Session Title: Imaging II
Session Date/Time: Sunday 29/09/2013 | 08:00-10:00
Paper Time: 08:16
Venue: Hall G1 (Level 2)
First Author: R.Smith USA
Co Author(s): M. Lee T. Ach Y. Tong D. Yoon
Lipofuscin is a complex mixture of fluorophores deposited in the retinal pigment epithelium (RPE) in normal eyes and an important disease marker. We describe hyperspectral autofluorescence (AF) imaging as a method to search for individual fluorophores ex vivo.
RPE-only flat-mounts from belts of normal chorioretinal human donor tissue were prepared by removing the retina and choroid. For spectral microscopy we used a Zeiss Axio Imager A2 microscope (Carl Zeiss, Jena, Germany) with Plan-Apochromat objectives, excitation wavelength 480 nm, and long-pass fluorescence emission filters. Hyperspectral AF images were captured using the Nuance FX camera (Caliper Life Sciences, US) at emissions between 590 and 700 nm in 10 nm intervals and then compared to the published spectra for A2E in solution and in cell culture  and other candidates .
The AF image (excitation 480 nm) shows a broad fluorescence peak around 600 nm consistent with lipofuscin. From the fluorescence emission spectra of pure A2E (excitation 480 nm, in cell culture and in solution, rescaled from ) and the RPE-only flat mount (excitation 480 nm), the spectral peak of A2E is 620 nm in solution and 590 nm in cell culture. The spectral peak from the RPE is at 600 nm with intensity steadily decreasing steadily from 600 nm to 700 nm and a shoulder at wavelengths >650, consistent with a mixture of A2E and lesser contributions from other bis-retinoid candidates .
We applied hyperspectral imaging in a preliminary search for important fluorophores of human RPE ex vivo. The long wavelength spectra recovered from peripheral RPE agreed most closely with A2E. A full investigation of complex fluorophore mixtures will aid our understanding of their biochemistry and distribution and could also be adapted to in vivo non-invasive research on the role of these compounds in chorioretinal degenerations. 1. Sparrow, J. R., Wu, Y., Kim, C. Y., Zhou, J., 2010. Phospholipid meets all-trans-retinal: the making of RPE bisretinoids. J Lipid Res 51, 247-261. PMID 19666736. 2. Sparrow, J. R., Gregory-Roberts, E., Yamamoto, K., Blonska, A., Ghosh, S. K., Ueda, K., Zhou, J., 2012. The bisretinoids of retinal pigment epithelium. Prog Ret Eye Res 31, 121-135. PMID 22209824."