Functional imaging of neurons with two-photon confocal microscopy using a Saturable-Bragg Reflector-modelocked compact solid state laser

Abstract

Two-photon excitation laser scanning microscopy (TPLSM) allows high resolution fluorescence imaging with minimal photodamage and photobleaching (for a review see [1]). TPLSM has recently been used to study, for example, information processing in dendritic spines, cell division in developing embryos, and calcium dynamics in auditory hair cells. TPLSM yields intrinsic submicron three-dimensional resolution (Fig. 1) and optical sectioning equivalent to one-photon confocal microscopy simply by spatial confinement of excitation. This eliminates counterproductive absorption outside the focal slice. In highly scattering samples, such as nervous tissue, the red or near-infrared light used for illumination in TPLSM leads to a much improved penetration depth and the absence of a detector pinhole permits ballistic and diffuse photons to contribute to the signal [1]. Because two-photon absorption depends on the square on the instantaneous light intensity, ultrashort pulses are essential for TPLSM to achieve efficient excitation while using low average power. The main impediment to a more widespread use of TPLSM have been the cost, utility, and space requirements for femtosecond modelocked lasers that are based on large-frame argon-ion pump sources. The use of passive modelocking schemes that directly produce pulses of < 100 fs duration is desirable to reduce overall complexity and optimize performance.