Original Paper

Analytical and Bioanalytical Chemistry

, Volume 405, Issue 4, pp 1311-1324

First online:

Open Access This content is freely available online to anyone, anywhere at any time.

The effect of optical substrates on micro-FTIR analysis of single mammalian cells

  • Katia WehbeAffiliated withDiamond Light Source, Harwell Science and Innovation Campus Email author 
  • , Jacob FilikAffiliated withDiamond Light Source, Harwell Science and Innovation Campus
  • , Mark D. FrogleyAffiliated withDiamond Light Source, Harwell Science and Innovation Campus
  • , Gianfelice CinqueAffiliated withDiamond Light Source, Harwell Science and Innovation Campus


The study of individual cells with infrared (IR) microspectroscopy often requires living cells to be cultured directly onto a suitable substrate. The surface effect of the specific substrates on the cell growth—viability and associated biochemistry—as well as on the IR analysis—spectral interference and optical artifacts—is all too often ignored. Using the IR beamline, MIRIAM (Diamond Light Source, UK), we show the importance of the substrate used for IR absorption spectroscopy by analyzing two different cell lines cultured on a range of seven optical substrates in both transmission and reflection modes. First, cell viability measurements are made to determine the preferable substrates for normal cell growth. Successively, synchrotron radiation IR microspectroscopy is performed on the two cell lines to determine any genuine biochemically induced changes or optical effect in the spectra due to the different substrates. Multivariate analysis of spectral data is applied on each cell line to visualize the spectral changes. The results confirm the advantage of transmission measurements over reflection due to the absence of a strong optical standing wave artifact which amplifies the absorbance spectrum in the high wavenumber regions with respect to low wavenumbers in the mid-IR range. The transmission spectra reveal interference from a more subtle but significant optical artifact related to the reflection losses of the different substrate materials. This means that, for comparative studies of cell biochemistry by IR microspectroscopy, it is crucial that all samples are measured on the same substrate type.


Cell separation by PCA due to the refractive index of the substrate used, revealing transmission artifact.


Single-cell analysis Synchrotron radiation IR microspectroscopy IR optical substrates Transmission Reflection PCA