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Methods Mol Biol. 2017;1663:139-152. doi: 10.1007/978-1-4939-7265-4_12.

Fully Automated Targeted Confocal and Single-Molecule Localization Microscopy.

Author information

1
High-Content Analysis of the Cell (HiCell) and Advanced Biological Screening Facility, BioQuant, Heidelberg University, Heidelberg, Germany.
2
Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany.
3
High-Content Analysis of the Cell (HiCell) and Advanced Biological Screening Facility, BioQuant, Heidelberg University, Heidelberg, Germany. Holger.Erfle@bioquant.uni-heidelberg.de.

Abstract

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.

KEYWORDS:

High content screening (HCS); Konstanz Information Miner (KNIME); Multiscale imaging; Online feedback image analysis; Single-molecule localization microscopy (SMLM); Super-resolution microscopy

PMID:
28924665
DOI:
10.1007/978-1-4939-7265-4_12
[Indexed for MEDLINE]

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