Fig. 43.1
(a) Schematic representation of Raman microscopic analysis for the freeze-dried samples (arrow) sandwiched between glasses. (b, c) Raman mapping image (b) and morphological image with toluidine-blue staining (c) for the same tissue area in IVCT-FS mouse eye. (d) Typical Raman spectrum obtained in the pink area shown in (b). (e) Raman spectrum obtained from the purified dry hemoglobin . Precise data have been reported in the previous paper (Terada et al. [5]). Bars: 10 μm
The beam line of a HeNe laser was focused on a position in a selected eyeball slice with the help of a bright-field microscope. Held up to the light, choroid and pigment epithelial layers were recognized as colored black owing to the presence of melanin granules. Raman spectra analyses were performed to test the suitability of the mouse eyeball slices for histological identification. Spectra obtained in this area could be categorized into four patterns (Fig. 43.1b), and typical spectrum in pink area is presented in Fig. 43.1d.
43.3 Embedding of Freeze-Dried Slices in Epoxy Resin to Visualize Tissue Structure
The eyeball slices analyzed for Raman microscopy were embedded in epoxy resin using the inverted capsule method . Briefly, the coverglass sandwiching the eyeball slice was removed, and a gelatin capsule filled with the epoxy resin was placed on the eyeball slice attached to the glass slide. After hardening the resin, the glass slide was detached by heating slightly. One-micrometer-thick sections were cut on an ultramicrotome stained with toluidine blue and observed with a bright-field microscope.
By comparing the image of the morphological tissue structures to that of the Raman mapping of four typical spectral patterns (Fig. 43.1b), it became clear that the distribution of colors reflected different tissue components: areas with the pattern shown by green, pink, red, and blue colors corresponded with the ocular skeletal muscle s, scleral connective tissues (Sc in Fig. 43.1c), choroid/pigment epithelium (Ch and PE in Fig. 43.1c), and rod/cone photoreceptor layer (RC in Fig. 43.1c), respectively.
43.4 Comparison of Raman Spectra in IVCT Tissues and Purified Proteins Prepared by Quick Freezing and Freeze Drying
The Raman spectra of isolated hemoglobin (Fig. 43.1e) showed specific patterns with several peaks. Some peaks and spectra patterns obtained from the purified hemoglobin and blood vessel s in the sclera (Fig. 43.1d, e) were almost similar. The Raman spectrum of freeze-dried purified rhodopsin was also similar to that obtained in rod/cone photoreceptor layer with IVCT-FD.
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