Single molecule measurements and biological motors

TIRF Application Examples

To illustrate what is possible with this technology, we present some example images and animations from our own research. We perform both prism-style (for in vitro experiments) and objective style (for cell experiments).

Using prism-style TIRF: single molecules in vitro

Cy3-EDA-ATP binding to myosin

Actin filaments diffusing in the evanescent wave.

The animation at left(not shown in real time) shows the use of single molecule fluorescence to observe intermolecular interactions. Single molecules of Cy3-EDA-ATP (the red spots) can be seen binding to, and releasing from, a myosin coated surface. This experiment is a proof of principle demonstration on the way to our goal of combining TIRF and optical tweezers measurements to study mechanochemical coupling in myosin. However, with this technique alone we can measure the rate of ADP release from different types of myosin. In this case a relatively high surface density of myosin was used because of the low occupancy of the nucleotide-bound state. The labelled nucleotide, in contrast, was present at less than 10 nM (at higher concentrations the background fluorescence is too high). See later section for more details.

The animation on the right is a demonstration of the properties of the evanescent wave. Actin filaments were labelled with Rhodamine phalloidin and added to a flow cell where they were visualised by TIRF. The very shallow penetration depth is evident from the way the filaments "flicker" as they diffuse up and down in the evanescent wave. This also illustrates the flexibility of the actin filaments.

Objective TIRF: Single molecules in living cells

One of the most exciting possibilities with TIRF is the ability to visualise single molecules in living cells. Here we illustrate how TIRF allows us to focus on the parts of the cell which are near to the surface, and to detect and monitor single molecules in this region.

Comparison of epifluorescence (left) vs. TIRF. The same cell (a myoblast) is visualised by the two techniques. The cell has been transfected with a construct expressing a myosin I GFP fusion construct. Note the greater detail of the structures in the cellular lamella visible in the TIRF image. Scale bar: 10 μm. Click on image for full size version.

This image is a still from a video showing the visualisation of single molecules in a living cell. Shown is the lamellar region of a myoblast expressing a myosin X pleckstrin homology domain GFP fusion protein. As the initially high background bleaches, discrete spots become visible. Note how these spots appear and disappear as the individual molecules bind to the membrane, are released, or possibly photobleach. Scale bar: 10 μm. Click on image for full-size AVI file.

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