Multi-Color Quantum Dot Tracking using a Hyperspectral Microscope Detects Protein Interactions below the Diffraction Limit
One method that cells use to sense and respond to their environment is through the binding of small ligands to proteins that reside in and pass through the cell's membrane. Often, this changes the affinity of these membrane proteins, causing them to bind together and this initiates a signaling process in the inside of the cell. This could, for example, result in cell division or movement. However, exactly how this happens and the dynamics of these interactions have not been well characterized since the spatial scale of these interactions (~ 10 nm) is below the resolution limit of the light microscope (~ 300 nm). Light microscopes together with fluorescent markers have long been used as a method to observe protein organization and dynamics in living cells but have a resolution limited by diffraction and the wavelength of visible light (400-700 nm).
When proteins are fluorescently labeled in such a way that isolated, individual fluorescent tags can be observed, their position can be estimated with much better precision (~ 10 nm) than the resolution of the microscope. However, this method doesn't work well when these labeled proteins come in close proximity, which is exactly case when studying protein interactions. The research group of Prof. Keith Lidke has developed a method using spectral imaging that is able to overcome this limitation. They designed and built a novel high-speed hyperspectral microscope (HSM) that can be used to image and track 8 spectrally distinct species of fluorescent quantum dots (QDs) labels at up to 30 frames per second. The distinct emission spectra of the QDs allows protein position determination with ~ 10 nm precision even when several fluorescently labeled proteins are clustered at spatial scales below the resolution limit. The microscope is now being used to measure the lifetime of protein-protein interactions in both normal cells and cells with mutated proteins that have been implicated in cancer.
For more information see the article at: Multi-Color Quantum Dot Tracking Using a High-Speed Hyperspectral Line-Scanning Microscope and the iXon 860 EMCCD powers direct video recording of Cell Signalling Complexes.
University of New Mexico
Department of Physics and Astronomy
1919 Lomas Blvd. NE
Albuquerque NM 87131-0001