Construction of a Two-photon Microscope for Video-rate Ca(2+) Imaging

We describe the construction of a video-rate two-photon laser scanning microscope, compare its performance to a similar confocal microscope, and illustrate its use for imaging local Ca(2+) transients from cortical neurons in brain slices. Key features include the use of a Ti-sapphire femtosecond laser allowing continuous tuning over a wide (700-1000 nm) wavelength range, a resonant scanning mirror to permit frame acquisition at 30 Hz, and efficient wide-field fluorescence detection. Two-photon imaging provides compelling advantages over confocal microscopy in terms of improved imaging depth and reduced phototoxicity and photobleaching, but the high cost of commercial instruments has limited their widespread adoption. By constructing one's own system the expense is greatly reduced without sacrifice of performance, and the microscope can be more readily tailored to specific applications.