Dual 2-photon microscope

We have developed a new 2P microscope (called “The Ultima”), in collaboration with Prairie Technologies that has two independently controlled sets of galvanometers. Using this microscope we can effect simultaneous 2P uncaging (at 720 nm) and 2P imaging (say at 900 nm) using two Ti:sapphire lasers. In addition, the software also controls a patch clamp amplifier, thus providing fully integrated imaging, uncaging and current measurements. We believe this approach will prove very powerful for the study of synaptic plasticity.

2P excitation permits highly localised uncaging, as the number of excited states varies as the flux density squared, and there is only sufficient flux density of photons in a focal volume of less than 1 fL to create an electronically excited state. Using this technique, single synapses in complex structures may be selectively stimulated:

When 2P uncaging is combined with 2P Ca imaging, one may quantify the efflux of Ca from the spine head into the dendrite:

(image by Jan Noguchi in the Kasai Lab, from Neuron (2005), 45, 609.)