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. 2023 Feb 27;136(4):jcs260728. doi: 10.1242/jcs.260728

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© 2023. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 6. — Fast4DReg can align 3D multichannel images. (A,B) A three-channel 3D calibration slide image was aligned using Fast4DReg. Merged images and line-intensity profiles (along the dashed white lines) are displayed to highlight the level of overlap between the three channels. (A) A z-projection is displayed to visualize the lateral misalignment corrected by Fast4DReg. (B) A y-projection of one of the calibration slide spots is displayed to illustrate the axial misalignment corrected by Fast4Dreg. (C,D) The drift table generated in A,B was then used to correct a 3D SIM image of a U2-OS cell expressing GFP-tagged lamellipodin (RAPH1, red) and MYO10–mScarlet (green), and labeled to visualize its actin cytoskeleton (blue). (C) A z-projection is displayed to visualize the lateral misalignment, evident in filopodia, corrected by Fast4Dreg. (D) A y-projection of one filopodium visualizes the axial misalignment corrected by Fast4Dreg. (E,F) A 3D SIM image of MCF10DCIS.com cells expressing RFP–Lifeact (magenta) and stained to visualize F-actin (green) was aligned using Fast4DReg directly. (E) A z-projection is displayed to visualize the lateral misalignment corrected by Fast4DReg. (F) A y-projection visualizes the slight axial misalignment corrected by Fast4DReg.