Raman Microscopy of Freeze‐dried Mouse Eyeball‐slice in Conjunction with the “in vivo Cryotechnique”

Abstract

The wavelength of Raman-scattered light depends on the molecular composition of the substance. This is the first attempt to acquire Raman spectra of a mouse eyeball removed from a living mouse, in which the eyeball was preserved using the "in vivo cryotechnique" followed by freeze-drying. Eyeballs were cryofixed using a rapid freezing cryotechnique, and then sliced in the cryostat machine. The slices were sandwiched between glass slides, freeze-dried, and analyzed with confocal Raman microscopy. Important areas including various eyeball tissue layers were selected using bright-field microscopy, and then the Raman spectra were obtained at 240 locations. Four typical patterns of Raman spectra were electronically mapped on the specimen images obtained by the bright-field microscopy. Tissue organization was confirmed by embedding the same eyeball slice used for Raman spectra into epoxy resin and the thick sections were prepared with the inverted capsule method. Each Raman spectral pattern represents a different histological layer in the eyeball which was mapped by comparing the images of toluidine blue staining and Raman mapping with different colors. In the choroid and pigment cell layer, the Raman spectrum had two peaks, corresponding to melanin. Some of the peaks of the Raman spectra obtained from the blood vessels in sclera and the photoreceptor layer were similar to those obtained from the purified hemoglobin and rhodopsin proteins, respectively. Our experimental protocol can distinguish different tissue components with Raman microscopy; therefore, this method can be very useful for examining the distribution of a biological structures and/or chemical components in rapidly frozen freeze-dried tissue.

Morphofunctional Merits of an <i>In Vivo</i> Cryotechnique for Living Animal Organs: Challenges of Clinical Applications from Basic Medical Research

“…The wavelength pattern of Raman-scattered light is known to reflect the molecular composition of the tissue structure. Therefore, we directly froze an anesthetized mouse eyeball, which was prepared by the freeze-drying method, using IVCT, and then examined retinal tissues in unstained specimens using confocal Raman microscopy [ 10 ]. The Raman wavelength showed a characteristic pattern in response to each retinal layer localized with specific molecules.…”

“…We also observed other specific wavelength patterns that corresponded to hemoglobin of erythrocytes flowing in sclera blood vessels and the photorecepter protein, rhodopsin, in the visual cell layer. Therefore, functional molecules in retinal tissues were identified without any staining in the IVCT-prepared specimens of living mice [ 10 ].…”