December 2002
Volume 43, Issue 13
ARVO Annual Meeting Abstract  |   December 2002
Two-Photon Excitation of RPE Melanin
Author Affiliations & Notes
  • RD Glickman
    Dept of Ophthalmology Univ of TX Hlth Sci Ctr SA San Antonio TX
  • BA Rockwell
    Air Force Research Laboratory Brooks AFB TX
  • GD Noojin
    Northrup Grumman Information Technology San Antonio TX
  • DJ Stolarski
    Northrup Grumman Information Technology San Antonio TX
  • ML Denton
    Northrup Grumman Information Technology San Antonio TX
  • N Kumar
    Dept of Ophthalmology Univ of TX Hlth Sci Ctr SA San Antonio TX
  • Footnotes
    Commercial Relationships   R.D. Glickman, None; B.A. Rockwell, None; G.D. Noojin, None; D.J. Stolarski, None; M.L. Denton, None; N. Kumar, None. Grant Identification: AFOSR Grant F49620-01-1-0211; and RPB, Inc.
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 680. doi:
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    • Get Citation

      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.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract: : 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.

Keywords: 504 oxidation/oxidative or free radical damage • 567 retinal pigment epithelium • 454 laser 

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