Abstract

We describe in detail a custom-built two-photon microscope based on a modified confocal scanhead (Olympus Fluoview) and mode-locked Ti:sapphire laser (Coherent Mira 900). This system has internal detectors as well as external whole-field detection and an electrooptical modulator for blanking the beam on flyback and effecting fast changes in excitation intensity. This microscope can be used in deep, scattering samples for quantitative measurements with a wide range of fluorophores (GFP, fura, calcium green, calcium orange, fluo-3, DiI, DiO, fluorescein, rhodamine), for fluorescent photobleaching recovery and for uncaging. Images obtained with this system can be deconvolved with the Estimation Maximization algorithm using the program XCOSM (freeware available at: http://www.ibc.wustl.edu/bcl/ xcosm/).