Spotlight 400 system

5 µm thick FFPE breast cancer samples were sectioned onto IR-transparent Barium Fluoride salt plates. Sections were deparaffinated by washing in xylene for 24 hours, changing the solvent every three hours. FT-IR spectroscopic images of the tissue sections were collected using a Perkin Elmer Spotlight 400 system, equipped with 16 elements linear focal plane array HgCdTe detector. For each pixel, four scans were averaged at 4 cm⁻¹ spectral resolution and 6.25 µm pixel size.
Image processing was performed in ENVI-IDL 4.8 with in-built and in-house written algorithms^{10 (link),43 (link)}. Images were stitched from the image tiles, baseline corrected, and normalized. The infrared images were converted to spectral metrics by calculating peak heights, areas and locations and taking ratios of these variables. Using these metrics, a previously developed breast supervised classifier was applied to the IR data to identify areas classified as collagen²⁸ .

For infrared imaging, the different cell types were plated on a calcium fluoride substrate at 1.5 × 10⁴ cells/mL and allowed to adhere. After 24 to 48 h of cell culture, CaF₂ substrates were removed from the culture medium and washed three times with Dulbecco’s phosphate buffer saline (DPBS). Cell fixation was performed using 4% paraformaldehyde (PFA) for 30 min at room temperature. Cells were then rinsed with DPBS and distilled water to remove PFA, then air-dried.
Cells (n = 10 cells per cell line) were analyzed by FTIR imaging using the Spotlight 400 system (Perkin Elmer, Courtaboeuf, France). After visual imaging and selection of cells of interest, FTIR imaging was acquired in transmission mode at a spatial resolution of 6.25 µm/pixel, in the spectral range 4000–800 cm⁻¹ and a spectral resolution of 4 cm⁻¹. Each pixel spectrum corresponded to 128 co-additions.

The right femurs and third lumbar vertebrae (L3) were removed and embedded in polymethyl methacrylate (PMMA). Longitudinal sections of 3-μm thickness were prepared using a microtome (Aparicio et al., 2002 (link)). FTIR images of the longitudinal sections were acquired using an FTIR imaging system with a mercury‑cadmium-telluride linear array detector (Spotlight 400 system, PerkinElmer, Waltham, MA, USA) in transmittance mode with a frequency range of 4000–680 cm⁻¹, a resolution of 4 cm⁻¹, and a pixel size of 25 × 25 μm. The background spectrum was obtained through a barium fluoride window. Thirty FTIR spectra extracted from each trabecular and cortical bone in the image were obtained with baseline collection and PMMA spectral subtraction using Spectrum 10 software (PerkinElmer) and subsequently used to evaluate the PO₄³⁻/amide I and CO₃²⁻/PO₄³⁻ in the femur and L3. The PO₄³⁻/amide I was calculated by integrating the area of the phosphate band (PO₄³⁻, 1181–906 cm⁻¹) and dividing it by the area of the amide I band (1712–1609 cm⁻¹). The CO₃²⁻/PO₄³⁻ was calculated by integrating the area of the carbonate band (CO₃²⁻, 890–851 cm⁻¹) and dividing it by the area of the PO₄³⁻ band.