Fast4DReg: A New Pipeline for 3D Video Drift Correction Pylvänäinen J.W et al, 2022 Fast4DReg is a Fiji macro for drift correction in 3D video and is able to correct drift in all x-, y- and/or z-directions. Fast4DReg creates intensity projections along both axes and estimates their drift using the NanoJ-Core (Laine et al., 2019) cross-correlation-based drift correction, and then translates the 3D video frame by frame (Figure 1). Additionally, Fast4DReg can be used for aligning multichannel 3D images which is particularly useful for instruments that suffer from a misalignment of channels. Datasets are available in Zenodo: 1 Software archive 1.1 Fast4DReg scripts (4) 1.2 Helper scripts (2) 1.3 Documentation from GitHub 2 Dataset 1: Synthetic drift -datasets 2.1 Synthetic drift files (3) 2.2 Expected results (6) 3 Dataset 2: the HUVEC monolayer -dataset 3.1 Biological dataset HUVEC channel 1 original 3.2 Biological dataset HUVEC channel 1 F4DReg 3.3 Biological dataset HUVEC channel 1 C3DD 4 Channel alignment 4.1 Dataset 3: Registration slide -dataset (2) 4.2 Dataset 4: Filopodia-dataset (2) 5 Videos 5.1 Video 1 5.2 Video 2 5.3 Video 3