Frequency-multiplexed aberration measurement for confocal microscopy

In single-photon confocal fluorescence microscopy, optical aberration affects both excitation and detection light paths, thus can severely degrade image quality. We incorporated an adaptive optics (AO) module into a confocal microscope and used a frequency-multiplexed aberration measurement method to measure and correct sample-induced aberration. We demonstrated that this method can measure aberration using signals from features of different sizes and recover diffraction-limited imaging performance. Applying our AO confocal microscope to imaging through and within living zebrafish larvae, as well as in the mouse brain in vivo, we showed that aberration correction can substantially improve confocal image brightness, resolution, and contrast.