  Temporal focusing two-photon microscopy enables high resolution imaging of
fine structures in vivo over a large volume. A limitation of temporal focusing
is that signal-to-background ratio and resolution degrade rapidly with
increasing imaging depth. This degradation originates from the scattered
emission photons are widely distributed resulting in a strong background. We
have developed Multiline Orthogonal Scanning Temporal Focusing (mosTF)
microscopy that overcomes this problem. mosTF captures a sequence of images at
each scan location of the excitation line, followed by a reconstruction
algorithm reassigns scattered photons back to the correct scan position. We
demonstrate mosTF by acquiring mice neuronal images in vivo. Our results show
remarkably improvements with mosTF for in vivo brain imaging while maintaining
its speed advantage.
