Figure 2.

Auto-focusing principle during scanning. First, a coarse scanning with large step size over a wide z range is performed. Then, the image at each z position is used to calculate the focus measure (F-metric). The best focus z position is then estimated as the peak location by fitting a Gaussian function to discrete F-metrics. Centered on this estimated best focus z position, a fine axial scanning with small step size is performed to capture the whole 3D information. Autofocusing is repeated for every lateral x–y scanning position and executed only in DAPI channel. The estimated best focus z position will be used across all channels. Chromatic aberration can be compensated by axial scanning.