Multifocal multiphoton microscopy

Multifocal multiphoton microscopy is a microscopy technique for generating 3D images, which uses a laser beam, separated by an array of microlenses into a number of beamlets, focused on the sample.¹ The multiple signals are imaged onto a CCD camera in the same way as in a conventional microscope. The image rate is determined by the camera frame rate, depending on the readout rate and the number of pixels and may range well above 30 images/s.²

By exploiting specific properties of pulsed-mode multiphoton excitation the conflict between the density of the foci, i.e. the degree of parallelization, and the axial sectioning has been resolved.³ The laser pulses of neighboring foci are temporally separated by at least one pulse duration, so that interference is avoided. This method is referred to as time-multiplexing (TMX). Moreover, with a high degree of time multiplicity, the interfocal distance can be reduced to such an extent that lateral scanning becomes obsolete. In this case axial scanning is sufficient to record a 3D-image.