Synchrotron infrared microspectroscopy of retinal layers

Abstract

Viewing a microscopic frozen section of a rat retina with differential interference contrast (DIC) reveals the layered structure. An infrared microspectrometer equipped with all reflecting infinity corrected optics provides such viewing capability just prior to probing individual layers or performing mapping experiments. Three-dimensional functional group images reveal the localized chemical microstructure of the layered tissue. This family of images is constructed from spectra scanned at each location in the area mapped. The excellent detail and sharpness of the transition between layers is achieved using a small projected image plane mask. Experimentation done with enhanced spatial resolution, achievable with the very bright synchrotron source that has no thermal noise and is non-divergent, provides high density (100%) rectangular maps from step sizes of 3 or 5 μm between points in the grid. In addition to the perspective provided by the high spatially resolved chemical images, mapping across layers revealed variation in layer contour and chemical composition complimenting the spectral information obtained by previous microspectroscopy investigations with a thermal (globar) source using a narrow but long mask to obtain spectra for each retinal layer. The mapping also showed consistent composition along layers providing baseline data for future investigation that may involve diseased states. Excellent spectra with 4 cm −1 resolution were extracted from any pixel obtained in the mapping process.