364  2001 American Society for Photobiology 0031-8655/01 $5.00+0.00 Photochemistry and Photobiology, 2001, 74(2): 364–368 Probing the Spatial Dependence of the Emission Spectrum of Single Human Retinal Lipofuscin Granules Using Near-field Scanning Optical Microscopy ¶ Nicole M. Haralampus-Grynaviski 1 , Laura E. Lamb 1 , John D. Simon* 1,2 , Jeffrey R. Krogmeier 3 , Robert C. Dunn 3 , Anna Pawlak 4 , Malgorzata Ro ´ zanowska 4 , Tadeusz Sarna 4 and Janice M. Burke 5 1 Department of Chemistry, Duke University, Durham, NC; 2 Department of Ophthalmology, Duke University Medical Center, Durham, NC; 3 Department of Chemistry, University of Kansas, Lawrence, KS; 4 Institute of Molecular Biology, Jagiellonian University, Krakow, Poland and 5 Department of Ophthalmology, Medical College of Wisconsin, Milwaukee, WI Received 22 February 2001; accepted 20 April 2001 ABSTRACT The emission spectra of single lipofuscin granules are ex- amined using spectrally resolved confocal microscopy and near-field scanning optical microscopy (NSOM). The emission spectrum varies among the granules examined revealing that individual granules are characterized by different distributions of fluorophores. The range of spec- tra observed is consistent with in vivo spectra of human retinal pigment epithelium cells. NSOM measurements reveal that the shape of the spectrum does not vary with position within the emissive regions of single lipofuscin granules. These results suggest that the relative distri- bution of fluorophores within the emissive regions of an individual granule is homogeneous on the spatial scale 150 nm. INTRODUCTION Lipofuscin is an autofluorescent heterogeneous material formed in tissues with high oxidative stress. In the case of the retinal pigment epithelium (RPE)² of the human eye li- pofuscin appears as intracellular yellow-brown granules which are roughly spherical in shape with a diameter on the order of 1 m (1–4). Lipofuscin found in the RPE is thought to result mainly from the life-long accumulation of residual lysosomal bodies that contain the nondigestible end-products of the phagocytosis of rod outer segments (5–11). This ma- terial is phototoxic, and strong supporting evidence links the phototoxicity of lipofuscin to diseases such as age-related macular degeneration (AMD) (2,4,12–14). Lipofuscin ac- cumulation in the RPE is also associated with Stargardt’s ¶Posted on the website on 30 April 2001. *To whom correspondence should be addressed at: P.M. Gross Chemical Laboratory, Duke University, P.O. Box 90346, Durham, NC 27708, USA. Fax: 919-660-1605; e-mail: jds@chem.duke.edu ² Abbreviations: A2E, N-retinylidene-N-retinylethanolamine; CCD, charge-coupled device; NSOM, near-field scanning optical mi- croscopy/microscope; RPE, retinal pigment epithelium. disease and Best’s macular degeneration (4). Unfortunately, the causative relationships between lipofuscin and these dis- eases as well as the mechanism behind the accumulation of lipofuscin, the structure of granules and the molecular con- stituents remain largely unknown. The ability with which lipofuscin photogenerates reactive oxygen species under blue-light illumination, causing dam- age to the RPE has produced intense interest in the blue- absorbing molecules contained in lipofuscin (15–17). In an effort to identify the number and type of chromophores Eld- red and Katz (18,19) isolated 10 fluorophores from chloro- form/methanol extracts of RPE cells. All fluorophores shared a common absorbance peak lying between 280 and 330 nm, but only the orange emitting fluorophores exhibited a second strong absorbance peak at 420 nm. In 1993 a pyridinium bis-retinoid, N-retinylidene-N-retinylethanolamine (A2E), was identified as a major contributor to the orange fluores- cence of lipofuscin granules (20). While the molecular struc- ture of A2E was not correctly determined until 1996 (21), the isolation and subsequent identification of A2E spurred extensive research efforts aimed at understanding its pho- tochemical effects on RPE cells (22). Studies showing A2E to be phototoxic have led to the suggestion that it is both a major hydrophobic component of lipofuscin and the domi- nant photoactive constituent under blue-light illumination (23,24). While attention has focused on this particular or- ange emitter, it is of interest to consider the mechanism of its formation in the cell. Parish et al. (25) proposed that A2E forms in RPE cells by an initial condensation reaction be- tween all-trans-retinaldehyde and phosphatidylethanolamine followed by the oxidation and hydrolysis of the phosphate group. This mechanism of formation has recently been con- firmed in studies by Mata et al. (26) and Liu et al. (27). Both studies demonstrate that many of the intermediates formed along the pathway of A2E synthesis absorb in the blue and could also contribute to the optical properties and phototoxicity of lipofuscin. This is important in the light of recent studies by Davies et al. (28) that demonstrate that A2E is not a major contributor to the phototoxicity of lipo- fuscin in RPE cells.