The flow of events: How the sequence of molecular interactions is seen by the latest, user‐friendly high throughput flow cytometric FRET

Abstract

F€ orster (or less precisely Fluorescence) Resonance Energy Transfer (FRET) is widely used by biologists to study the clustering of biomolecules tagged appropriately with chromophores in order to decipher the inner workings of the cell. FRET is a collision-free interchromophoric relaxation process that transfers energy nonradiatively from an initially excited donor to a ground-state acceptor in a distance-dependent way. FRET occurs over interatomic distances due to resonance-based dipole-dipole interaction of chromophores without transmission of photons from donor to acceptor species. Therefore, it is inaccurate to use fluorescence with the acronym FRET, since fluorescence involves emission of photons. At distances below 1 nm, collisions between the donor and acceptor would prevail, whereas at distances higher than 10 nm, photon emission by the donor would dominate, so FRET occurs only in the near field, i.e., in the range of 1-10 nm (1-3). Measuring the efficiency of FRET can serve as a "spectroscopic ruler" within this range, and provide absolute quantitative data describing interand intramolecular interactions (4).The application of FRET in biological sciences has seen an incredible expansion lately with almost 9000 hits retrieved by PubMed using FRET as a unique keyword. While the number of published papers applying FRET fluctuated between zero and one annually before 1985, >1000 publications could be found in the PubMed database in the year of 2015. This stark contrast clearly demonstrates the astonishing expansion rate of FRET applications. Partly because of this tremendous increase in the number of FRET studies, two international discussion meetings were devoted solely to FRET in life sciences. The FRET1 meeting was held from the 27th till the 30th of March in 2011 (http://www.fret.uni-duesseldorf.de/cms/fret_1.html), and the FRET 2 conference took place between the 3rd and the 6th of April in 2016 (http://www.fret.uni-duesseldorf.de/cms), both of them in G€ ottingen. Both FRET meetings were very successful, and the participants of the first meeting compiled an excellent book about "FRET from theory to applications" (5).This enormous increase in the application of FRET in biological sciences is partially due to the increase in the number of methods for evaluating FRET quantitatively, including improvements in intensity-and lifetime-based FRET methods. The accuracy of the calculation of FRET efficiency based on intensity ratios could be increased computationally even in samples having low signal to noise ratios by applying maximum likelihood estimation (MLE) (6,7). The improvement occurs because the MLE method takes the Poisson statistics inherent to photon-based detection into consideration (6,7). Not only intensity ratios but changes in the fluorescence lifetime can be used to estimate the FRET efficiency. Fluorescence

Analysis of GPI-Anchored Receptor Distribution and Dynamics in Live Cells by Tag-Mediated Enzymatic Labeling and FRET

“…There are many ways to measure FRET [ 10 , 11 , 12 , 13 , 14 ]. Here, we use methods based on confocal microscopy and flow cytometry.…”

“…However, heteroFRET is compatible with conventional equipment for detecting and analyzing the fluorescence of both the donor and acceptor in two different channels. Detailed information regarding the different types of FRET measurements can be found in several reviews [ 10 , 11 , 12 , 13 , 14 ].…”

Cell and small animal models for phenotypic drug discovery

“…Fortunately, there are numerous methods such as flow cytometry,15,16 laser scanning fluorescence,17 microfluidics18 and electrophysiology methodologies19 readily available for conducting high-throughput PDD screening. A simple cell painting approach20 allows extensive morphological profiling, with over 1,500 morphological features to be extracted and analyzed, making it a very effective tool for developing a multiplexed PDD setup.…”

Advanced FRET normalization allows quantitative analysis of protein interactions including stoichiometries and relative affinities in living cells

“…1b and Supplementary Figs 1, 2). The experimental design of this method is comparatively easy and, while mostly used in microscopy, can be applied to many different set-ups, including high-throughput methods such as slide- 15,16 and flow-based cytometry 17–19 . These methods allow the assessment of FRET in entire cell populations, which highly increases the statistical power and permits a more precise extraction of underlying variables from a dataset.…”