Air objective na 0.90

Raman spectroscopy measurements were conducted using a Confocal Raman Microscope System Alpha 300R (WITec, Ulm, Germany) with a UHTS300 spectrometer and DV401 CCD detector with 600/mm grating. The WITec spectrometer was calibrated with a mercury-argon lamp. A Nd:YAG laser (532 nm wavelength) was used as an excitation source. A 100× air objective (NA 0.90; Nikon Instrument, Melville, NY) was used for focusing the 532 nm excitation laser to the sample. The laser at the objective was 10 mW, as measured by an optical power meter (Thorlabs, Newton, NJ). The lateral resolution of the microscope was about 296 nm according to the Abbe’s diffraction formula. Cell samples were frozen using a four-stage Peltier (Thermonamic Electronics Corp, Jiangxi, China) and a series 800 temperature controller (Alpha Omega Instruments Corp, Lincoln, RI). About 1 to 3 μl of cell suspension was placed on the stage, covered with a piece of mica (TED PELLA, Redding, CA), and sealed with Kapton tape (Dupont, Wilmington, DE) to prevent sample evaporation/sublimation.
The temperature of the cooling stage was maintained at 1°C, and it took several seconds to cool the sample from 1°C to the seeding temperature of −6°C, at which point ice was nucleated in samples using a liquid nitrogen cooled needle, cooled at 1°C/min to a holding temperature of −50°C and held for 20 min before imaging.