Professor Andrew Plested
Leibniz Research Institute for Molecular Pharmacology, Humboldt University Berlin
The Plested lab investigates the characterization of receptors relevant to neuronal systems. They apply multiple techniques to image isolated neurons, including a combination of electrophysiology and fluorescence live-cell imaging.
One of their areas of study involves measuring Foerster Resonance Energy Transfer (FRET) upon electrical stimulation of the neurons. They label individual relevant structures and receptors with suitable fluorophores and observe the FRET signal in the dendritic region of cells of interest. This enables them to obtain information about the state, localization, and behavior of channels, and particularly their colocalization in the nanometer range.
The individual interactions the group is interested in observing only last for a very brief period of time (10-100 ms). Therefore, to capture the weak signal arising from the energy transfer from a donor fluorophore to an acceptor molecule, very fast imaging is required. This means the number of photons available for detection is very limited and an extremely sensitive imaging device is essential.
Their currently used EMCCD camera – although sensitive enough for detection – is only able to image a very small field of view which makes experimental measurements a time-consuming exercise. FRET measurements are also limited by the 30 ms read time per frame of the EMCCD camera, achieving just 30 fps (corresponding to ~15 fps FRET) at full field. Furthermore, binning had to be used to increase frame rate, reducing spatial resolution.
The Prime 95B lets us look at 5× the FOV at about twice the speed, but with the same sensitivity as our previous EMCCD system.