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. Author manuscript; available in PMC: 2020 Dec 1.
Published in final edited form as: Nat Methods. 2020 Jun 1;17(7):741–748. doi: 10.1038/s41592-020-0851-7

Extended Data Fig. 2. Real time 3D movement correction performance and AOL microscope field of view.

Extended Data Fig. 2

a: Example of 1- and 5-μm fluorescent beads distributed in agarose in a 400×400 μm FOV using the maximum scan angle for the AOL with an Olympus XLUMPlanFLN 20X objective lens. Fall off in intensity of image in corners due to reduced AOL light transmission efficiency at large angles.

b: Example of maximum intensity projection of a 400×400×400 μm Z-stack of localised expression of TdTomato (Magenta) and GCaMP6f (Green) in L2/3 pyramidal cells in motor cortex. Note that the expression levels were higher at the center of the FOV, contributing to a larger fall off in brightness (n=4 mice).

c: The trade-off between the imaging overhead of RT-3DMC and the feedback period for small 10 ×10 pixel reference patches with a single axial scan and larger 18 ×18 pixel patches with 3 axial scans. The dotted lines show the overhead for a reference scan cycle of 2 ms can vary between 17–30%.

d: Relationship between the maximum error and the feedback time in mouse and zebrafish. The graph assumes a maximum brain speed of 0.34 μm/ms in the mouse and 1.02 μm/ms maximum speed for 99.5% of swimming bouts in the zebrafish. To maintain a sub-micrometer error (dotted line) the feedback time should be less than 3 ms and 1 ms in the mouse and zebrafish, respectively.