Lasercheck

The Raman spectra were measured by a Renishaw in Via Raman spectrometer (controlled by WiRE 3.4 software, Renishaw, UK) connected to a Leica microscope (Leica DMLM, Leica Microsystems, Buffalo Grove, IL, USA), equipped with a 785 nm near-IR laser that was focused through a 63 × NA = 0.90 water immersion objective (Leica Microsystems, USA). The laser intensity before and after travelling through the 50× objective was 110 mW and 29.4 mW, respectively (measured with LaserCheck, Coherent Inc., Portland, OR, USA). The standard calibration peak for the spectrometer with silicon mode at a static spectrum was 520.5 ± 0.1 cm^-1. Cells cultured with blank media, or media with G-CSF for 10 days and 7 weeks were seeded on MgF2 (United Crystals Co., Port Washington, NY, USA) for 24h prior to Raman spectra collection. The exposure time was 10 s for one accumulation at 100% laser power for all of the cell samples. A total of 50 spectra (randomly 5 spots for 5 spectra per cell) were acquired for each group in the fingerprint region (600 −1800 cm⁻¹) which has been proven effective in evaluation of molecular changes of cancer cells ^{13 (link), 21 (link)–24 (link)}

The RR measurements were carried out as previously described.^{1 (link)} Briefly, the 413.1 nm excitation from a Kr ion laser (Spectra-Physics, Mountain View, CA) was focused to an ~30 μm spot on the spinning quartz cell rotating at ~1000 rpm. The scattered light, collected at a right angle to the incident laser beam, was focused on the 100 μm wide entrance slit of a 1.25 m Spex spectrometer equipped with a 1200 grooves/mm grating (Bausch & Lomb, Analytical Systems Division, Rochester, NY), where it was dispersed and then detected by a liquid nitrogen-cooled CCD detector (Princeton Instruments, Trenton, NJ). A holographic notch filter (Kaiser Optical Systems, Ann Arbor, MI) was used to remove the laser line. The Raman shifts were calibrated with indene. The laser power was adjusted by neutral density filters, and the power at the sample point was measured by a handheld laser power meter (LaserCheck, Coherent Inc., Santa Clara, CA).
The IR spectra were recorded at 4 cm⁻¹ resolution on an FTIR instrument (Magna-IR 560, Nicolet, Madison, MI) with a CaF₂ IR cell (200 μm path). For the IR dark–light experiments, the 413.1 nm laser beam was introduced into one end of an optical fiber, and the IR cell surface (~1 cm diameter) was illuminated by the laser beam dispersed from the other end of the optical fiber.