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 imagi ng applications when photon shot noise is limiting. Working on brain slices with excitation powers of 8-10 mW at the specimen plane, the resting signa l from cerebellar Purkinje cell somas loaded with 10 mu M Oregon Green 488 BAPTA-1 averaged 4 detected photons/mu s; axons of interneurons loaded with 200 mu M of this indicator yielded about 1 photon/mu s. To obtain satisfac tory images at high time resolution, long pixel dwell times are required an d data collection should be restricted to as few pixels as necessary. Furth ermore, a large proportion of total measurement time (duty cycle) should be available for data collection. We therefore developed a method for scannin g small regions of interest with line repetition rates two to four times hi gher than conventional ones and a duty cycle of 70%. We also compared the p erformance 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/mu s. the photon counting avalanche photodiode shows the be st signal to noise ratio. At larger fluxes, photomultipliers or intensified photodiodes are superior. (C) 1999 Elsevier Science B.V. All rights reserv ed.