Atomic Force Microscopy and Near-Field Scanning Optical Microscopy Measurements of Single Human Retinal Lipofuscin Granules

Clancy, Christine M. R.; Krogmeier, Jeffrey R.; Pawlak, Anna; Rozanowska, Malgorzata Barbara; Sarna, Tadeusz; Dunn, Robert C.; Simon, John D.

doi:10.1021/jp0030544

Cited by 33 publications

(21 citation statements)

References 17 publications

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“…Prior studies have shown that the emission across a single LF granule is invariant with respect to the Ϸ150-nm resolution limit of near-field scanning optical microscopy (NSOM). Significant variation in the emission maxima and spectral shape from different individual LF granules was observed by using both NSOM and spectrally resolved confocal microscopy (52,53). The variation in the emission maxima observed in the present study is shown in Table 1.…”

Section: Emission Behavior Of Individual Lipofuscin Granulessupporting

confidence: 59%

“…The emission spectra of 91 granules were collected and analyzed. As previously reported, significant variations in the emission maxima were observed (52,53). Prior studies have shown that the emission across a single LF granule is invariant with respect to the Ϸ150-nm resolution limit of near-field scanning optical microscopy (NSOM).…”

Section: Emission Behavior Of Individual Lipofuscin Granulessupporting

confidence: 57%

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Spectroscopic and morphological studies of human retinal lipofuscin granules

Haralampus-Grynaviski

Lamb²,

Clancy³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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The emission properties of ocular lipofuscin granules isolated from human retinal pigment epithelial cells are examined by using steady-state fluorescence spectroscopy and spectrally resolved confocal microscopy. The shape of the emission spectrum of a thick sample of lipofuscin granules dried on glass varies with excitation energy. The polarization of this emission is wavelength-dependent, exhibiting significant polarization near the excitation wavelength and becoming mostly depolarized over the majority of the emission spectrum. These results show that the yellow-emitting fluorophores [e.g., A2E (2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]-pyridinium)] are excited as a result of energy transfer within the granules and therefore are not the dominant blue-absorbing chromophores within lipofuscin granules. Atomic force microscopy images show lipofuscin granules to be an aggregated structure. Bulk and in vivo emission measurements must therefore take into account the effect of Raleigh scattering. When corrected for scattering, the emission spectrum of a thick lipofuscin deposit or intracellular lipofuscin resembles that for A2E. The sum of the emission spectra of a collection of individual granules also resembles the emission spectrum of A2E, but the spectrum of individual granules varies significantly. This result suggests that the agreement between the emission spectra of lipofuscin and A2E is fortuitous, and the collective data indicate the presence of several blue-absorbing chromophores in lipofuscin and show A2E is not the dominant yellow-emitting fluorophore in many of the granules studied.L ipofuscin (LF) is a common morphological result of the aging process and is manifested as a heterogeneous complex of fluorescent, lipid-protein aggregates found in the cytoplasm of postmitotic cells (1-5). In the retinal pigment epithelium (RPE) of the human eye, the formation of LF is attributed to the accumulation of indigestible end-products from the phagocytosis of photoreceptor outer segments (1, 6-8). LF accumulates in RPE cells as clusters of granules and can occupy Ϸ20% of the cytoplasmic space by 80 yr of age (9). LF exhibits a yellow fluorescence ( max Ϸ 600 nm) upon blue-light excitation (10). In vitro experiments also show that blue-light excitation of cultured RPE cells ʈ fed LF generates a variety of reactive oxygen intermediates (including hydrogen peroxide, singlet oxygen, and superoxide radical anion), which renders LF phototoxic to cultured RPE cells (11)(12)(13).Determining the molecule(s) responsible for the aerobic photoreactivity and emissive properties of LF is the focus of current research. In a groundbreaking paper in 1988, Eldred and Katz (14) analyzed chloroform:methanol (2:1, vol͞vol) extracts of RPE cells and separated several emissive bands by using TLC. A variety of yellow-emitting fluorophores were found. To date, only two isomers of a pyridinium bis-retinoid, A...

