Time-multiplexed multifocal multiphoton microscope.

We resolve the classical conflict between parallelization and axial resolution in three-dimensional fluorescence microscopy through time-multiplexed multifocal multiphoton excitation. A rotating array of microlenses on a disk splits ultrafast laser pulses in such a way that an array of high-aperture foci are created in the sample. Two rigidly mounted corotating glass disks with suitable arrays of holes ensure that adjacent foci illuminate the sample at different time points. Recordings of biological specimens demonstrate elimination of out-of-focus haze for densely packed foci and concomitant substantial improvement of contrast and resolution.