Fundus autofluorescence mostly originates from bisretinoid fluorophores in lipofuscin granules, which accumulate in retinal-pigment-epithelium cells with age. The dynamics of accumulation, photo-oxidation, and photodegradation of bisretinoids during aging or in the presence of pathology have been insufficiently investigated. Changes in spectral properties and composition of human lipofuscin-granule fluorophores with age and pathology have now been investigated by a high-performance liquid chromatography method using spectrophotometric and fluorescent detectors connected in series. It was found that: (i) N-retinylidene-N-retinylethanolamine (A2E) fluorescence intensity is not predominant in the chloroform extract of human-cadaver-eye retinal pigment epithelium studied; bisretinoid photo-oxidation and photodegradation products have much higher fluorescent properties; (ii) the relative emission maximum in the fluorescence spectrum of suspended retinal-pigment-epithelium cells obtained from an individual human-cadaver eye without pathology is irrespective of donor age and falls within the range 575 ± 15 nm; in two cadaver eyes with signs of age-related macular degeneration, emission maxima were shifted by 23-36 nm towards the shortwave region; and (iii) the ratio of bisretinoid photo-oxidation and photodegradation products to unoxidized bisretinoids in the chloroform extract of cadaver-eye retinal pigment epithelium increases with donor age, from 0.69 ± 0.03 to 1.32 ± 0.04. The differences in fluorescence properties between chloroform extracts obtained from cadaver eyes with and without signs of age-related macular degeneration could be used to increase the potential of fundus autofluorescence imaging as a noninvasive diagnostic method.
The coherent 11-cis-retinal photoisomerization dynamics in bovine rhodopsin was studied by femtosecond time-resolved laser absorption spectroscopy at 30-fs resolution. Femtosecond pulses of 500, 535, and 560 nm wavelength were used for rhodopsin excitation to produce different initial Franck-Condon states and relevant distinct values of the vibrational energy of the molecule in its electron excited state. Time evolution of the photoinduced rhodopsin absorption spectra was monitored after femtosecond excitation in the spectral range of 400-720 nm. Oscillations of the time-resolved absorption signals of rhodopsin photoproducts represented by photorhodopsin(570) with vibrationally-excited all-trans-retinal and rhodopsin(498) in its initial state with vibrationally-excited 11-cis-retinal were studied. These oscillations reflect the dynamics of coherent vibrational wave-packets in the ground state of photoproducts. Fourier analysis of these oscillatory components has revealed frequencies, amplitudes, and initial phases of different vibrational modes, along which the motion of wave-packets of both photoproducts occurs. The main vibrational modes established are 62, 160 cm(-1) and 44, 142 cm(-1) for photorhodopsin(570) and for rhodopsin(498), respectively. These vibrational modes are directly involved in the coherent reaction under the study, and their amplitudes in the power spectrum obtained through the Fourier transform of the kinetic curves depend on the excitation wavelength of rhodopsin.
Ultrafast reverse photoreaction of visual pigment rhodopsin in the femtosecond time range at room temperature is demonstrated. Femtosecond two-pump probe experiments with a time resolution of 25 fs have been performed. The first рump pulse at 500 nm initiated cis-trans photoisomerization of rhodopsin chromophore, 11-cis retinal, which resulted in the formation of the primary ground-state photoproduct within a mere 200 fs. The second pump OPEN ACCESSMolecules 2014, 19 18352 pulse at 620 nm with a varying delay of 200 to 3750 fs relative to the first рump pulse, initiated the reverse phototransition of the primary photoproduct to rhodopsin. The results of this photoconversion have been observed on the differential spectra obtained after the action of two pump pulses at a time delay of 100 ps. It was found that optical density decreased at 560 nm in the spectral region of bathorhodopsin absorption and increased at 480 nm, where rhodopsin absorbs. Rhodopsin photoswitching efficiency shows oscillations as a function of the time delay between two рump pulses. The quantum yield of reverse photoreaction initiated by the second pump pulse falls within the range 15% ± 1%. The molecular mechanism of the ultrafast reversible photoreaction of visual pigment rhodopsin may be used as a concept for the development of an ultrafast optical molecular switch.
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