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Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy
Published on: January 18, 2017
High-throughput quantification of posttranslational modifications in situ by CA-FLIM
Hernán E Grecco1, Pedro Roda-Navarro, Sven Fengler
1Department of Systemic Cell Biology, Max Planck Institute for Molecular Physiology, Dortmund, Germany.
Methods in Enzymology
|September 28, 2011
Summary
Quantifying protein posttranslational modifications (PTMs) is crucial for understanding cell signaling. Cell array-based fluorescence lifetime imaging microscopy (CA-FLIM) offers a rapid and accurate method for in situ PTM quantification in multiple proteins.
Area of Science:
- Cell Biology
- Biochemistry
- Microscopy
Background:
- Signal transduction relies on protein posttranslational modifications (PTMs).
- Understanding cellular function requires quantifying PTM levels in complex signaling networks.
- Accurate in situ quantification of multiple PTMs is essential for systems biology approaches.
Purpose of the Study:
- To detail the protocol for cell array-based fluorescence lifetime imaging microscopy (CA-FLIM).
- To establish CA-FLIM as an efficient method for high-throughput PTM quantification.
- To enable rapid analysis of signaling network dynamics.
Main Methods:
- Development and implementation of the CA-FLIM protocol.
- Utilizing cell arrays for parallelized sample preparation and imaging.
- Employing fluorescence lifetime imaging microscopy for sensitive PTM detection.
Main Results:
- CA-FLIM enables accurate in situ quantification of PTM levels for multiple proteins.
- The entire process from cell array preparation to data analysis takes less than 2 days.
- A single 384-spot cell array can be imaged and analyzed in under 2 hours.
Conclusions:
- CA-FLIM is a powerful and time-efficient technique for studying signaling networks.
- This protocol facilitates high-throughput analysis of PTMs in response to stimuli.
- CA-FLIM significantly advances the ability to investigate cellular signaling mechanisms.
