Purchase this article with an account.
RD Glickman, BA Rockwell, GD Noojin, DJ Stolarski, ML Denton, N Kumar; Two-Photon Excitation of RPE Melanin . Invest. Ophthalmol. Vis. Sci. 2002;43(13):680.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: Previously we reported that cultured RPE cells exposed to near infrared (NIR) ultrashort pulse laser exposures suffered more DNA breakage than after continuous wave (CW) exposures delivering the same radiant exposure. A multiphoton absorption process in an intracellular chromophore was proposed as a possible mechanism for DNA interaction. As an initial test of this hypothesis, we measured fluorescence of RPE chromophores after two-photon excitation. Methods: Melanosomes were isolated from bovine RPE cells by methods previously reported (Dontsov et al., Free Rad Biol Med 26:1436-1446, 1999). Aqueous suspensions of RPE melanosomes were made and placed in glass fluorimeter cells. Single-photon excitation of the melanosomes was achieved by exposing them to the 406 nm CW output of a Krypton-ion laser at average power output of 200 mW. Two-photon excitation was accomplished with a Ti:Sapphire regenerative laser with a 1 KHz output train of ∼48-fsec pulses at 810 nm. Average power of the ultrashort pulses was varied from 100 to 200 mW. Fluorescence was measured orthogonally to the excitation beam with a fiber optic-coupled photodiode array spectrometer (Ocean Optics). Results: Single-photon excitation of the RPE melanosomes produced a fluorescence peak at about 525 nm. Two-photon excitation produced a white-light continuum in the melanosome sample, with a greenish fluorescence that declined rapidly below about 500 nm superimposed on the continuum. In both cases, the intensity of the fluorescence depended upon the irradiance of the sample and the concentration of pigment granules. There was an indication that degradation of the melanoproteins was associated with greater fluorescence. Conclusions: Multiphoton absorption occurs when the photon density is high enough to produce multiple absorption in a chromophore within a critical time period, causing the electronic transition of the chromophore to an excited state. NIR photons are not energetic enough to excite melanin fluorescence in single-photon mode, however, the demonstration that melanin fluoresces during two-photon excitation supports - but does not yet prove - our hypothesis that melanin excitation results in cellular DNA damage. Further experiments are underway to elucidate this possible interaction.
This PDF is available to Subscribers Only