Rainer Pick

Multifocal Multiphoton Microscopy.

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 1.4mum , 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.

Nonlinear absorption extends confocal fluorescence microscopy into the ultra-violet regime and confines the illumination volume

It is shown that two-photon absorption confines the illumination volume and present quantitative evidence that an additional confocal arrangement of the detector further improves the resolution by 48%. The axial resolution in a confocal fluorescence microscope using two-photon absorption with infra-red light is comparable to that achievable with ultra-violet light half the wavelength. An important advantage of two-photon microscopy over single-photon microscopy is that absorption is almost confined to the observed volume. This means no photodamage is caused outside the observed volume.