Correction of dispersive line shape artifact observed in diffuse reflection infrared spectroscopy and absorption/reflection (transflection) infrared micro-spectroscopy Melissa Romeo, Max Diem * Departments of Chemistry and Biochemistry City University of New York, Hunter College 695 Park Avenue New York, NY 10021, USA Available online 23 May 2005 Abstract We report a computational method to remove or reduce dispersion artifacts from infrared microspectral data collected in transflection (reflection/absorption) mode. This artifact occurs along the edges of tissue samples, in particular if the tissue does not adhere well to the substrate. The method proposed for the removal of the artifact is similar to the phase correction used in standard Fourier transform infrared spectroscopy. # 2005 Elsevier B.V. All rights reserved. Keywords: Fourier transform infrared micro-spectroscopy; Dispersion artifact 1. Introduction The majority of Fourier-transform infrared (FT-IR) measurements are being carried out in transmission mode, in which the IR beam probing the sample passes through the analyte in solid, liquid or gaseous forms. Recently, new sampling methodologies have become popular, among them attenuated total reflection (ATR) and diffuse reflection infrared Fourier transform spectroscopy (DRIFTS), due to the ease of sample preparation. In ATR, the interaction of the evanescent wave at the interface of a highly refractory material samples the analyte, which is in close physical contact with the interface. ATR spectra can be converted to spectra collected in transmission mode by a simple calibration for the penetration depth of the evanescent wave [1]. DRIFT spectra may be contaminated by contributions of the specular reflection, which may result from crystalline surfaces if the sample is not sufficiently finely ground, or from metallic surfaces below the analyte if the sample thickness is insufficient. This contamination manifests itself by superposition of dispersive and absorptive line shapes (vide infra). A completely analogous artifact may also be observed in infrared micro-spectroscopy (IR-MSP). In this technique, the IR beam sampling the analyte is focused and collected using low numerical aperture Cassegrain objec- tives [2]. The off-axis rays of light, which impinge on the samples at angles very far from the normal, contribute strongly to the reflection contribution, particularly if sample substrates with a high refractive index (for example, ZnSe) are utilized. Bio-medical applications of IR-MSP are often carried out using ‘‘low-e’’ sample substrates and ‘‘reflection/absorp- tion’’ or ‘‘transflection’’ sampling methodology. ‘‘low-e’’ substrates consist of glass slides with a thin silver coating and a transparent overcoat to protect the silver layer. These substrates are completely reflective in the mid-IR spectral region. An IR-beam passing a thin sample will be reflected by the silver layer, and passes the sample once more, experiencing twice the attenuation due to a single pass. The IR beam is subsequently collected by the same Cassegrain objective used for focusing the beam, and is analyzed in reflection geometry of the IR microscope. www.elsevier.com/locate/vibspec Vibrational Spectroscopy 38 (2005) 129–132 * Corresponding author. Fax: +1 212 772 5332. E-mail address: mdiem@hunter.cuny.edu (M. Diem). 0924-2031/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vibspec.2005.04.003