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.
At a glance
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- Video 3: Raw data of yeast cells expressing Cse4-GFP. (18.88 MB, Download)
- Lateral field of view 20×20 μm. Focus step between successive planes Δz = 380 nm. (Voxel size: x,y = 120 nm, z = 380 nm.) Data were acquired at a speed of one multifocus image (exposure time 100 ms) every 3 s. Scale bar, 1.5 μm.
- Video 4: 3D reconstruction of Supplementary Video 3, visualizing the 3D dynamics of yeast centromeres during anaphase. (19.71 MB, Download)
- Upper movie, xy view; lower movie, xz view. Scale bar, 1 μm.
- Video 5: 3D view of single trajectories of RNA polymerase II (magenta) diffusing in the nucleus of U2OS cells transfected with lamin B1–GFP as a nuclear membrane marker (green). (17.2 MB, Download)
- Upper movie, xy view; lower movie, xz view. Only trajectories longer than five frames are displayed as segments. Data were acquired at a speed of 35 volumes s–1. (Voxel size: x,y = 120 nm, z = 380 nm.)
- Video 6: Examples of 3D trajectories, exhibiting slow diffusion, rapid diffusion and mixed behavior. (7.22 MB, Download)
- Data were acquired at a speed of 35 volumes s–1.
- Video 7: The unc-47 GABAergic motor neurons of this C. elegans embryo express a green fluorescent protein. (2.79 MB, Download)
- This movie shows the raw multifocus data, a set of nine simultaneously formed focal planes (60×60 μm at 2-μm separation covering a depth of 18 μm) arranged in a 3×3 array on the camera. Data were recorded at 9 Hz for 5.5 min and are displayed at 10× this speed. Scale bar, 20 μm.
- Supplementary Text and Figures (7 MB)
Supplementary Figures 1–3 and Supplementary Notes 1–4