We expect to observe minimal cellular phototoxicity during two-photon excitation (TPE) since the fluorescence excitation is confined to a small volume surrounding the beam focus. however, little is known about the limitations and possible damage mechanisms of this process. We are currently generating an action spectrum of photodamage by monitoring the onset of DNA synthesis inhibition in cultured HeLa cells. After exposures of controlled doses of pulsed illumination from a mode-locked Ti:sapphire laser in an imaging raster pattern, cell proliferation is assayed by measuring, via immunofluorescence labeling, the amount of bromodeoxyuridine incorporated into cellular DNA. Data are analyzed to determine the intensity and dose dependence of the synthesis inhibition. Two-photon doses (W2s) are defined as the square of the peak power at the sample multiplied by the total dwell time of the focal volume in a cell. The typical doses to form a ratio image of cellular calcium using Indo-1 fluorescence excited at 700 nm with 230 fs pulses and an average power of 5 mW is about 0.03 W2s. The dose for the onset of DNA synthesis inhibition in unloaded cells is approximately 0.3 W2s at 700 nm with 10 to 35 mW average powers and 165 to 200 fs pulses. At 740 and 780 nm, this threshold occurs near 5 W2s under similar conditions. These initial results support observations indicating that biological samples are not adversely affected by low doses of TPE.
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