Nicolet continuum microscope

Synchrotron radiation Micro-Fourier Transform Infra-red Analysis (Micro-FTIR) was carried out at the IRIS beamline (BESSY II) at Helmholtz-Zentrum, Berlin (Germany) on a Thermo Nicolet Continuum™ microscope equipped with a mercury–cadmium–telluride (MCT) detector. The sample was mounted on a diamond cell on a motorized microscope stage and raster scanned through the synchrotron beam with a diameter of 15 µm collecting a grid-like pattern of IR spectra spaced in 10 μm increments. Measurements were performed in transmission mode at a magnification ×32 using confocal objectives. Infrared spectra were registered between 4000 and 650 cm⁻¹ with a spectral resolution of 4 cm⁻¹. An accumulation of 128 scans per point was used. Background spectra were collected under identical conditions with only the BaF₂ window on which the sections were placed. The spectrum and mapping acquisition was performed by using the OMNIC Atlμs™ software (Waltham, MA, USA) [42 (link)]. The sample preparation protocol consisted of the microtoming at 10 µm of the samples with an embedding-free approach as described in the literature [43 (link)].

Optical reflectance of the NOAAD array is measured using FTIR micro spectroscopy system configured using a Thermo Scientific Nicolet Continuum microscope and a Thermo Scientific Nicolet 8700 spectrometer. The spectrometer utilizes a CaF2 beamsplitter, while the microscope is equipped with an internal MCT-a detector to increase signal to noise ratio.

Samples collected from the gradient tubes were deposited on a CaF₂ plate and rinsed three times with deionized water. Fourier transform infrared spectromicroscopy (FTIR) analyses were performed using a Thermo Nicolet Continuum microscope linked to a Nexus 670 FTIR spectrometer. The infrared beam was collimated by a 100 × 100 μm window and focused on the samples. Infrared absorbance spectra were collected on a liquid nitrogen-cooled MCTA detector to minimize electronic noise and water absorption in the detector. Spectra were acquired in transmitted mode between 4,000 and 400 cm⁻¹, and each spectrum obtained represents an integration of 150 spectral scans, with a wavenumber resolution of 4 cm⁻¹. Background corrections were applied to the data following each measurement to compensate for instrumental noise and contributions from atmospheric CO₂ and H₂O by dividing the absorbance of the sample spectrum by the background spectrum at each data point. The spectrum of an agarose reference powder (Fisher Scientific) was acquired using the same method.