Fig. 1.

The INVAPP /Paragon movement index algorithm is a fully-automated high-throughput system able to determine motility and growth rate. (A) Schematic of the INVAPP setup (B) Principle of the algorithm: thresholding of moving pixels by statistical analysis of variance of each pixel through time. Histogram shows the distribution of pixel variance over time. Data was from obtained from a movie of all wells in a single 96-well plate containing wild-type C. elegans. The distribution is obtained by counting the number of pixels that fall into each bin of log pixel variance over time. Blue vertical line indicates mean pixel variance. The green vertical line indicates mean plus standard deviation of pixel variance; the blue shaded portion of the histogram indicates pixels that exceed this threshold so are deemed to be ‘motile’. (C) Image of 96-well plate containing C. elegans adults processed by the INVAPP/Paragon movement index system. Dark pixels are those categorized as moving by the algorithm. (D) Increasing the number of C. elegans worms per well leads to increase in reported movement index. Boxplot bars indicate 95% confidence interval. Dataset contains 16 wells per group from a single 96-well plate. (E) Movement index algorithm is able to quantify C. elegans growth in 96-well plates. Movement index increases with growth. Synchronised L1 population refed on day 0. Decrease in movement index in 25 °C group on Day 4 reflects completion of the C. elegans lifecycle and exhaustion of the bacterial food source. Boxplot notches indicate 95% confidence interval, n = 192 wells of worms for each of the 20 °C and 25 °C groups. (F) Absence of edge effects in this assay – analysis of a 1920-well C. elegans growth dataset shows no difference of the normalised movement score for 96-well plate outer edge wells (the wells found in columns 1 and 12 or rows A and H) compared to the score for inner wells (the other wells in the plate). Dataset is from a total of 20 96-well plate movies obtained from C. elegans grown at 20 °C, 25 °C and 26 °C to show that this assay is robust over a range of experimentally useful temperatures. Movement index for each well is normalised by dividing by the mean movement index for all wells of that plate. The blue bar indicates median. (G) No edge effects or other inhomogeneity across the plate – heat map shows average normalised movement index for each well location. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)