Journal of Lipid Research, Vol 36, 1139-1146, Copyright © 1995 by Lipid Research, Inc.
New approach to assess the cholesterol distribution in the eye lens: confocal Raman microspectroscopy and filipin cytochemistry
HJ Duindam, GF Vrensen, C Otto, GJ Puppels and J Greve
The Netherlands Ophthalmic Research Institute, Amsterdam.
Confocal Raman microspectroscopy (CRM) is a non-invasive, non- destructive,
and sensitive analytical tool for the study of some aspects of the
molecular organization of cells and tissues with high spatial resolution.
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 CRM-filipin approach
to study the distribution of cholesterol in the human 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 sensitive, allowing
detection of cholesterol in the femtogram range. It has an excellent
spatial resolution (0.2-0.5 micron 3) when using point measurements. Due to
the intrinsic anisotropy of membranes in the eye lens and therefore of the
cholesterol distribution, a line-scan approach has to be adopted when
fiber-to-fiber changes in cholesterol are of interest. The distribution of
filipin along the optical axis of four human eye lenses was compared with
data from the literature. The combined CRM-filipin approach is a highly
specific and sensitive method for the study of cholesterol within cells and
tissues. The spatial resolution 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.
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