We present a real-time, direct-view multiphoton excitation fluorescence microscope that provides three-dimensional imaging at high resolution. Using a rotating microlens disk, we split the near-infrared light of a mode-locked titanium–sapphire laser into an array of beams that are transformed into an array of high-aperture foci at the object. We typically scan at 225 frames per second and image the fluorescence with a camera that reads out the images at video rate. For 1.4 aperture oil and 1.2 water immersion lenses at 780-nm excitation we obtained axial resolutions of 0.84 and 1.4μm , respectively, which are similar to that of a single-beam two-photon microscope. Compared with the latter setup, our system represents a 40–100-fold increase in efficiency, or imaging speed. Moreover, it permits the observation with the eye of high-resolution two-photon images of (live) samples.
© 1998 Optical Society of America
Jörg Bewersdorf, Rainer Pick, and Stefan W. Hell, "Multifocal multiphoton microscopy," Opt. Lett. 23, 655-657 (1998)