Analysis of biological tissues using scanning near-field infrared microspectroscopy

Infrared absorption microspectroscopy is a useful technique to analyze biological tissues, as it can rapidly and non- destructively provide quantitative information about the molecular composition of tissue on a small spatial scale. At the Stanford Picosecond Free Electron Laser Center, a Scanning Near-field Infrared Microscope (SNIM) with the Free Electron Laser (FEL) as its illumination source has been used for in situ microspectroscopic characterization of constituents in human atherosclerotic tissue. The system consists of a Near-field Scanning Optical Microscope utilizing a tapered chalcogenide fiber as the scanning probe. The Stanford mid-infrared FEL provides high power infrared radiation that can be easily coupled into the chalcogenide fiber and whose wavelength is continuously tunable from 3 to 15 micrometers. With the FEL, the SNIM can acquire an image at a single wavelength of a 200 micrometer square region with 2 micrometer spatial resolution in under 30 minutes. It can also obtain infrared spectra at sub- wavelength resolution. The SNIM was used to examine unstained, frozen microtone sections of human atherosclerotic lesions. Spectra from localized regions in the sample were taken and analyzed to determine the distribution of various protein, lipid, and mineral constituents among the tissue microstructures. These findings were compared with results obtained by polarization microscopy and traditional histological staining techniques. The molecular information obtained in these studies can potentially lead to a greater understanding of atherosclerosis. Moreover, they demonstrate the usefulness of SNIM towards micrometer-scale vibrational microspectroscopy.