Unsupervised fluorescence lifetime imaging microscopy for high content and high throughput screening.
Proteomics and cellomics clearly benefit from the molecular insights in cellular biochemical events that can be obtained by advanced quantitative microscopy techniques like fluorescence lifetime imaging microscopy and Förster resonance energy transfer imaging. The spectroscopic information detected at the molecular level can be combined with cellular morphological estimators, the analysis of cellular localization, and the identification of molecular or cellular subpopulations. This allows the creation of powerful assays to gain a detailed understanding of the molecular mechanisms underlying spatiotemporal cellular responses to chemical and physical stimuli. This work demonstrates that the high content offered by these techniques can be combined with the high throughput levels offered by automation of a fluorescence lifetime imaging microscope setup capable of unsupervised operation and image analysis. Systems and software dedicated to image cytometry for analysis and sorting represent important emerging tools for the field of proteomics, interactomics, and cellomics. These techniques could soon become readily available both to academia and the drug screening community by the application of new all-solid-state technologies that may results in cost-effective turnkey systems. Here the application of this screening technique to the investigation of intracellular ubiquitination levels of alpha-synuclein and its familial mutations that are causative for Parkinson disease is shown. The finding of statistically lower ubiquitination of the mutant alpha-synuclein forms supports a role for this modification in the mechanism of pathological protein aggregation.
Esposito
Alessandro
A
Cell Biophysics Group, European Neuroscience Institute-Göttingen, Waldweg 33, 37073 Göttingen, Germany. aesposito@quantitative-microscopy.org
Dohm
Christoph P
CP
Bähr
Matthias
M
Wouters
Fred S
FS
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D009154:Mutation; D051381:Rats; D012984:Software; D025801:Ubiquitin / Q000378:metabolism; D051844:alpha-Synuclein / Q000235:genetics / Q000378:metabolism
D025801:Ubiquitin; D051844:alpha-Synuclein