Fast multicolor 3D imaging using aberration-corrected multifocus microscopy

An epifluorescence microscope is turned into a multifocus microscope for fast three-dimensional imaging of live biological samples. Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z scanning and is often too slow to capture biological events. We report an aberration-corrected multifocus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multifocus system enables high-resolution 3D imaging in multiple colors with single-molecule sensitivity, at speeds limited by the camera readout time of a single image.