Several acquisition modes are supported to adapt to the organization of the sample, and the Image analysis workflows used for target detection of new applications can be simply developed and loaded as ImageJ macro functions independent from the main software. For increased flexibility, the software is also compatible with Leica confocal microscopes 1 ( Supplementary Data S7.1). only to sample surface) with the target acquisition settings for the application.ĪutoScanJ is compatible with Micro-Manager ( Supplementary Data S0.0) and it brings a new dimension to Intelligent Microscopy by enabling the real-time detection of events of interest in live samples. The first one is designed to selectively image objects of interest in fixed samples while the second can map a 3D sample at coarse resolution and restrict posterior image acquisition (e.g. Micro-Manager offers two plugins ( Pinkard et al., 2016 Micro-Manager, 2020) to perform Intelligent Microscopy. For instance, Micro-Manager ( Edelstein et al., 2010) is a versatile open source software to control camera-based microscopes which naturally integrates with ImageJ, one of the most widely used bioimage analysis software. Open source software for image acquisition ( Edelstein et al., 2010) and image analysis ( de Chaumont et al., 2012 Schneider et al., 2012 McQuin et al., 2018 Berg et al., 2019) are overall more interoperable and easier to deploy anywhere. In practice however, the inherent fragmentation of the market makes it difficult to share reproducible Intelligent Microscopy protocols across laboratories, and no turnkey solution is currently provided for the selective imaging of events of interest in live samples. The proprietary acquisition software of commercial microscopes now often offer advanced protocols combining imaging modalities, mosaic scans, high content screening, and partial support for Intelligent Microscopy ( Leica, 2020 Nikon, 2020 Zeiss, 2020). We refer to this technique as Intelligent Microscopy. Image acquisition can then naturally be re-organized in two sequential scans: 1) a scan covering a large sample area with resolution adjusted to allow the reliable detection of the targets of interest (primary scan) and 2) a higher resolution scan with fields of views selectively centered on the targets of interest (secondary scan). Fortunately, it often turns out that only part of the data acquired is useful to an experimenter interested in rare events or sparsely spread objects. Additionally, for live microscopy, guaranteeing sample integrity also puts hard limits on acquisition speed and maximum light dose. High-resolution fluorescence microscopy can generate an overwhelming amount of data and require a prohibitive acquisition time when imaging a wide sample area. Overall, AutoScanJ helps to optimize microscope usage by autonomous operation, and it opens up new experimental avenues by enabling the real-time detection and selective imaging of transient events in live microscopy. The target detection functions for these applications are provided and can be used as a starting point and a source of inspiration for new applications. We illustrate five different application scenarios with the system ranging from samples fixed on micropatterned surfaces to live cells undergoing several rounds of division. The software is straightforward to set up and new custom image analysis workflows to detect targets of interest can be simply implemented and shared with minimal efforts as independent ImageJ macro functions. AutoScanJ is compatible with motorized fluorescence microscopes controlled by Leica LAS AF/X or Micro-Manager. For fixed samples, the software can dramatically reduce the amount of data acquired and the acquisition duration in situations where statistically few targets of interest are observed per field of view. For live samples, our software can sequentially detect biological events from their onset and further image them at high resolution, an action that would be impractical by user operation. We developed AutoscanJ, a suite of ImageJ scripts enabling to image targets of interest by automatically driving a motorized microscope at the corresponding locations. 4Center for Genomic Regulation, CRG, Barcelona Institute of Science and Technology, BIST, Barcelona, Spain. 3Genetics of Male Fertility Group, Cell Biology Unit, Faculty of Biosciences, Autonomous University of Barcelona, Bellaterra, Spain.2Molecular Imaging Platform, Molecular Biology institute of Barcelona IBMB-CSIC, Barcelona, Spain.1Institute for Research in Biomedicine, IRB Barcelona, Barcelona Institute of Science and Technology, BIST, Barcelona, Spain.Sébastien Tosi 1*, Anna Lladó 1, Lídia Bardia 1, Elena Rebollo 2, Anna Godo 3, Petra Stockinger 4 and Julien Colombelli 1
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