PMC

Trapping of individual particles of TMV. (a) Measured (Right) and pseudofree (Left) trajectories of 13 trapped particles of TMV. Each particle was trapped for 6.8 s (2,000 video frames at 3.4 ms per frame). The pseudofree trajectories are offset for clarity. (b) Diffusion coefficients along the x and y axes for the 13 particles trapped in a. If there were no statistical errors in the measurements, the data points would fall along the line. The rms deviation (rmsd) of D_x from D_y, 〈(2(D_x − D_y)/(D_x + D_y))²〉^1/2, is 9%, whereas the rmsds of D_x and D_y from their ensemble-averaged values are 28% and 26%, respectively, indicating heterogeneity in the ensemble above the noise level of the measurement. The square indicates the value from dynamic light scattering.

Glass microfluidic cell for the ABEL trap. (a) Trapping region, showing the patterned glass cell. Molecules are trapped in the center. Four channels ≈17 μm deep (the regions shaped like a bird’s beak) extend to the edge of the image and terminate in macroscopic electrodes. (Scale bar, 100 μm.) (b) The microfluidic cell sits above the oil-immersion objective of an inverted optical microscope capable of observing single molecules. The lower part of the cell is formed by a glass or fused silica slide. The top and bottom of the cell can be separated for cleaning or surface-treatments. The aqueous solution (blue) containing fluorescent biomolecules (red dots) sits above the coverslip and is confined by the cell material (shown transparent in this image). Molecules in the trapping region are confined to a thin fluid layer several hundred nanometers thick, preventing diffusion out of the focal plane of the microscope. The voltages applied across the electrodes provide the electrokinetic forces to counteract Brownian motion.

Trapping of individual fluorescent objects. (a) Histogram of intensities of individual trapped fluorescently labeled vesicles. The vesicles had an average diameter of 100 nm and were composed of egg-phosphatidylcholine with 1 part in 10⁵ of the fluorescent lipid N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine. A total of 26 vesicles were trapped, for a cumulative 2,690 video frames (at 6.5 ms per frame). Intensities were computed from a sliding average with a 26-ms window. Peaks indicate vesicles containing zero, one, or two fluorophores, as well as an unresolved contribution from more highly labeled vesicles. Some intensity values are negative because the background subtraction was set to yield zero mean intensity when the trap was empty. (Inset) A typical trajectory showing two-step photobleaching (arrows) of a vesicle containing two fluorophores. The vesicle was trapped with only one active fluorophore for ≈700 ms (between arrows). (b–d) Time-averaged images of trapped single molecules and CdSe nanocrystals. (b) A single molecule of B-phycoerythrin (average of 500 images taken over 2.2 s). (c) GroEL labeled with Cy3 (average of 10,000 images taken over 45 s). During this interval, several single molecules were sequentially trapped, eventually photobleached, and released from the trap. (d) A single CdSe fluorescent nanocrystal (average of 20,000 images taken over 90 s). (Scale bar in b–d, 2 μm.)