A common goal of fluorescence microscopy is to collect data on specific biological events. Yet, the event-specific content that can be collected from a sample is limited, especially for rare or stochastic processes. This is due in part to photobleaching and phototoxicity, which constrain imaging speed and duration. We developed an event-driven acquisition (EDA) framework, in which neural networkbased recognition of specific biological events triggers real-time control in an instant structured illumination microscope (iSIM). Our setup adapts acquisitions on-the-fly by switching between a slow imaging rate while detecting the onset of events, and a fast imaging rate during their progression. Thus, we capture mitochondrial and bacterial divisions at imaging rates that match their dynamic timescales, while extending overall imaging durations. Because EDA allows the microscope to respond specifically to complex biological events, it acquires data enriched in relevant content.
words)The processing of microscopy images constitutes a bottleneck for large-scale experiments. A critical step is the establishment of cell borders ('segmentation'), which is required for a range of applications such as growth or fluorescent reporter measurements. For the model organism budding yeast (Saccharomyces cerevisiae), a number of methods for segmentation exist. However, in experiments involving multiple cell cycles, stress, or various mutants, cells crowd or exhibit irregular visible features, which necessitate frequent manual corrections. Furthermore, budding events are visually subtle but important to detect. Convolutional neural networks (CNNs) have been successfully employed for a range of image processing applications. They require large, diverse training sets. Here, we present i) the first set of publicly available, high-quality segmented yeast images (>10'000 cells) including mutants, stressed cells, and time courses, ii) a corresponding U-Net-based CNN, iii) a Python-based graphical user interface (GUI) to efficiently use the system, and iv) a web application to test it (www.quantsysbio.com). A key feature is a cell-cell boundary test which avoids the need for additional input from fluorescent channels. A bipartite graph matching algorithm tracks cells in time with high reliability. Our network is highly accurate and outperforms existing methods on benchmark images recorded by others, suggesting it transfers well to other conditions. Furthermore, new buds are detected early with high reliability. We apply the system to detect differences in geometry between wild-type and cyclin mutant cells. Our results indicate that morphogenesis control occurs unexpectedly early in the cell cycle and is gradual, demonstrating how the efficient processing of large numbers of cells uncovers new biology. Our system can serve as a resource to the community, expanded continuously with new images. Furthermore, the techniques we develop here are likely to be useful for other organisms as well.2 Dietler et al.YeaZ: A CNN for highly accurate, label-free segmentation of yeast images Abstract (150 words)The identification of cell borders ('segmentation') in microscopy images constitutes a bottleneck for large-scale experiments. For the model organism Saccharomyces cerevisiae, current segmentation methods face challenges when cells bud, crowd, or exhibit irregular features. Here, we present i) the first set of publicly available, high-quality segmented yeast images (>10'000 cells) including mutants, stressed cells, and time courses, ii) a corresponding convolutional neural network (CNN), iii) a graphical user interface and a web application (www.quantsysbio.com) to efficiently employ, test, and expand the system. A key feature is a cell-cell boundary test which avoids the need for fluorescent markers. Our CNN is highly accurate, including for buds, and outperforms existing methods on benchmark images, indicating it transfers well to other conditions. To demonstrate how efficient, large-scale image processing uncove...
In fluorescence microscopy, the amount of information that can be collected from the sample is limited, often due to constraints imposed by photobleaching and phototoxicity. Here, we report an event-driven acquisition (EDA) framework, which combines real-time, neural network-based recognition of events of interest with automated control of the imaging parameters in an instant structured illumination microscope (iSIM). On-the-fly prioritization of imaging rate or experiment duration is achieved by switching between a slow imaging rate to detect the onset of biological events of interest and a fast imaging rate to enable high information content during their progression. In this way, EDA allows the data capture of mitochondrial and bacterial divisions at imaging rates that match their dynamic timescales, while extending the accessible imaging duration, and thereby increases the density of relevant information in the acquired data.
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