peltier as it cools.
3. A copper heat sink is adhered to the bottom surface of the
Peltier using VIO thermal paste.
4. A wooden case is used for holding the heat sink. The wooden
case also serves as insulation between the heat sink and the
scanning stage. The Peltier, copper heat sink, and wooden case
are fixed on the motor-driven scanning stage with
masking tape.
5. A circulating bath (Thermo Fisher Scientific, Massachusetts,
USA) pumps 30% propylene glycol aqueous solution through
the copper heat sink to remove heat absorbed by the heat sink
from the Peltier cooling device.
6. A piece of 5 mm  5 mm mica sheet (Ted Pella, California,
USA) is used for covering the sample.
2.3 Setup
for Preventing
Condensation
on the Sample
1. Parafilm is used for wrapping around the objective along with
the cooling stage to generate an enclosed space.
2. Tubing is used for connecting a pressurized nitrogen gas cylinder to the enclosed space.
3. Nitrogen gas is used for purging the enclosed space to prevent
water condensation on the sample surface at low temperatures.
The whole experimental setup is shown schematically with a
zoomed-in view of the cooling stage (see Fig. 1).
2.4 Cell Culture
1. Jurkat cells (ATCC, TIB-1522).
2. Standard supplies for cell culture including a biosafety cabinet
and a CO 2 incubator.
Raman Cryomicroscopic Imaging
353
Instruments, NY, USA).
3. A 532 nm Nd:YAG laser (WITec, Ulm, Germany) as the excitation source (see Note 1).
4. A handheld digital power meter (Thorlabs, New Jersey, USA)
for measurements of laser power.
5. A personal computer installed with Raman spectra and image
acquisition software (e.g., “WITec Control” from WITec).
6. Software for Raman spectra and image analysis (e.g., “Project
FOUR” from WITec).
2.2 TemperatureControlled Cooling
Stage and Sample
Handling
1. A temperature-controlled cooling stage consists of a four-stage
Peltier (Thermonamic Electronics Corp., Jiangxi, China) and a
series 800 temperature controller (Alpha Omega Instruments
Corp., Rhode Island, USA) (see Note 2).
2. A type T thermocouple (OMEGA Engineering, Connecticut,
USA) is used for measuring the temperature on the top surface
of the Peltier as it cools.
3. A copper heat sink is adhered to the bottom surface of the
Peltier using VIO thermal paste.
4. A wooden case is used for holding the heat sink. The wooden
case also serves as insulation between the heat sink and the
scanning stage. The Peltier, copper heat sink, and wooden case
are fixed on the motor-driven scanning stage with
masking tape.
5. A circulating bath (Thermo Fisher Scientific, Massachusetts,
USA) pumps 30% propylene glycol aqueous solution through
the copper heat sink to remove heat absorbed by the heat sink
from the Peltier cooling device.
6. A piece of 5 mm  5 mm mica sheet (Ted Pella, California,
USA) is used for covering the sample.
2.3 Setup
for Preventing
Condensation
on the Sample
1. Parafilm is used for wrapping around the objective along with
the cooling stage to generate an enclosed space.
2. Tubing is used for connecting a pressurized nitrogen gas cylinder to the enclosed space.
3. Nitrogen gas is used for purging the enclosed space to prevent
water condensation on the sample surface at low temperatures.
The whole experimental setup is shown schematically with a
zoomed-in view of the cooling stage (see Fig. 1).
2.4 Cell Culture
1. Jurkat cells (ATCC, TIB-1522).
2. Standard supplies for cell culture including a biosafety cabinet
and a CO 2 incubator.
Raman Cryomicroscopic Imaging
353