We expect to observe minimal cellular phototoxicity during multiphoton excitation (MPE) 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. It is possible that the damage arises from various MPE events, or from one-photon excitation (OPE) events.

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 (BrdU) incorporated into cellular DNA (Gratzner). Cells that were synthesizing DNA during the BrdU loading are defined as 'living' cells and are identified

by their highly fluorescent nuclei, as shown in the top figure. Data are analyzed to determine the intensity and dose dependence of the synthesis inhibition. Doses are calculated for two-photon excitation (TPE) events and 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. (See bottom figure for data collected to date.) The typical dose 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 0.01 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 (Nichols).

These initial results support observations indicating that biological samples are not adversely affected by low doses of MPE. Continuations of these studies include: completing the action spectrum of DNA synthesis inhibition up to 1000 nm and determining the excitation order (OPE or MPE) leading to cellular damage from this data; a quantitative comparison of the amount of damage arising from MPE with that from comparable OPE UV-wavelengths; the determination of the damage mechanisms, as well as the reactive species involved.