Invia qontor raman microscope

Raman spectra and maps were acquired using a Renishaw InVia Qontor Raman microscope. The Raman microscope was calibrated to the 520 cm⁻¹ mode from a silicon wafer prior to measurements each day. The excitation laser power was measured and adjusted to prevent differences in the laser power throughout the course of experiments. Raman measurements were made with a 532 nm laser, 1800 l/mm grating, and 0.1 second acquisitions. The measurements were made with either high (10 mW) or moderate (1 mW) laser power.
Raman maps of cells were acquired by point mapping in 1 micrometer steps in a x-y raster pattern, taking ~1–2 minutes per map. Volume maps were acquired with the same x-y resolution and a 2-micrometer z step. A 63x, 0.9 N.A. water dipping objective (Leica) was used. Dark field images were acquired in transmission using an oil dark field condenser (Olympus, U-DCW) and Type-F immersion oil (Olympus).
Data was exported from the Raman instrument’s WiRe software, converted into MATLAB files, and a publicly available peak fitting code for MATLAB³¹ was used to fit Raman peaks that did not overlap with background or other analytes. Peaks fit in the calibration experiments were fit in the cell maps to predict concentrations of lycopene and tween from point to point across the cell map.

Raman measurements were obtained on two different Raman spectrometers. A custom Snowy Range IM-52 spectrometer with a 638 nm laser was used in calibration experiments. A Renishaw InVia Qontor Raman microscope with three excitation lasers (532 nm, 633 nm, and 785 nm) was used for Raman maps of cellular samples. A 63x dipping objective was used (LEICA 63x catalog # 506148​) for cell imaging experiments. Cell maps were acquired the same day, and the Renishaw spectrometer was calibrated with a silicon reference. There was no visible evidence of cell damage in the maps used (other maps with different acquisition parameters had resulted in visible rupture of cells). Raman maps were collected in the spectral range between 719.12 and 1816.9 cm⁻¹ using a 1200 l/mm grating and a single 10 s accumulation per pixel. Confocal images were acquired with 5 mW power at the sample. Linefocus images were acquired with 10 mW power at the sample.