Fast scanning and efficient photodetection in a simple two-photon microscope.

Two-photon laser scan microscopy carries many advantages for work on brain slices and bulk tissue. However, it has very low signal levels compared to conventional fluorescence microscopy. This is disadvantageous in fast imaging applications when photon shot noise is limiting. Working on brain slices with excitation powers of 8-10 mW at the specimen plane, the resting signal from cerebellar Purkinje cell somas loaded with 10 microM Oregon Green 488 BAPTA-1 averaged 4 detected photons/micros; axons of interneurons loaded with 200 microM of this indicator yielded about 1 photon/micros. To obtain satisfactory images at high time resolution, long pixel dwell times are required and data collection should be restricted to as few pixels as necessary. Furthermore, a large proportion of total measurement time (duty cycle) should be available for data collection. We therefore developed a method for scanning small regions of interest with line repetition rates two to four times higher than conventional ones and a duty cycle of 70%. We also compared the performance of several photodetectors and found the optimum choice to depend strongly on the photon flux during a given application. For fluxes smaller than 5 photons/micros, the photon counting avalanche photodiode shows the best signal to noise ratio. At larger fluxes, photomultipliers or intensified photodiodes are superior.