It’s purpose is to immobilize single molecules, proteins or nanoparticles in solution by active canceling their three dimensional Brownian motion. The nano-object is monitored by fluorescence imaging. The position of the nano-object is determined with respect to a target position and used for a feedback voltage applied to four electrodes. The electric field or the induced electroosmotic flux pushes the nano-object back and compensates the Brownian motion.
A microfluidic device constricts the diffusion of the nano-object to the x and y
dimensions. The chamber is made of transparent PDMS. Four 20 µm deep channels with the electrodes are arranged perpendicular to confine the nano-object within the trap region with only 1 µm thickness.
Widefield illumination of the trap region and a fast sensitive CCD camera to detect the position of the fluorescent nano-object was sufficient to immobilize a single DNA molecule, a tobacco mosaic virus (300 nm length), a liposome (100 nm radius) or even a 10 nm polystyrene bead. For trapping smaller objects like single fluorophores or soluble proteins, faster versions of the ABEL trap have been developed.
Application to a single FoF1-ATP synthase
(monitoring conformations of a single membrane transporter)
In collaboration with AEC and WEM we modify the ABEL trap to immobilize a single liposome with one embedded membrane proteine. Therefore the FoF1-ATP synthase is labeled with two fluorophores to monitor the internal rotary motion by confocal single-molecule FRET. and is transported to the target position in the ABEL trap.
Actually we can trap 100 nm liposomes (see image on the left: the liposome is confined for seconds before the fluorescence intensity falls below a trapping threshold due to photobleaching; then the liposome diffuses away in the upper right direction) and are implementing the combined FRET @ ABELtrap experiment.
Movie of an Atto680-labeled liposome trapped for several seconds in the ABEL trap (Flash file):
The faster versions of the ABEL trap now use a confocal laser pattern and single-photon-based feedback from APDs. Localization analysis and electrode feedback are implemented in a FPGA chip. Fluorescent lipids, which might interfere with the single-molecule FRET measurements of proteins, are not required anymore because the feedback utilizes the photons of the single FRET fluorophors. We have built a modified ‘hardware ABELtrap’ and have captured 20-nm fluorescent beads for more than 8 seconds in contrast to their mean diffusion time of 10 ms within the PDMS microfluidics. A time trajectory of an ABELtrapped fluorescent 20-nm bead is shown below.
Now we study the conformational dynamics of single FoF1-ATP synthases at different ATP concentrations for the first time.