Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy
| Autor: | Yuriy Alexandrov, Edward J. Murray, Clifford Talbot, James McGinty, Sunil Kumar, Dominic Alibhai, Emmanuelle Thinon, Paul M. W. French, Remigiusz A. Serwa, Vincenzo da Paola, Mark A. A. Neil, Edwin Garcia, Ian Munro, Frank Stuhmeier, Lucien West, Douglas J. Kelly, Edward W. Tate, Christopher Dunsby, Sean C. Warren, Frederik Görlitz |
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| Přispěvatelé: | Biotechnology and Biological Sciences Research Council (BBSRC), Wellcome Trust, Engineering & Physical Science Research Council (E, Medical Research Council (MRC) |
| Rok vydání: | 2017 |
| Předmět: |
Fluorescence-lifetime imaging microscopy
Computer science General Chemical Engineering PROTEIN 02 engineering and technology Biosensing Techniques RESONANCE ENERGY-TRANSFER gag Gene Products Human Immunodeficiency Virus fluorescence microscopy 0302 clinical medicine open source BIOSENSOR Chlorocebus aethiops Fluorescence microscope Fluorescence Resonance Energy Transfer HCA 030212 general & internal medicine LIVING CELLS General Neuroscience Optical Imaging 021001 nanoscience & nanotechnology TIME Multidisciplinary Sciences High-content screening COS Cells Science & Technology - Other Topics 0210 nano-technology Biological system STANDARDS FLIM Biophysics automated microscopy General Biochemistry Genetics and Molecular Biology drug discovery 03 medical and health sciences Open source data Animals Humans Issue 119 Science & Technology General Immunology and Microbiology Open source Förster resonance energy transfer Microscopy Fluorescence Time course NADH FRET Biosensor Software |
| Zdroj: | Journal of Visualized Experiments : JoVE |
| ISSN: | 1940-087X |
| Popis: | We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in μ Manager. The application of FLIMfit, an open source MATLAB- based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set. |
| Databáze: | OpenAIRE |
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