Abstract

Laser scanning microscopy combined with two-photon excitation of fluorescence holds great promise in imaging biological systems. This two-photon excitation laser scanning microscopy (TPLSM) [1] yields intrinsic submicron three-dimensional resolution with much reduced background fluorescence and thus reduced photodamage. Although the advantages of TPLSM as compared to wide field fluorescence microscopy and confocal microscopy have been demonstrated in a number of applications [2], the large cost and utility requirements of mode locked Ti:sapphire laser systems and other femtosecond light sources have kept TPLSM out of reach for most biology labs. We demonstrate here that a recently developed compact solid state laser that is mode locked with a Saturable Bragg Reflector (SBR) [3] is well-suited for TPLSM. A SBR-modelocked Cr:LiSAF laser was pumped with a 0.5 W, 670 nm diffraction-limited MOPA (SDL), providing 90 fs pulses at 860 nm with CW power of 25-44 mW per beam (Fig. la). A single beam was directed into a laser scanning microscope consisting of a pair of galvomirrors, a relay lens, a dichroic mirror, a Zeiss water-immersion objective (63 x 0.9 NA), and a photomultiplier tube for the detection of fluorescence photons [2]. Rat cortical brain slices (300 μm thick) were prepared using standard techniques. For anatomical imaging, neocortical pyramidal cells that were deeply embedded in the tissue were dialyzed and voltage clamped using whole-cell electrodes containing 500 μM fluorescein dextran (MW = 3 kD). TPLSM imaging at low magnification (Fig. 1B) revealed primary and secondary dendrites and the initial segment of the axon. At high magnification single dendritic spines, the smallest neuronal compartments, became apparent (Fig. 1C, arrow). A series of images acquired at different focal planes (Δz = 1.6 μm) demonstrates the sectioning capabilities of the microscope (Fig. 1D-F). For functional imaging of physiological calcium responses, neurons were dialyzed with electrodes containing the calcium indicator Ca-green-1 (300 μM, Molecular Probes). Ca-green is a fluorophore that undergoes a large fluorescence intensity change in response to Ca²⁺ binding. Intracellular free calcium concentration changes evoked by single action potentials could easily be detected (Fig. 1G).

© 1996 Optical Society of America