Multiline Orthogonal Scanning Temporal Focusing (mosTF) Microscopy for Scattering Reduction in High-speed Brain Imaging.

Temporal focusing two-photon microscopy enables high resolution imaging of fine structures 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 . Our results show remarkably improvements with mosTF for brain imaging while maintaining its speed advantage.