Single Molecule FRET
Fluorescence Microscopy is an essential tool for modern biological experimentation. It allows for non-ambiguous detection for molecules of interest while significantly improving the effective resolving power of diffraction limited optics. For our in vitro, single molecule experiment, we use total internal reflection microscopy (TIRF), which further eliminates background noises by exciting only a single layer on the sample substrate and is used for our smFRET studies.
A) Our optical setup: 532 nm and 638 nm lasers are used for TIRF excitation. The image is collected through a 60X water immersion, 1.2 N.A. objective and then split by a Dualview optical splitter (645 nm dichroic mirror). The donor and acceptor signals then pass through optical filters (donor: 585/70 bandpass; acceptor: 655 longpass) before detection by an emCCD camera. B) Total internal reflection at the quartz:water interface produces an evanescent wave that excites only those molecules within ~200 nm of the slide surface.
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Image credit: Jake
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Förster resonance energy transfer (FRET), or more commonly (improperly) known as fluorescence resonance energy transfer (FRET), is a nonradiative, electric dipole-dipole interaction between two molecules that involves transfer of energy from one (called donor) to the other (called acceptor). In particular, single-molecule FRET (or smFRET) has emerged as a useful tool for detecting changes in the separation between two molecules of 10-100Å, a scale pertinent to molecular biological events.
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Image from Wikipedia
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The following figures illustrates the distance-FRET relationship and how a typical experiment looks like. In a FRET experiment, the donor is excited by laser, meaning that they gain energy. When a donor is close to an acceptor, FRET occurs, and the energy transfer efficiency increases as the distance between them decreases, resulting in a drop of donor’s energy (as indicated by its fluorescence intensity) and an increase of the acceptor’s energy (so that the acceptor, which was not excited, now is excited and becomes fluorescent). Different wavelength filters are used to distinguish the emissions from donors and acceptors, respectively. The FRET is then calculated from the donor intensity (time traces from a spot of interest, usually the location of a fluorescent molecule that’s tagged on the targets such as protein or DNA) and acceptor intensity.
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Image from German Cancer Research Center
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Image credit: Jake
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With smFRET, we are able to probe the following aspects of protein-DNA conformations and dynamics –
Protein-DNA Interactions:
- Protein Stoichiometries
- Dynamics of Protein Assembly
- Complex Conformational Changes
- Mobility of Proteins on DNA
- Protein Conformational Changes
Protein-DNA Interactions:
- Protein Stoichiometries
- Dynamics of Protein Assembly
- Complex Conformational Changes
- Mobility of Proteins on DNA
- Protein Conformational Changes
Our current and past studies using smFRET can be found below, or navigated through the ‘Single Molecule FRET’ category on the left sidebar.
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