Supplementary Fig. 2: Parameter optimization for MOVIE-focus.
From: Advanced CUBIC tissue clearing for whole-organ cell profiling
(a) Optimized six parameters of MOVIE-focus is shown in this table by comprehensive autofocus simulation by using 400,000 images datasets (400 of different light-sheet positions at every 0.35 μm) which was acquired by our LSFM at the 5 μm step size covering 5000 μm depth, using a PI stained mouse brain. Several parts of parameter optimization are shown as an example in (b)-(i). In these panels, only one or two parameters from six parameters are changed. (b)The relationship between block size for image analysis and mean error from ideal focus positions. Error bars show standard deviation of error. (c) The relationship among the block size for image analysis of MOVIE-focus and minimum calculation time for 80% focus accuracy. The focus accuracy was calculated from the ideal focus position, which is determined by the light-sheet position with the highest DCTS score. Although block size 128×128 and 64×64 exhibit similar error in panel (b), the minimum calculation time of block size 128×128 is less than 10 ms per image and faster than that of block size 64×64. (d) The relationship between analyzed area and mean error from ideal focus positions. Error bars show standard deviation of error. (e) The relationship between amplitude of light-sheet oscillation and mean error from ideal focus positions. Error bars show standard deviation of error. (f) The relationship between focus value threshold and mean error from ideal focus positions. Error bars show standard deviation of error. (g) The relationship between focus value threshold and tracking ability. (h) The relationship between block selection order and mean error from ideal focus positions. “Descending” and “Ascending” indicate block selections from top or bottom, respectively. Error bars show standard deviation of error. (i) The relationship between block selection distance (distance function used in block selection; Mean, L1-norm and L2-norm) and mean error from ideal focus positions. Error bars show standard deviation of error.
