two-photon microscopy
 

Dual 2-photon microscope

In 2002 I asked  Prairie Technologies to make a new 2P microscope (now called “The Ultima”) that had two independently controlled sets of galvanometers. This microscope enables 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. The "one-laser" version of this approach was initially developed by Winfried Denk in his pioneering 2P uncaging paper published in 1994. Masanori Matsuzaki and Haruo Kasai (University of Tokyo) refined Denk's method with our landmark 2001 and 2004 publications using MNI-Glu. In my view the key to the success of these studies was the fact that the caged compound was designed to be 2P sensitive. Denk had to use a caged transmitter that was really only meant for one-photon photolysis.

In the past few years many labs around the world have purchased an Ultima 2P microscope. Subsequently, Olympus has introduced their own versions of a dual laser 2PM, according to a collaboration with Prof. Kasai. 2P excitation permits highly localised uncaging, as  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.

On moving to Sinai in 2010 we assembled a three laser, 2PM that allows uncaging at two wavelengths whilst imaging at a third. Importantly new software from Prairie enables uncaging with two or three lasers in an arbitrary way while imaging and electrophysiological recording. Using this new microscope we are studying neuronal integration of bimodal chemical signaling with the new caged compounds we make.