Figure 1.

a. Schematic diagram of the two-photon unit of the microscope. After expanding the beam in a telescope to match the back aperture of the microscope objective the laser beam passes the scan unit, is focused by the scan lens on an intermediate image plane and re-collimated by the field lens to match the infinity corrected microscope optics. Fluorescence and SHG is collected by three PMTs in the microscope’s non-descanned detection port. Emission and SHG wavelengths are selected by appropriate bandpass and dichroic filters.

b. Schematic diagram of the confocal unit of the microscope. The confocal illumination laser enters the unit through a fiberoptic port, is collimated and passes polarization optics which converts linear polarization to circular polarization. After reflecting on a dichroic mirror the beam path is merged with the two-photon system by two beam steering mirrors. Reflected light is separated from the illumination light through a polarizing beam splitter cube and imaged on the PMT through a confocal pinhole. Fluorescence passes the dichroic mirror and is imaged similarly on a PMT.