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Issue 4, 2012
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Electric field standing wave artefacts in FTIR micro-spectroscopy of biological materials

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Abstract

FTIR absorption micro-spectroscopy is a widely used, powerful technique for analysing biological materials. In principle it is a straightforward linear absorption spectroscopy, but it can be affected by artefacts that complicate the interpretation of the data. In this article, artefacts produced by the electric-field standing-wave (EFSW) in micro-reflection-absorption (transflection) spectroscopy are investigated. An EFSW is present at reflective metallic surfaces due to the interference of incident and reflected light. The period of this standing wave is dependent on the wavelength of the radiation and can produce non-linear changes in absorbance with increasing sample thickness (non-Beer–Lambert like behaviour). A protein micro-structure was produced as a simple experimental model for a biological cell and used to evaluate the differences between FTIR spectra collected in transmission and transflection. By varying the thickness of the protein samples, the relationship between the absorbance and sample thickness in transflection was determined, and shown to be consistent with optical interference due to the EFSW coupled with internal reflection from the sample top surface. FTIR spectral image data from MCF 7 breast adenocarcinoma cells was then analysed to determine the severity of the EFSW artefact in data from a real sample. The results from these measurements confirmed that the EFSW artefact has a profound effect on transflection spectra, and in this case the main spectral variations were related to the sample thickness rather than any biochemical differences.

Graphical abstract: Electric field standing wave artefacts in FTIR micro-spectroscopy of biological materials

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Publication details

The article was received on 21 Oct 2011, accepted on 13 Dec 2011 and first published on 09 Jan 2012


Article type: Paper
DOI: 10.1039/C2AN15995C
Citation: Analyst, 2012,137, 853-861
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    Electric field standing wave artefacts in FTIR micro-spectroscopy of biological materials

    J. Filik, M. D. Frogley, J. K. Pijanka, K. Wehbe and G. Cinque, Analyst, 2012, 137, 853
    DOI: 10.1039/C2AN15995C

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