Authors:
Randolph D. Glickman
Department of Ophthalmology, University of Texas Health Science Center, San Antonio, Texas 78229-3900

Thomas E. Johnson
Department of Environmental and Radiological Health Sciences, Colorado State University, Ft. Collins, Colorado 80523

Gary D. Noojin
David J. Stolarski
Michael L. Denton
Northrup Grumman Warfighter and Applications Department, San Antonio, Texas 78228-1330

Neeru Kumar
Department of Ophthalmology, University of Texas Health Science Center, San Antonio, Texas 78229-3900

Benjamin A. Rockwell
US Air Force Research Laboratory, AFRL/HEDO, Brooks City-Base, Texas 78235-5214

Although laser-induced breakdown has the lowest energy threshold in the femtosecond domain, and appears to be responsible for production of threshold retinal lesions by ultrashort pulse lasers, previous findings suggested a role for multiphoton absorption in retinal damage. In this study, we investigated if near infrared ultrashort laser pulses produced DNA damage by exciting short wavelength-absorbing chromophores via multiphoton absorption. The experiments were carried out in nonpigmented and artificially pigmented hTERT-RPE cells, a line of human-derived retinal pigment epithelial (RPE) cells. Cells were exposed to 0.25 s pulses of CW and mode-locked 810 nm laser emissions, at irradiances equivalent to 0.5 and 1.0 times the ED₅₀ for threshold damage in the primate eye, and DNA strand breaks were measured using the comet assay (single cell gel electrophoresis). At 0.5 X ED₅₀, increased DNA strand breakage occurred after the ultrashort (mode-locked) exposures, but not the CW exposures, compared to control cells. At 1.0 X ED₅₀, both the mode-locked- and CW-exposed cells exhibited increased DNA damage, compared to controls. These differences, although significant, were inconsistent, because they were found only in the comet assay tail length parameter, but not the tail moment parameter. Moreover, the amount of DNA strand breakage was clearly dependent on the amount of melanin pigmentation in the cells, regardless of laser pulsewidth. A theoretical analysis found that the number of photons delivered to the RPE melanosome during a single 33–250 fs pulse, at an irradiance equivalent to the ED₅₀, was insufficient to produce multiphoton excitation. In addition, the equivalent DNA strand breakage in pigmented RPE cells after ultrashort and CW exposures indicated that multiphoton absorption is not a major contributor to the ultrashort pulse laser damage threshold in the near infrared optical spectrum.