High-speed two-photon imaging

The small size of neuronal dendrites and spines combined with the high speed of neurophysiological signals, such as transients in membrane potential or ion concentration, necessitates that any functional study of these structures uses recording methods with both high spatial and high temporal resolutions. In this regard, conventional two-photon microscopy, in combination with fluorescent indicators sensitive to physiological parameters, has proved to be only a partial solution by providing near-diffraction-limited spatial resolution even when imaging structures deep inside light-scattering tissue. This is because the relatively slow beam-scanning methods used in most conventional two-photon microscopes severely limit the extent to which functional data can be recorded. Here, we detail developments to create high-speed two-photon imaging systems that overcome this limitation and discuss important considerations that must be taken into account when attempting to construct such systems.