A custom confocal and two-photon digital laser scanning microscope

We describe a custom one-photon (confocal) and two-photon all-digital (photon counting) laser scanning microscope. The confocal component uses two avalanche photodiodes (APDs) as the fluorescence detector to achieve high sensitivity and to overcome the limited photon counting rate of a single APD (~5 MHz). The confocal component is approximately nine times more efficient than our commercial confocal microscope (fluorophore fluo 4). Switching from one-photon to two-photon excitation mode (Ti:sapphire laser) is accomplished by moving a single mirror beneath the objective lens. The pulse from the Ti:sapphire laser is 109 fs in duration at the specimen plane, and average power is ~5 mW. Two-photon excited fluorescence is detected by a fast photomultiplier tube. With a x63 1.4 NA oilimmersion objective, the resolution of the confocal system is 0.25 μm laterally and 0.52 μm axially. For the two-photon system, the corresponding values are 0.28 and 0.82 μm. The system is advantageous when excitation intensity must be limited, when fluorescence is low, or when thick, scattering specimens are being studied (with two-photon excitation).