Reflection contributions to the dispersion artefact in FTIR spectra of single biological cells

Abstract

Fourier transform infrared spectra of a single cell in transflection geometry are seen to vary significantly with position on the cell, showing a distorted derivative-like lineshape in the region of the optically dense nucleus. A similar behaviour is observable in a model system of the protein albumin doped in a potassium bromide disk. It is demonstrated that the spectrum at 10 any point is a weighted sum of the sample reflection and transmission and that the dominance of the reflection spectrum in optically dense regions can account for some of the spectral distortions previously attributed to dispersion artefacts. Rather than being an artefact, the reflection contribution is ever present in transflection spectra and it is further demonstrated that the reflection characteristics can be used for cellular mapping.

Infrared spectral histopathology (SHP): a novel diagnostic tool for the accurate classification of lung cancer

“…The experimental methods utilized in this study represent the most automated procedures for data acquisition and preprocessing. The pre-processing steps, in particular, are based on most recent research and understanding of confounding effects in IR micro-spectroscopy, 53,54 and use procedures (such as segmenting of second derivative data by HCA, and subsequent analysis by ANN) that have been shown by several research groups 11,49,55,56 to produce the most reliable data and most reproducible diagnostic algorithms. In addition, the software and procedure developed during this study for pathology-based annotation are novel, and allow a more reliable and reproducible classification of the training spectra.…”

Molecular pathology via IR and Raman spectral imaging

“…Mie-scattering is a well-known effect in classical physics and occurs when the light incident on spherical sample particles has about the same wavelength as the size of the particles (see Section 3.3). In addition to producing broad, undulating background features superimposed on the absorption spectra of the sample, Mie scattering also can mix the reflective and absorptive components of the interaction of radiation with the sample; this mixing, which has been referred to as resonance-Mie (RMie) scattering [55,63,64] can cause severe distortion of the absorptive line shapes observed in IR spectroscopy. This distortion causes an apparent shift in the band position (up to 40 cm -1 in extreme cases), and severe distortions of observed intensities.…”

“…This distortion causes an apparent shift in the band position (up to 40 cm -1 in extreme cases), and severe distortions of observed intensities. The interference between absorption and scattering effects has been the subject of several recent papers [64,65], and several approaches have been developed to account for them [55,65,66]. In more recent spectral images reported later in this review, these artifacts have been accounted for in the pre-processing methods described in Section 3.3.…”

Spectroscopic and chemometric approaches to radiobiological analyses

“…As FTIRM in cytometry requires the transmission of the IR radiation through the cell, mounting substrates such as zinc-selenide (ZnSe) and calcium-fluoride (CaF 2 ) disks are commonly used for 'transmission mode' measurements and glasses coated with materials reflecting in the IR are commonly used for 'transflection mode' measurements (such as MirrIR (Kevley Technologies)) [64]. Spectra may, due to optical transmission effects exhibit a broad oscillating baseline that is attributable to Mie scattering [62,65].…”