| We report the construction of a Scanning Near-field Infrared Microscope (SNIM) and its successful operation towards infrared microspectroscopic analysis of biological tissue. Infrared microspectroscopy is a useful technique that can rapidly identify and localize the chemical constituents within biological tissues and cells on the basis of the vibrational spectroscopic signatures of their organic functional groups. Such analysis may be particularly useful if carried out using near-field optics so as to achieve subwavelength resolution unavailable to Fourier Transform InfraRed (FTIR) microscopy. We constructed SNIM by adapting a conventional scanning near-field microscope for use in the infrared. In particular, we employ infrared-transmitting chalcogenide fibers to serve as the fiber optic probes, and a free electron laser, a laser tunable in the mid-infrared wavelength range, is used as the illumination source. SNIM has acquired mid-infrared images of metal, semiconductor, and biological samples. We have demonstrated resolution down to 2.5 micrometers, and the device appears to be sensitive to differences in sample absorption of less than 0.1 OD. We used SNIM to examine unstained microtome sections of human atherosclerotic lesions, both by infrared imaging and by localized vibrational spectroscopy. In this way, we were able to identify and localize protein, lipid, and mineral components within the tissue. The studies of atherosclerotic tissue illustrate the usefulness of SNIM towards in situ vibrational microspectroscopic investigation of biological systems. In addition, SNIM has demonstrated the ability to perform infrared microscopic imaging in a liquid medium, and we have successfully used the device to take images of living cells in a liquid environment. We believe that SNIM provides a unique opportunity to study the cellular processes of living cells and bacteria by spectroscopic means. |
