Thermo nicolet nexus ftir spectrometer

Fixed cells analyses in FTIR-ATR were carried out in a Thermo Nicolet Nexus 670 FTIR spectrometer (Thermo Scientific, Madison, WT, USA). Fixed cells (300,000 to 350,000 cells) were dried directly in the Ge crystal of the Attenuated Total Reflectance (ATR) for 20 to 30 min. Spectra were recorded in the range of 4000 to 800 cm⁻¹ with a maximum resolution of 6 cm⁻¹, and 150 scans per spectrum were collected. Five spectra were recorded for each treatment population.
Fixed cells were observed using the FTIRM end station in the line ID-21 at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France, using a Thermo Nicolet Continuum (Thermo Scientific, Madison, WT, USA) microscope coupled to a Thermo Nicolet Nexus FTIR spectrometer (Thermo Scientific, Madison, WT, USA). The IR microscope was equipped with a 32x objective, a motorized sample stage, and a liquid nitrogen-cooled 50 μm mercury cadmium telluride detector. Fixed cells were mounted in BaF₂ windows of 1 mm height. Spectra were recorded over the range of 4000 to 850 cm⁻¹, the spectral resolution was set to 6 cm⁻¹, and 120 scans per spectrum were collected. Spectra of 30 individual cells were recorded for each treatment.

FTIR spectra in transmission mode were collected from 4000 to 400 cm⁻¹ using the KBr pellet technique on a Thermo Nicolet Nexus FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed with Thermo Scientific Omnic Software ver. 8.3.

FTIR microscopy analyses were performed at beamline ID21 at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France^{44 (link)}. The beamline is equipped with a Thermo Nicolet Continuum (Thermo Scientific, Madison, WT, USA) microscope coupled to a Thermo Nicolet Nexus FTIR spectrometer (Thermo Scientific, Madison, WT, USA) with a 32x objective, a motorized sample stage, and a liquid nitrogen-cooled 50 µm HgCdTe detector. Maps were acquired in transmission mode using a 10 × 10 µm² beam, step size of 8 µm. Spectra were recorded as an average of 64 scans per spectrum, over a range of 4000 to 850 cm⁻¹ and with a spectral resolution of 4 cm⁻¹.
The OMNIC software was used to transform spectra from maps of skin and lymph node samples to second derivatives using Savitsky-Golay of second polynomial order with 21 smoothing points^{45 (link), 46 (link)}. Unscrambler X software (Version 10.3, CAMO Software, Oslo, Norway) was used for further statistical analysis. Principal component analysis (PCA) was performed on the mean-centered data using the spectral regions from: 1800 to 1350 cm⁻¹ (related to proteins) and 3200 to 2800 cm⁻¹ (related to lipids)^{47 (link), 48 (link)}. PCA was performed separately for skin and lymph node samples. Score plots and loading plots obtained by PCA analysis as well as mean values from the regions of interest were used for data interpretation.