Development of a spectrally resolved multifocal multiphoton microscope

Multifocal Multiphoton Microscopy (MMM) can acquire three-dimensional (3D) fluorescence microscopic images of samples by multiphoton excitation with the advantages of high speed, reduced photobleaching, enhanced penetration depth and high signal-to-noise ratio. As fluorescence spectrum can provide information about the components of the sample, it is becoming increasingly popular in biomedicine to combine fluorescence spectrum measurement with multidimensional fluorescence microscopy. In this paper, we present the development of a spectrally resolved multifocal multiphoton microscope (SR-MMM). A microlens array is employed in the MMM system to produce 2D excitation foci on the sample for simultaneous two-photon excitation and a pair of galvo mirrors is used to scan the excitation foci across the sample. A liquid crystal tunable filter (LCTF) is mounted in front of a high-speed cooled CCD camera and is used to change the detection wavelength of the MMM system. Depth-resolved and spectrum-resolved two-photon excitation fluorescence images of a few samples are obtained with the SR-MMM system.