Fully Automated Targeted Confocal and Single-Molecule Localization Microscopy
Single-molecule localization microscopy (SMLM) enables imaging of biological structures in the nanometre range. Long measurement times are the consequence of this kind of microscopy due to the need of acquiring thousands of images. We built a setup that automatically detects target structures using confocal microscopy and images them with SMLM. Utilizing the Konstanz Information Miner (KNIME), we were able to connect a confocal microscope with an SMLM unit for targeted screening. In this process, we developed KNIME plugins to communicate with the microscope components and combined them to a workflow. Thus, measuring biological nanometre-sized structures in a sufficient number to get statistical significance becomes feasible. For proof of principle HIV-1 assembly complexes in HeLa cells derived from transfection of replication deficient viral construct were imaged by a fully automated screen.
Key wordsSuper-resolution microscopy Single-molecule localization microscopy (SMLM) Konstanz Information Miner (KNIME) High content screening (HCS) Multiscale imaging Online feedback image analysis
The authors thank Michael Berthold and the KNME/KNIP developers for their support with the creation of the KNIME nodes. This work was funded within the project CancerTelSys (grant number 01ZX1302) in the e:Med program, within the project RNA-Code (grant number 031A298) in the e:Bio program and within the project HD-HuB (grant number 031A537C), all of the German Federal Ministry of Education and Research (BMBF). The Advanced Biological Screening Facility is also supported by the CellNetworks-Cluster of Excellence (grant number EXC81).
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