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Section: Emission Behavior Of Individual Lipofuscin Granulessupporting

confidence: 59%

Section: Emission Behavior Of Individual Lipofuscin Granulessupporting

confidence: 57%

Spectroscopic and morphological studies of human retinal lipofuscin granules

Haralampus-Grynaviski

Lamb²,

Clancy³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

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“…Such shape and characteristic sizes were typical of all lipofuscin granules studied. A similar shape of the granules was also described in [7]. Figure 1b shows the image obtained during scanning of the central region of the granule by means of phase contrast.…”

Section: Methodssupporting

confidence: 70%

Heterogeneity of Structure and Fluorescence of Single Lipofuscin Granule from Retinal Pigment Epithelium of Human Donor Eyes: Study with the Use of Atomic Force Microscopy and Near-Field Microscopy

Petrukhin

Астафьев

Zolotavin

et al. 2005

Dokl Biochem Biophys

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“…The lipofuscin of RPE is amassed within organelles of the lysosomal compartment of the cells; because of their ultrastructural appearance, these bodies are known as lipofuscin granules (49)(50)(51)(52). Other macromolecules phagocytosed by the RPE are degraded by lysosomal enzymes to small molecules that leave the lysosome assisted by permeases ( 53 ).…”

Section: All-trans -Retinal and Phospholipidmentioning

confidence: 99%

Phospholipid meets all-trans-retinal: the making of RPE bisretinoids

Sparrow

Kim

et al. 2010

Journal of Lipid Research

168

174

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segments ( 1 ). The lipid phase of the disc membrane is dominated by phospholipids (87 mol % of total lipid in human outer segment membrane) with the major species being phosphatidylcholine (PC) (32.5 mol %), phosphatidylethanolamine (PE) (37.6 mol %), and phosphatidylserine (PS) (12.1 mol %) ( 2 ). A remarkable feature of the phospholipids of the outer segment disc membrane is their unusually high content of long-chain polyunsaturated (e.g., 22:6 n -3) fatty acids; in human outer segments, 22:6 n -3 accounts for 34.2% of the fatty acid in PE, 19.5% of the fatty acid in PC and 34.1% of the fatty acid in PC ( 2 ). Indeed, docosahexaenoic acid (DHA) is more abundant in photoreceptor outer segments that in any other mammalian cell membrane ( 2 ). These unsaturated acyl chains provide the high fl uidity necessary for proper signal transduction in the disc membrane.Following absorption of a photon, the 11-cis -retinal chromophore of visual pigment rhodopsin isomerizes to all-trans -retinal and initiates a series of conformational rearrangements leading to formation of metarhodopsin II, a 380-nm absorbing species that consists of all-trans -retinal bound to opsin by a deprotonated Schiff base linkage ( Fig. 1 ). Metarhodopsin II is distinguished by its ability to activate the G protein transducin ( 3 ). This activation initiates the phototransduction cascade leading to hyperpolarization of the photoreceptor cell and altered neurotransmitter release. Movement of phospholipid species during the rhodopsin to metarhodopsin II transition has been reported by multiple groups ( 4, 5 ). PHOSPHOLIPID ASSOCIATIONS WITH RHODOPSINThroughout the outer segment compartment of the photoreceptor cell, visual pigments (rhodopsin in rods and cone pigments) are densely packed within the plasma membrane and disc membranes; low levels of rhodopsin are also present in the plasma membrane of the rod inner

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Atomic Force Microscopy and Near-Field Scanning Optical Microscopy Measurements of Single Human Retinal Lipofuscin Granules

Cited by 33 publications

References 17 publications

Spectroscopic and morphological studies of human retinal lipofuscin granules

Spectroscopic and morphological studies of human retinal lipofuscin granules

Heterogeneity of Structure and Fluorescence of Single Lipofuscin Granule from Retinal Pigment Epithelium of Human Donor Eyes: Study with the Use of Atomic Force Microscopy and Near-Field Microscopy

Phospholipid meets all-trans-retinal: the making of RPE bisretinoids

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