Improved Signal Processing in Multi-Photon Imaging

Extract

Multi-photon excitation has been used in microscopy for nearly a decade, providing a number of demonstrated advantages over other methods for fluorescence imaging. Because excitation is achieved using longer, less energetic light, photodamage and photobleaching of the sample are reduced. Furthermore, since excitation occurs only at the focal point, this approach allows the practical collection of three-dimensionally resolved fluorescence images of live cells. However, due to the small two-photon cross-section of most fluorophores, pulsed lasers are required to generate detectable signal levels. This is due to the quadratic dependence of twophoton absorption on the instantaneous power of the laser. Typically, these lasers are pulsed at very high repetition frequencies, on the order of 10⁶ pulses per second with pulse durations of a few hundreds of femtoseconds. For example, a titanium:sapphire (Ti:S) laser mode-locked at 76 Mhz can provide up to 100,000 watts of instantaneous power and is ideal for exciting two-photon events.