Femtosecond near-infrared laser pulse induced strand breaks in mammalian cells.

Multiphoton laser scanning microscopy (MPLSM) is based on non-resonant simultaneous absorption of two or three near infrared (NIR) photons by a fluorophore in the subfemtoliter focal volume of a high numerical aperture (N.A. 1.3) objective. The higher penetration depth of NIR radiation enables optical sectioning across thick biological specimens and because of the absence of efficient single photon absorbers in the NIR spectral region of 700 to 1200 nm there is hardly any out-of-focus photodamage and photobleaching. Recent in vitro studies (14) have demonstrated that irradiation of supercoiled plasmid DNA with intense multiphoton NIR 810 nm of 140 fs pulse width, 76 MHz pulse repetition rate results in single strand breaks as a result of simultaneous absorption of three or more photons. Herein, we have investigated the influence of 800 nm NIR 170 fs laser pulses, 80 MHz pulse repetition frequency at mean powers of 2 to 20 mW on nuclear DNA of unlabelled PtK2 cells. In situ TdT-mediated dUTP-nick end labelling (TUNEL) revealed that cells exposed to the NIR irradiation above > or =5 mW mean laser power alone contained TUNEL-positive nuclei. The intensity of TUNEL fluorescence was relatively higher at increased mean NIR laser power. These results provide evidence that DNA strand breaks also occur in vivo when mammalian cells are exposed to high average power > or =5 mW NIR irradiation during MPLSM possibly due to multiphoton absorption process. Because intense DNA fragmentation is one of the hall marks of programmed cell death it is hypothesised that NIR induced cell death is by apoptosis.