The ability to perform optical sectioning is one of the great advantages of laser-scanning microscopy. This introduces, however, a number of difficulties due to the scanning process, such as lower frame rates due to the serial acquisition process. Here we show that by introducing spatiotemporal pulse shaping techniques to multiphoton microscopy it is possible to obtain full-frame depth resolved imaging completely without scanning. Our method relies on temporal focusing of the illumination pulse. The pulsed excitation field is compressed as it propagates through the sample, reaching its shortest duration at the focal plane, before stretching again beyond it. This method is applied to obtain depth-resolved twophoton excitation fluorescence (TPEF) images of drosophila egg-chambers with nearly 105 effective pixels using a standard Ti:Sapphire laser oscillator.

By introducing spatiotemporal pulse shaping techniques to multiphoton microscopy it is possible to obtain video-rate images with depth resolution similar to point-by-point scanning multiphoton microscopy while mechanically scanning in only one dimension. This is achieved by temporal focusing of the illumination pulse: The pulsed excitation field is compressed as it propagates through the sample, reaching its shortest duration (and highest peak intensity) at the focal plane before stretching again beyond it. This method is applied to produce, in a simple and scalable setup, video-rate two-photon excitation fluorescence images of Drosophila egg chambers with nearly 100,000 effective pixels and 1.5 microm depth resolution.

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We achieve depth-resolved polarization microscopy by measuring third-harmonic generation induced by a tightly focused circularly polarized beam. In crystals exhibiting strong birefringence this signal is dominated by positively phase-matched third-harmonic generation. This process occurs in only optically anisotropic media, in which the birefringence compensates for the phase mismatch between the fundamental and the third harmonic induced by dispersion. Both the intensity and the polarization of the emitted signal provide information on the local optical anisotropy. We demonstrate the technique by imaging biogenic crystals in sea urchin larval spicules.

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