Spotlight 400 microscope

Fourier transform mid-infrared imaging was used for collecting spectra with transflection optical setup. The feasibility of this approach has been tested on the separation of ethanol fixed cell lines, this has been reported previously [31 (link)]. Spotlight 400 microscope (Perkin Elmer Inc., Waltham, MA, United States) was connected to Spectrum 400 spectrophotometer used for scanning images. The Mercury Cadmium Tellurite (MCT) detector collected spectra with 32 scans with a resolution of 16 cm⁻¹ and data interval of 8 cm⁻¹ were recorded for each spectrum in the mid-infrared wavelength range between 4000 and 648 nm. The 250 μm × 600 μm images were scanned (Figure 1) by pixel size 6.25 μm × 6.25 μm. A single image contained 40 × 96 pixels and resulted in 3,840 spectra on a 0.15 mm² area. The acquisition time for each selected area was 46 min.

The wavelength range of NIR is defined from 780 to 2500 nm (12820–4000 cm⁻¹ – since due to the dispersed Fourier-transform (FT) spectrophotometers the wavenumber is typically measured in units of cm⁻¹), the wavelengths of MIR are between 2500 nm and 25,000 nm (4000–400 cm⁻¹) and the FIR range is between 25 and 1000 μm (400–10 cm⁻¹). The higher the wavenumber, the higher the energy of the light. NIR and MIR photons elevate the chemical bonds to higher energy level, causing deformation motions (e.g. angular changes).
Fourier transform mid-infrared imaging was used for collecting spectra with transflection optical setup. Spotlight 400 microscope (Perkin Elmer Inc., Waltham, Massachusetts, USA) was connected to Spectrum 400 spectrophotometer used for scanning images. The Mercury Cadmium Tellurite (MCT) detector collected spectra with 4000–648 cm⁻¹ wavelength range using step 4 cm⁻¹, resolution 8 cm⁻¹. The 300 μm × 550 μm images were scanned by pixel size 6.25 μm × 6.25 μm and 32 scans per pixel. A single image contained 48 × 88 pixels and resulted 4224 spectra. The same size areas were selected to image the three cell lines attached on three separate slides.