Differences in infrared spectroscopic data of connective tissues in transflectance and transmittance modes

• IR data collected in transflectance are significantly different from transmittance.
• Thickness variation causes spectral variation between transflectance and transmittance.
• Scattering artifacts causes spectral variation between transflectance and transmittance.

Fourier transform infrared imaging spectroscopy (FT-IRIS) has been used extensively to characterize the composition and orientation of macromolecules in thin tissue sections. Earlier and current studies of normal and polarized FT-IRIS data have primarily used tissues sectioned onto infrared transmissive substrates, such as salt windows. Recently, the use of low-emissivity (“low-e”) substrates has become of great interest because of their low cost and favorable infrared optical properties. However, data are collected in transflectance mode when using low-e slides and in transmittance mode using salt windows. In the current study we investigated the comparability of these two modes for assessment of the composition of connective tissues. FT-IRIS data were obtained in transflectance and transmittance modes from serial sections of cartilage, bone and tendon, and from a standard polymer, polymethylmethacrylate. Both non-polarized and polarized FTIR data differed in absorbance, and in some cases peak position, between transflectance and transmittance modes. However, the FT-IRIS analysis of the collagen fibril orientation in cartilage resulted in the expected zonal arrangement of fibrils in both transmittance and transflectance. We conclude that numerical comparison of FT-IRIS-derived parameters of tissue composition should account for substrate type and data collection mode, while analysis of overall tissue architecture may be more invariant between modes.

Figure optionsDownload as PowerPoint slide