Multifocal multiphoton microscopy: a fast and efficient tool for 3-D fluorescence imaging

Multifocal multiphoton microscopy (MMM) is an efficient and technically simple method for generating multiphoton fluorescence images. Featuring the high axial resolution of confocal and multiphoton scanning microscopes, MMM also achieves high speed in 3-D microscopy. In this paper, examples of fast-mode 3-D microscopy are given including imaging of fixed brain tissue as well as living PC12 cells. The imaging speed of MMM is solely determined by the fluorescence photon flux, so that in practice, for brightly fluorescent specimens, a whole stack of about 50 images of 30–70 μm diameter can be acquired within a few seconds. MMM represents a significant advance towards high speed nonlinear optical tomography of fluorescent specimens.