Fourier-Transform Infrared microspectroscopy (microFT-IR) is nowadays considered a valuable tool for investigating biochemical changes occurring in cells during the interaction with external agents . In particular, microFT-IR has been usefully applied in the analysis of the complex biological processes occurring during X-ray radiation-cell interaction [2, 3].
Different experimental approaches are available for FT-IR spectra collection (transmission, attenuated total reflection (ATR) and transflection modes) on cells samples. Transflection-mode FTIR spectroscopy has become particularly used for this kind of samples due to the relatively low cost of substrates compared to transmission windows, and a higher absorbance due to a double pass through the same sample approximately doubling the effective path length. Recently, some questions have been raised about the role of transmitted and reflected components of the infrared beam in transflection mode [4,5].
For this reason, we investigated two different transreflection approaches for collecting spectra from cells samples exposed to X-ray. In the former approach, cells were grown on MirrIR slides (25x25 mm2) (Kevley Technologies, Chesterland, Ohio), a specific reflection FT-IR spectroscopy microscope slide; for the second approach cell pellets were prepared.
In both cases, SH-SY5Y neuroblastoma (American Type Culture Collection, Manassas, VA, USA) cells were used. X-ray exposure was performed at room temperature using a Gilardoni MGL 200/8D machine operating at 250 kVp and 6 mA (dose rate 60 cGy / min) at doses of 2 and 4 Gy.
After X-ray exposure, the cells grown on MirrIR slides were fixed in a 3.7% formaldehyde PBS solution for 20 min at room temperature, and, then, briefly washed in distilled water for 3 s to remove the residue PBS from the surface of the cells. Subsequently, the samples were dried under ambient conditions and stored in a desiccator until analysis. To obtain cell pellets, X-ray irradiated cells were centrifuged for 8 minutes at 1,500 rpm. The supernatant was aspirated and the pellet resuspended in 300 μl of NaCl 0.9% until spectra acquisition.
For the acquisition of the IR absorption spectra a Spectrum One FTIR (PerkinElmer, Shelton, CT, USA) spectrometer, equipped with a Perkin Elmer Multiscope system infrared microscope and an MCT (mercury cadmium telluride) detector was used.
Significant spectra were obtained by using both the approaches in the 4000 - 600 cm-1 spectral range from exposed and not-exposed samples. The main contribution from proteins, lipids, carbohydrates and DNA were clearly evidenced and assigned. Particular care has paid in considering how the transmission and reflection infrared beam component can affect the obtained spectra.
The results of this investigation can be particularly useful in evaluating the effective reliability of low-cost metallic substrates that can really contribute to significantly spread the use of microFT-IR.
 Baker MJ, Trevisan J, Bassan P, Bhargava R, Butler HJ, Dorling KM, Fielden PR, Fogarty SW, Fullwood NJ, Heys KA, Hughes C, Lasch P, Martin-Hirsch PL, Obinaju B, Sockalingum GD, Sulé-Suso J, Strong RJ, Walsh MJ, Wood BR, Gardner P, Martin FL. 2014. Using Fourier transform IR spectroscopy to analyze biological material. Nat Protoc. 9:1771–1791.
 Gault N, Lefaix JL. 2003. Infrared microspectroscopic characteristics of radiation-induced apoptosis in human lymphocytes. Radiation Research. 160:238-250.
 Meade A, Clarke C, Byrne H, Lyng F. 2010. Fourier transform infrared microspectroscopy and multivariate methods for radiobiological dosimetry. Radiation Research. 173:225-237.
 Bassan P, Lee J, Sachdeva A, Pissardini J, Dorling KM, Fletcher JS, Henderson A, Gardner P. 2013 The inherent problem of transflection-mode infrared spectroscopic microscopy and the ramifications for biomedical single point and imaging applications. Analyst. 138: 144-157
 Mayerhöfer TG, Pahlow S, Hübner U, Popp J. 2018 Removing interference-based effects from the infrared transflectance spectra of thin films on metallic substrates: a fast and wave optics conform solution. Analyst. 143: 3164-3175