In JoVE (1)
Articles by Frederik Görlitz in JoVE
Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy Frederik Görlitz*1, Douglas J. Kelly*1, Sean C. Warren1, Dominic Alibhai2, Lucien West3, Sunil Kumar1, Yuriy Alexandrov1, Ian Munro1, Edwin Garcia1, James McGinty1, Clifford Talbot1, Remigiusz A. Serwa4, Emmanuelle Thinon4, Vincenzo da Paola3, Edward J. Murray5, Frank Stuhmeier6, Mark A. A. Neil1, Edward W. Tate4, Christopher Dunsby1,7, Paul M. W. French1 1Photonics Group, Department of Physics, Imperial College London, 2Institute for Chemical Biology, Department of Chemistry, Imperial College London, 3MRC Clinical Sciences Centre, Hammersmith Hospital, 4Chemical Biology Section, Department of Chemistry, Imperial College London, 5Retroscreen Virology Ltd, 6Pfizer Global Research and Development, Pfizer Limited, Sandwich, Kent, UK, 7Centre for Histopathology, Imperial College London We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in µManager and data analysis is undertaken in FLIMfit.
Other articles by Frederik Görlitz on PubMed
STED Nanoscopy with Fluorescent Quantum Dots Nature Communications. May, 2015 | Pubmed ID: 25980788 The widely popular class of quantum-dot molecular labels could so far not be utilized as standard fluorescent probes in STED (stimulated emission depletion) nanoscopy. This is because broad quantum-dot excitation spectra extend deeply into the spectral bands used for STED, thus compromising the transient fluorescence silencing required for attaining super-resolution. Here we report the discovery that STED nanoscopy of several red-emitting commercially available quantum dots is in fact successfully realized by the increasingly popular 775 nm STED laser light. A resolution of presently ∼ 50 nm is demonstrated for single quantum dots, and sub-diffraction resolution is further shown for imaging of quantum-dot-labelled vimentin filaments in fibroblasts. The high quantum-dot photostability enables repeated STED recordings with >1,000 frames. In addition, we have evidence that the tendency of quantum-dot labels to blink is largely suppressed by combined action of excitation and STED beams. Quantum-dot STED significantly expands the realm of application of STED nanoscopy, and, given the high stability of these probes, holds promise for extended time-lapse imaging.
Plasmonic Nanoprobes for Stimulated Emission Depletion Nanoscopy ACS Nano. Nov, 2016 | Pubmed ID: 27794591 Plasmonic nanoparticles influence the absorption and emission processes of nearby emitters due to local enhancements of the illuminating radiation and the photonic density of states. Here, we use the plasmon resonance of metal nanoparticles in order to enhance the stimulated depletion of excited molecules for super-resolved nanoscopy. We demonstrate stimulated emission depletion (STED) nanoscopy with gold nanorods with a long axis of only 26 nm and a width of 8 nm. These particles provide an enhancement of up to 50% of the resolution compared to fluorescent-only probes without plasmonic components irradiated with the same depletion power. The nanoparticle-assisted STED probes reported here represent a ∼2 × 10(3) reduction in probe volume compared to previously used nanoparticles. Finally, we demonstrate their application toward plasmon-assisted STED cellular imaging at low-depletion powers, and we also discuss their current limitations.