Acousto-optic multiphoton laser scanning microscopy (AO-MPLSM) for structural and functional imaging in living brain slices

The intrinsic optical sectioning, reduced light-scattering, and reduced photodamage of multiphoton laser-scanning microscopy (MPLSM) has generated great interest for this technique in experimental Neuroscience, as it enables to study both structure and function of fine neuronal processes within living brain tissue. At present, virtually all MPLSM systems employ galvanometric beam positioning. Due to this inertia-limited approach, single-dimension line scans are employed to achieve frame rates sufficient for functional imaging. Although such line scans allow adequate sampling rates (<= 1kHz), two significant drawbacks remain. First, the majority of scan time is wasted by illuminating regions of no interest, while sacrificing signal integration time at sites-of-interest. Second, the sites from which signals can be recorded are limited to those along a single line. Alternatively, acousto-optic (AO) beam positioning with high-resolution TeO2 deflectors allows inertia-free skipping between arbitrary sites within the field-of-view in < 15 mu s. This achieves high sampling rate recording at multiple, non-adjacent sites quasi-simultaneously (1-5kHz frame rate, 12-60 sites). Such a multi-site optical recording system would greatly advance studying complex neuronal function, by enabling membrane potential or calcium transients to be observed throughout the complex geometry of neuronal dendrites. This paper presents images and functional recordings from living neurons within brain slices, acquired with AO-MPLSM. Our novel imaging system allows a user to collect structural images first and subsequently select sites of interest for fast functional imaging. To demonstrate the system's power, we present high-speed recordings (1kHz) from > 10 sites within the dendrites of pyramidal neurons in acute brain slices, at signal-to-noise ratios comparable to line-scan systems.