NEW APPROACH TO ASSESS THE CHOLESTEROL DISTRIBUTION IN THE EYE LENS -CONFOCAL RAMAN MICROSPECTROSCOPY AND FILIPIN CYTOCHEMISTRY

Confocal Raman microspectroscopy (CRM) is a non-invasive, non-destruct ive, and sensitive analytical tool for the study of some aspects of th e molecular organization of cells and tissues with high spatial resolu tion. Filipin, a polyene antibiotic, specifically binds to cholesterol , and its molecular structure predicts it to be Raman-active. The aim of the present study was to assess the potentialities of a combined CR M-filipin approach to study the distribution of cholesterol in the hum an eye lens. Paraformaldehyde-fixed human lenses were sliced (0.7 mm), incubated with filipin, and analyzed by CRM. Filipin proved to give a specific Raman signal at 1586 cm(-1), hardly interfering with signals from lens proteins. The CRM-filipin approach proved to be extremely s ensitive, allowing detection of cholesterol in the femtogram range. It has an excellent spatial resolution (0.2-0.5 mu m(3)) when using poin t measurements. Due to the intrinsic anisotropy of membranes in the ey e lens and therefore of the cholesterol distribution, a line-scan appr oach has to be adopted when fiber-to-fiber changes in cholesterol are of interest. The distribution of filipin along the optical axis of fou r human eye lenses was compared with data from the literature. The com bined CRM-filipin approach is a highly specific and sensitive method f or the study of cholesterol within cells and tis sues. The spatial res olution is high and can be adapted to the desired discriminative power . The gross distribution of filipin along the optical axis obtained in this study is similar to that found in biochemical studies.