High efficiency beam splitter for multifocal multiphoton microscopy

In this article we present the development of a multibeam two-photon laser scanning microscope. A new type of beam splitter to create the multitude of laser beams is described, This type of beam splitter has higher transmissi on and generates more uniform beams than can be achieved with the microlens approach used by other groups. No crosstalk exists between the different f oci due to small temporal delays between the individual beams. The importan ce of dispersion compensation to obtain maximum efficiency of the microscop e is discussed. With optimum compensation the fluorescence signal was raise d by a factor of 14. Different modes of detecting the fluorescence signals and their effect on imaging speed and resolution are discussed.