Infrared microscopy for the study of biological cell monolayers. I. Spectral effects of acetone and formalin fixation

Infrared spectroscopy of biological cell monolayers grown on surfaces is a poorly developed field. This is unfortunate because these monolayers have potential as biological sensors. Here we have used infrared microscopy, in both transmission and transflection geometries, to study air-dried Vero cell monolayers. Using both methods allows one to distinguish sampling artefactual features from real sample spectral features. In transflection experiments, amide I/II absorption bands down-shift 9/4 cm(-1), respectively, relative to the corresponding bands in transmission experiments. In all other spectral regions no pronounced frequency differences in spectral bands in transmission and transflection experiments were observed. Transmission and transflection infrared microscopy were used to obtain infrared spectra for unfixed and acetone- or formalin-fixed Vero cell monolayers. Formalin-fixed monolayers display spectra that are very similar to that obtained using unfixed cells. However, acetone fixation leads to considerable spectral modifications. For unfixed and formalin-fixed monolayers, a distinct band is observed at 1740 cm(-1). This band is absent in spectra obtained using acetone-fixed monolayers. The 1740 cm(-1) band is associated with cellular ester lipids. In support of this hypothesis, two bands at 2925 and 2854 cm(-1) are also found to disappear upon acetone fixation. These bands are associated with C-H modes of the cellular lipids. Acetone fixation also leads to modification of protein amide I and II absorption bands. This may be expected as acetone causes coagulation of soluble cellular proteins. Other spectral changes associated with acetone or formalin fixation in the 1400-800 cm(-1) region are discussed.