Real time imaging of the excitation volume of a multiphoton microscope

Knowledge of the excitation profile in a confocal or multiphoton microscope can improve the image resolution, e.g. by using deconvolution, pixel reassignment or adaptive optics strategies. Here we demonstrate a method by which the scanning beam can be used to place a stationary, virtual 'guide star' at any chosen location in the sample, during imaging. This can then be used to directly image the excitation profile. The major advantage of our easy-to-install method, compared to competing methods, is that it can work for non-descanned multiphoton microscopy, the method of choice for deep tissue or ultraviolet imaging. Our experimental results reproduce diffraction theory based calculations in a minimally-scattering sample, and provide detailed information about the aberrated excitation profile in a highly scattering sample.