Continuum xl ftir microscope

S-μFTIR was performed at the SMIS beamline at the synchrotron SOLEIL (L’Orme des Merisiers, 91192 Gif Sur Yvette Cedex, France). For microspectroscopy, the FACS-sorted cells were seeded onto clean 1-mm CaF₂ windows (Crystran Ltd., Dorset, UK) and left to adhere on the surface. After two days of growth, cells were fixed in 4% paraformaldehyde (PFA) solution (Solveco, Rosersberg, Sweden) for 20 min at room temperature, washed 3 × 10 min in PBS, and then rinsed for 10 min in sterile water.
Single point microscopy was performed using a ThermoFisher Scientific Continuum XL FTIR microscope with a 32× magnification, 0.65 NA Schwarzschild objective. FTIR spectra were acquired in transition mode with a spectral range between 4000 and 1000 cm⁻¹ at 4 cm⁻¹ spectral resolution with 8 µm × 8 µm aperture dimensions using 256 co-added scans. Background spectra were collected from a clean area of the same CaF₂ window. All measurements were made at room temperature.

For FTIR micro-spectroscopy analyses, fresh frozen mouse brain tissues containing human grafts were cut into 16 μm thick sections on a cryostat. Sections were mounted on the 1 mm thick CaF2 round 10 mm spectrophotometric windows. Infrared spectra were taken from RANDOM areas of the section at the SMIS beamline of the SOLEIL synchrotron (SMIS beamline, France) using a Thermo Fisher Scientific Continuum XL FTIR microscope through a 32 × magnification, 0.65 NA Schwarzschild objective. For the collection, parameters were at spectral range 1000–4000 cm⁻¹, in transmission mode at 4 cm⁻¹ spectral resolution, with 10 µm × 10 µm aperture dimensions, using 256 codded scans. Background spectra were collected from a clean area of the same CaF2 window. All measurements were made at room temperature. For analysis of FTIR spectra OPUS software (Bruker) and Orange (University of Ljubljana) were used and included atmospheric compensation. Derivation of the spectra to the second order using Savitsky-Golay of 3^rd polynomial order 3 with 9 smoothing points, was used to unmask the number of discriminative features and to eliminate a contribution of a baseline.

The peptide solution was processed and placed on clean BaF₂ glass. The FTIR spectra were recorded using a Nicolet 6700 FTIR spectrometer with a Continuum XL FTIR microscope (Thermo Fisher Scientific, United States) at a spectral resolution of 4 cm⁻¹. Infrared spectra were recorded between 1800 and 1200cm⁻¹. All resulting spectra were corrected for the blank background around sample absorption. Spectra were processed using the OMNIC 9.2 (Thermo Fisher Scientific, United States) for smoothing and normalization.