Simultaneous multiplane in vivo nonlinear microscopy using spectral encoding.

Conventional point-by-point imaging schemes for laser scanning microscopy limit acquisition speeds, particularly when imaging three-dimensional volumes. We report a novel approach that achieves parallelization of multiple fields of view through the use of spectral encoding. By focusing two or more beams of different wavelengths at different positions within a suitable tissue, fluorescence or second/third harmonic generation emissions from these regions can be uniquely separated. We demonstrate that this approach can allow simultaneous in vivo imaging of fluorescence in two planes within the living rodent cortex, and of second harmonic generation in fresh tissue.