FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

Abstract

Förster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo [1][2] . Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest [3][4] . Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells 5 , while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium 6 , cAMP [7][8] , inositol phosphates 9 and cGMP [10][11] . Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a β-adrenergic receptor agonist and blocker.

In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

“…The cells were washed once with 300 ml of FRET buffer (in mM: 144 NaCl, 5.4 KCl, 1 MgCl 2 , 1 CaCl 2 , 10 HEPES, pH 7.3), and 300 ml of the same buffer were added to the chamber. FRET measurements were performed using an inverted fluorescent microscope (Nikon Ti) and ImageJ software 39 . The FRET donor CFP was excited at 440 nm using a CoolLED light source.…”

Direct monitoring of cAMP at the cardiac ryanodine receptor using a novel targeted fluorescence biosensor mouse

“…were isolated by enzymatic digestion during Langendorff perfusion, plated on laminin coated round glass coverslides and used for FRET or Ca 2+ measurements as previously described. 4,30,31 Transverse aortic constriction (TAC). All animal experiments were performed in accordance with institutional (Tierschutzbüro Universitätsmedizin Göttingen) and governmental (LAVES Niedersachsen, BGV Hamburg) guidelines.…”

A Software Tool for High-Throughput Real-Time Measurement of Intensity-Based Ratio-Metric FRET

“…Our microscopy system is based on the same principles as shown by J. U. Sprenger et al [13], but with the addition of a mechanised sample stage and focus drive (Märzhäuser Wetzlar, Wetzlar, Germany) that allow higher throughput acquisition of multiple fields of view (FOV) in one experiment. Individual cell positions (X, Y and Z) are recorded within the Micro-Manager prior to an experiment, allowing each image capture to be cell centered and in focus.…”

“…Moreover, the ROI is selected separately between MultiFRET and the old macro, subjecting this to human error where the areas selected for analysis may be several pixels off. , data were acquired with the MultiFRET plugin and re-analysed with the 'offline' macro according to [13]. E macro data were obtained using the "online" macro and analysed using the "offline" macro according to [13], whereas MultiFRET data were acquired and analysed using MultiFRET with a different set of samples from the macro data.…”

“…This provides additional flexibility, allowing experimental procedures to be carried out in response to changes in the FRET ratio. As a low-cost alternative, Julia U. Sprenger et al (2012) built a customised epifluorescence FRET imaging system and developed a non-proprietary ImageJ macro, which displays real-time ratio-metric data obtained from a Micro-Manager controlled time-lapse acquisition of images of two fluorescence channels [13]. This macro gives a rough estimation of FRET ratios in real-time during the experiment, but it does not save the ratio data, and the method still requires a separate analysis of the image-stacks once they are saved.Here we report a microscope system incorporating a motorized sample stage and describe a new software plug-in for Micro-Manager named MultiFRET, which allows for higher throughput real-time ratio-metric experiments.…”