Methods

1. Single-molecule Förster Resonance Energy Transfer (smFRET)

Single-molecule Förster Resonance Energy Transfer is a well-established technique to measure distances between two fluorophores with high spatial and temporal resolution. Theodor Förster showed in 1948 that the energy from an excited donor fluorophore can be transferred to an acceptor fluorophore as long as the emission spectrum of the donor overlaps with the absorption spectrum of the acceptor and both fluorophores are in close proximity (<10 nm). The proportion of transferred energy E depends strongly on the distance r between the two fluorophores (E is proportional 1/r^6) making the technique useful to study, for example, conformational changes and dynamics of enzymes such as DNA polymerases.

FRET_scheme(A) A donor fluorophore (green) is excited by a light source and attached to a flexible biomolecule. As long as the acceptor dye (red) is far away (>10 nm) from the donor, the emission spectrum of the donor is not significantly altered (zero or low FRET). (B) If the distance between the fluorophores decreases, more energy is transferred from the donor to the acceptor and the entire emission spectrum shows a significant contribution of the acceptor flurophore emitting lower energy photons. (C) FRET efficiency plotted as a function of the distance between the fluorophores for various Förster radi (R0). The Förster radius can be calculated for specific dye pairs and is normally between 5 and 7 nm.

2. Alternating Laser Excitation (ALEx)

A very important addition to the concept oES_histogramf single-molecule FRET was the development of ALEx: Instead of exciting only the donor fluorophore and measuring the energy transfer to the acceptor, the acceptor is directly excited in order to probe its existence.  If there is, for example, no red fluorescence after red excitation, the acceptor is already bleached and is not participating in FRET any more. The detection of red fluorescence after red excitation allows the introduction of a new parameter called stoichiometry, which relates the green fluorescence after green excitation to the overall fluorescence emission after green and red excitation. The figure shows an ES histogram allowing the clear separation of species which can not easily be resolved on the E histogram alone.

3. Confocal microscopy for detection of single molecules

The laser-based excitation is alternating between green and a red excitation. The key component in confocal microscopy is the dichroic (or polychroic) mirror or which allows the convolution of a diffraction limited excitation volume with a detection volume. The efficient rejection of out-of-focus light is required to achieve high signal-to-noise ratios sufficient for detection of single molecules. A immersion objective with high numerical aperture focuses the excitation laser light into a very small volume (~1fl), where the laser light excites randomly diffusing fluorophores. The red shifted fluores- cence light is collected by the same objective and can now, due to its longer wavelength, pass the dichroic mirror, which is fully reflective for the excitation laser light.  After spatially filtering of the beam using a pinhole, the fluorescent light is then separated with an additional dichroic mirror and focussed onto two avalanche photodiodes.

4. Total internal reflection fluorescence microscopy (TIRF)

In TIRF microscopy the laser light is focussed into the rim of backfocal plane of the objective in a way that the laser light is totally reflected at the glass/ water interface. An evanescent field is created whose intensity decays within a few hundred nanometres. Thereby, only a small volume is illuminated within the solution. The striking feature of TIRF is the possibility to excite fluorophores close to the surface over an area of roughly 50 by 50 micrometer and image the fluorescence using an emCCD camera. In contrast to confocal microscopy, which allows only the detection of one molecule at a time, we can detect hundreds of molecules in parallel with TIRF microscopy. The setup in Wageningen uses a four-colour laser engine and a three-channel detection as shown below.

TIRF_microscopeWageningen