A custom confocal and two-photon digital laser scanning microscope

We describe a custom one-photon (confocal) and two-photon all-digital (phot on counting) laser scanning microscope. The confocal component uses two ava lanche photodiodes (APDs) as the fluorescence detector to achieve high sens itivity and to overcome the limited photon counting rate of a single APD (s imilar to 5 MHz). The confocal component is approximately nine times more e fficient than our commercial confocal microscope (fluorophore fluo 4). Swit ching from one-photon to two-photon excitation mode (Ti: sapphire laser) is accomplished by moving a single mirror beneath the objective lens. The pul se from the Ti: sapphire laser is 109 fs in duration at the specimen plane, and average power is similar to 5 mW. Two-photon excited fluorescence is d etected by a fast photomultiplier tube. With a x63 1.4 NA oil-immersion obj ective, the resolution of the confocal system is 0.25 mu m laterally and 0. 52 mu m axially. For the two-photon system, the corresponding values are 0. 28 and 0.82 mu m. 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).