Micropilot: automation of fluorescence microscopy-based imaging for systems biology

Abstract

Quantitative microscopy relies on imaging of large cell numbers but is often hampered by time-consuming manual selection of specific cells. The 'Micropilot' software automatically detects cells of interest and launches complex imaging experiments including three-dimensional multicolor time-lapse or fluorescence recovery after photobleaching in live cells. In three independent experimental setups this allowed us to statistically analyze biological processes in detail and is thus a powerful tool for systems biology.

Figure 1

Schematic workflow of Micropilot. (a) After autofocussing different positions to find the best focal plane (yellow frame), low-resolution prescan images (optionally maximum z-dimension projections, gray frames) are presented to the automatic classification. If a cell is selected, a complex imaging protocol is executed; otherwise the system continues to prescan. After completion of the complex imaging protocol, the system loops back to prescan mode, continuing at the sample position where it stopped for the complex imaging mode. (b) Communication steps executed by the different microscope systems (red outlines) and the Micropilot software (blue outlines). The microscope sends the image path either via windows registry or socket interface to Micropilot. In the synchronous modes, each positive classification launches the complex imaging mode. In the asynchronous mode, microscope and Micropilot send and receive messages via transmission control protocol or internet protocol (TCP/IP), allowing classification of several different positions before launching the complex imaging protocol for a list of positions. (c) After reading the low-resolution image, Micropilot segments, extracts the feature set per object and classifies the cells during scanning to return eventually the positions of interest. After the criteria are met (time or number of positions) Micropilot deploys the complex imaging and the microscope switches back to prescan mode (a).

Figure 2

Assays of SEC31 and H2B-tubulin HeLa cells. (a) Examples for Hoechst-labeled (blue; DNA label) and SEC31-labeled (green) cells representing null or artifact and anaphase or telophase cells (insets, close-up images). Scale bars, 10 µm. (b) Confusion matrix of the prediction shows true positives (TP) horizontally against the predicted class vertically for cells. At edges the total numbers of the cells are given (overall total, 10,793 cells) corresponding to PPV = TP / (TP + false positives) and sensitivity = TP / (TP + false negatives). (c) Examples of null or artifact (left) and anaphase or telophase (right) cells stained with Hoechst (blue) and ERES spot (green) (50 slices of 0.2 µm). Scale bar, 10 µm. (d) Number of ERES spots of 91 anaphase cells to late-telophase cells plotted versus volume of nuclei, with exponential fit plotted. Red and blue data points correspond to the nuclei in the left and right images in c, respectively. (e) Example of negative control experiment (time resolution, 3 min; 30 slices of 1 µm; maximum projections) started after prophase recognition. Times indicated are after prophase recognition. Scale bar, 10 µm. (f) Spindle lengths after treatment with scrambled siRNA. (g) Example images after treatment with siRNA to CENPE, showing centrosome poles (arrows; left) for the first recognizable metaphase (acquisition as in e). Scale bar, 10 µm. (h) Normal mixture modeling of pole-pole distances in metaphase from 71 movies after treatment with siRNA to CENPE resulted in three distributions, which are shown as colored curves.

Figure 3

Examples and measurements of automatic FRAP on CBX1-EGFP cells. (a) After the automatic selection of an interphase or prophase cell with a trained prophase SVM classifier, a prebleached image was taken, followed by bleaching of the predefined upper half of the nucleus and subsequent time-lapse imaging with 2-s time resolution for 60 s (values in the lower images indicate time relative to bleaching). Scale bar, 5 µm. (b) Normalized intensities for CBX1-EGFP measured during fluorescence relaxation after photobleaching in interphase and prophase cells. We measured, normalized, averaged and plotted over time fluorescence intensities in bleached region of the nucleus. Error bars, s. (c) Recovery rates as box plots for interphase and prophase cell populations.