Hyperion 3000

Spectra were recorded in transmission mode on a Bruker Tensor 27 spectrometer equipped with a Hyperion 3000 microscopy accessory and a liquid N₂ cooled 64 × 64 mercury cadmium telluride (MCT) focal plane array (FPA) detector. The entire setup was placed on a vibration-proof table. Spectra were recorded in the region 950–4000 cm⁻¹, with 4 cm⁻¹ spectral resolution and 32 scans co-added in double sided, forward-backward mode. FPA frame rate was 3773 Hz and integration time 0.104 ms, with offset and gain optimized for each sample between 180–230 and 0–1, respectively. Background was recorded on a clean, empty spot on the CaF₂ carrier and automatically subtracted. Fourier transformation was carried out using a zero filling factor of 2 and Blackman-Harris 3-term apodization function. Phase correction was set to the built-in Power mode with no peak search and a phase resolution of 32. Spectra were recorded using OPUS (version 6.5 and 7, Bruker Optics GmbH, Ettlingen. Germany), cut to the fingerprint region of 950–2000 cm⁻¹ and exported as.mat files for subsequent multivariate image analysis. White light images were recorded with a Sony ExwaveHAD color digital video camera mounted on the top of the microscope and exported as jpg files. Spectra used to construct the models are provided as Supplementary Dataset 1.

Immobilized films of recombinant His₆-tagged ERK2^309–357 (His₆-EDI), purified from BL21 by His-tag affinity purification^{13 (link)}, were prepared by immobilization of the His₆-EDI from a 3.8 mg/mL solution in binding-buffer on gold substrates (Ssens, Netherlands). After immobilization, the samples were rinsed with deionized water. IR Microscopy was performed using a Bruker Hyperion 3000 FTIR microscope with a Grazing Angle Objective at spectral resolutions of 4 cm⁻¹ in dry-air purged environment. A photovoltaic mercury cadmium telluride detector served for maximum linearity of the detected IR signals. AFM-IR was performed using a commercially available AFM-IR setup by Anasys Instruments (nanoIR2-FS) equipped with a tunable p-polarized MIR quantum cascade laser (QCL) by Daylight Solutions (MIRcat) with a spectral resolution of 1 cm⁻¹ at 20 cm⁻¹ per second sweep rate in ambient conditions. Grazing angle IR (GIR) spectrum is displayed reversed and baseline-corrected.

The samples were fabricated in the ISO7 cleanroom at UPNA facilities evaporating Ti on 4H-SiC wafer with an Angstrom Engineering e-Beam evaporator. The six samples are shown in the Fig. 5a. Normal incidence measurements of the samples reflection were performed via Fourier Transform Infrared Spectroscopy (FTIR) with a microscope Hyperion 3000, and the oblique incidence measurements with the coupled accessory A513/Q in the Bruker Vertex 80v spectrometer.