Microscope: Fluorescence, Single-Molecule /Patch Clamp
- Simultaneously quantifies molecular interactions and functional outcomes
- Uses 200x magnification
- Allows single-channel and whole-cell recording
At EMSL, researchers use the single-molecule fluorescence/patch clamp microscope to combine high-sensitivity fluorescence imaging simultaneously with physiological measurements to identify real-time molecular interactions and conformational changes of cell-membrane receptors and their functional consequences.
This system is one of the few worldwide that provides the sensitivity needed to quantify molecular interactions at the level of single or few molecules simultaneously with the functional outcome of these interactions. Constructed by EMSL scientists using commercial components, the system consists of a wide-field microscope and a set of lasers coupled to a high-sensitivity charge-coupled device camera, combined with a patch-clamp amplifier for single-channel and whole-cell recording.
Using 200X magnification and several laser lines with a precise power control, single or a few molecules can be imaged at small areas of the cell membrane. The conformational changes within the receptor complex, and the molecular interactions of the receptor with its ligands and modulators, are quantitatively identified by fluorescence resonance energy transfer (FRET). Correlating FRET efficiency with the ionic-current for ion channels, or with calcium imaging for other receptors, can identify the underlying mechanisms of ion channels and receptors function.
EMSL researchers have used this technology to study the N-methyl-D-aspartate (NMDA) receptor, a ligand-gated calcium channel, in order to identify the molecular interactions that govern the channel kinetic behavior. The precise activity of the NMDA receptor is crucial to the formation of neuronal connections during development, and to the formation of neuronal networks during learning and memory. This technology has the potential to identify the mechanisms that determine the precise behavior of the receptor, which cannot be observed using any other approach.
In addition, this technology has been used to study the epidermal growth factor receptor (EGFR). Using the single-molecule fluorescence approach, the molecular interactions between the receptor and other molecules at the cell membrane were identified and quantified in real time and in living cells. The EGFR plays an important role in mediating the flow of information from the extracellular to the intracellular environment, thus controlling cell fate. Identifying the cellular behavior of this receptor at the single-molecule level has unraveled mechanisms of action that were hidden using other approaches.
Researchers external to EMSL may use this system for their research, provided they are experienced using patch clamp techniques and are familiar with single molecule fluorescence imaging.
The study of individual proteins in the membrane of living cells is enabled by the presence of a tissue culture facility adjacent to the microscopes. The facility supports the growth of eukaryotic primary cells or cell-lines, including human cell lines. In addition to a bio-hood, the facility includes CO2 incubators, a centrifuge, 37°C water bath,n and a shaker.
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