What are the fluorescence propertied of aging pigment in the human eye? I first broadened my research scope to work on the vertebrate eye in the early 1980's. I applied my microspectrophotometric techniques to a unique inconsistency in the human visual system: Why does lipofuscin, which accumulates in aging retinal pigment epithelium, appear yellow under UV excitation while spectrofluorometry had shown a blue emission? Working together with Dr.G. E. Eldred and Prof. L. Feeney-Burns, Department of Ophthalmology, Universityof Missouri, I showed that a substantial body of literature on this important cellular aging product was in error because of inadequate optical calibration.This was my springboard to further research in vertebrate vision.
When I started my job at Missouri in 1979, so did Lynette
Feeney-Burns. She gave a seminar I attended about lipofuscin,
the aging pigment. Lipofuscin fluoresced yellow but the spectra
she showed peaked around 450 nm. I told her I could not believe
that and that I knew how to do that work correctly and that the
emission should peak around 580 nm if it looked yellow. My student,
Greg Miller, and her post-doc, Graig Eldred, joined the project.
We went to a chemist for assistance, and, after "preaching"
about the limitations of your apparatus, gave us a curve from
his spectrofluorimeter that peaked in the blue. "OK?"
"No, we're not leaving" Graig held a UV lamp next to
the cuvette and it fluoresced bright yellow. Many adventures later
we publisned that the fault was that benchtop fluorimeters had
monochromators and photomultipliers optimized for short wavelengths
and that they were simply wrong, in this case by over 100 nm.
Our consultant bemoaned that Eldred and Stark got a Science paper
out of "the notorious insensitivity of spectrofluorimeters
to long wavelengths." My former mentor, Jerry Wasserman,
said "You're not going to make yourself very popular since
you're saying that, not only was everybody else wrong, but that
they were also idiots." When I gave seminars, people would
comment, "That explains what happened in the field of so-and-so
(or for my protein or whatever) -- the fluorescence looked one
color, but the spectral data did not correlate." Eventually,
companies made spectrofluorimeters tthat were better corrected
at long wavelengths.
Papers on lipofuscin, the aging pigment:
Eldred, G.E., Miller, G.V., Stark, W.S. and Feeney-Burns, L. Lipofuscin: Resolution of discrepant fluorescence data. Science, 1982, 216, 757-759. PubMed
Stark, W.S., Miller, G.V., and Itoku, K.A. Calibration of microspectrophotometers as it applies to the detection of lipofuscin and the blue and yellow emitting fluorophores in situ. Chap. 42 In: Methods in Enzymology: (series eds.:S.P. Colowick and N.O. Kaplan): Vol. 105 Oxygen Radicals in Biological Systems (vol. ed. L. Packer) New York: Academic Press, 1984 pp. 341-347. (Invited chapter.). PubMed
Ultraviolet (UV) excited pale yellow emission in retinal pigment epithelial (rpe) cells from 82 year old donor (original color photograph used for Stark et al., 1984 chapter).
A scanning electron micrograph(SEM) which shows the association of the retinal pigment epithelial (rpe) cells with the rod cells in a fracture through a rat retina
of the orders-of-magnitude sensitivity error from spectrofluorometers
which led researchers to be over 100 nm too short on the emission
spectrum of lipofuscin (from Eldred et al., 1982)
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This page was last updated May 26, 2005