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...

Time-resolved spectroscopic techniques are used to determine the primary photoprocesses of A2E in solution. Comparison of the absorption and excitation spectrum of A2E in methanol solution indicates excitation at 400 nm populates the S2 excited state. Transient absorption signals decaying with a time constant of 0.9 ps were observed probing around 800 nm. These signals are attributed to the S2→S n transition and reveal the S2→S1 relaxation occurs on the subpicosecond time scale. Transient absorption data probing at shorter wavelengths (480 and 550 nm) are attributed to the S1→S n absorption. These signals exhibit an exponential decay with a time constant of 11 and 13 ps, respectively. Time-resolved emission measurements of the corresponding S0←S1 decay reveal a nonexponential decay; however, >95% of the signal amplitude is described by an exponential decay with a time constant of 12.4 ps. Both time-resolved emission and absorption experiments therefore indicate repopulation of the ground electronic state occurs with a time constant of ∼12 ps. A weak transient absorption probing in the blue (400 nm) persists onto the nanosecond time scale and is attributed to the T1→T n absorption of A2E. Photoacoustic spectroscopy establishes the quantum yield for intersystem crossing of A2E in methanol solution is at most 0.03. The emission quantum yield of A2E in ethanol is determined to be 0.01, and so, nonradiative relaxation is the dominant primary event. The quantum yield for the generation of singlet molecular oxygen following 355 nm excitation of A2E in acetonitrile was determined to be 0.02, consistent with a low production of the excited triplet state. These results establish A2E is not an efficient photogenerator of reaction oxygen species in solution.

Urocanic acid, UCA, is characterized by two electronic transitions in the UV-B (280-320 nm) which comprise its broad absorption spectrum and give rise to wavelength-dependent isomerization quantum yields. The absorption spectrum of UCA extends into the UV-A (320-400 nm). Given the UV-A component of sunlight is significantly greater than the UV-B component it is hypothesized even weak UV-A photochemistry of UCA could be important for in vivo responses to UV radiation. Degenerate pump-probe experiments performed on t-UCA at several wavelengths in the UV-A reveal an excited-state absorption that undergoes a rapid, approximately 1 ps decay. Photoacoustic experiments performed on both the cis and trans isomers reveal the formation of a long-lived intermediate following UV-A excitation. The efficiency and action spectra for this latter photoactive process are presented and are similar for both isomers of UCA. Cholesterol hydroperoxide assays designed to investigate the nature of the UV-A photoreactivity of t-UCA confirm the production of reactive oxygen species. The bimolecular rate constant for the quenching of singlet oxygen by t-UCA is determined to be 3.5 x 10(6) M(-1) s(-1). Taking into consideration recent theoretical calculations and jet expansion studies of the electronic structure of gas-phase t-UCA, a model is proposed to explain the isomerization and photoreactivity of t-UCA in solution over the UV-A region.