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Fluorescence microscopy is a ubiquitous and powerful tool for the biologist mainly due to the availability of a wealth of highly specific fluorescent probes. Multiphoton (two or more photon) excitation fluorescence microscopy is an optical sectioning technique that offers significant advantages over other optical sectioning techniques in terms of improved viability of living material and the ability to penetrate deeper into specimens. The use of a longer excitation wavelength (typically twice that of the excitation peak of the fluorophore) increases the penetration of the excitation into the sample, yet essentially eliminates single-photon excitation in the bulk of the sample. In order to attain the high peak-power densities necessary for the production of multiphoton events while keeping mean power levels below damaging levels, ultrashort-pulsed excitation sources are used. Some sources, such as mode-locked, Ti:sapphire lasers, can produce pulses less than 100 fs. Pulses this short need to be pre-chirped in order to compensate for the group velocity dispersion of the microscope optics so that the pulse width is maintained at the sample. Without such pre-compensation we show that the average power required to produce a fixed level of two-photon excited signal, using typical microscope optics, is fairly constant from 60fs to 250fs. We argue that the choice of pulse width is an important consideration for a biological imaging system since varying the source pulse width may be used to change the relative amounts of two- and three- photon excitation. With a pre-chirped (compensated) system, if the pulse length is quadrupled then twice the power will be required to attain the previous level of two-photon excited fluorescence, but only half the three-photon excitation (or absorption) will be produced. Pulse widths may be varied on compensated systems by adjusting the pre-compensation. This may be used to favor three-photon excitation of UV-excited fluorophores, or, on the other hand, it may be desirable to reduce levels of three-photon excitation during two-photon imaging of live samples using 700 nm - 800 nm radiation as deleterious excitation of endogenous fluorophores or absorption by (and therefore damage to) proteins and nucleic acids could occur. Variable pulse widths may therefore prove to be an important parameter for live cell studies. Alternatively, for a given range of applications, a simpler and cheaper fixed-pulse length source with the desired characteristics may be chosen.
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