There are several ways to study particles smaller than the diffraction limit if you pin them down to a substrate — atomic force microscopy is but one possible technique to use. Counting or measuring such tiny particles while they are moving about freely in solution, however, does not work so well. Light-scattering methods are used but lack high sensitivity. Fluorescence correlation spectroscopy and particle tracking with video imaging and software analysis are feasible, but these techniques are not as effective as they could be because particles can move in and out of the imaging area.A schematic illustrates a setup used to conduct both particle tracking and fluorescence correlation spectroscopy experiments on nanoparticles confined within small droplets. Restricting the particles prevented them from wandering out of the detection area. Images reprinted with permission of the American Chemical Society.Now researchers at the University of Washington in Seattle, led by Daniel T. Chiu, have developed a technique to study nanometer-scale particles by first trap-ping them in a very small volume of solution — droplets only about 10 μm in diameter.The investigators placed several aqueous droplets with a scattering of their target particles — rat synaptic vesicles or nanobeads — onto a coverslip, then identified those droplets that had between one and five particles to measure. They excited the bead or the dye-tagged vesicles with a 488-nm beam from a Coherent laser or with a 633-nm beam from a Coherent HeNe laser. The researchers designed the system to split and recombine the beams to provide both confocal and epifluorescence illumination. They used a Nikon microscope with a 1.45-NA objective to direct the beam and to collect the emissions.For particle tracking, the scientists used a CCD camera made by Roper Scientific of Tucson, Ariz.; for fluorescence correlation spectroscopy, they used an avalanche photodiode from PerkinElmer Optoelectronics of Fremont, Calif., along with an autocorrelator from Correlator.com of Bridgewater, N.J.Using a CCD camera, researchers tracked the diffusion path (gray lines) of a fluorescent bead that was confined within a 20-μm-diameter aqueous droplet.According to the researchers, the particle-tracking technique works best with bright particles and with particles larger than about 100 nm, which are less susceptible to photobleaching or to damage caused by the constant illumination of the imaging area.On the other hand, correlation spectroscopy is better suited to smaller, dimmer particles because a smaller region of the droplet is illuminated and, thus, the sampling rate can be higher.Images acquired with the particle-tracking technique showed the path of a single bead as it moved across the droplet — not the size and shape. Therefore, the investigators had to calculate the optimum exposure time and frame rate to maximize detection sensitivity and to minimize blurring. In the fluorescence correlation spectroscopy experiments, they found that several factors affected accurate sizing. One was the proximity of the particles to the inside wall of the droplet: The wake caused by the particle in motion increases the friction between the particle and the wall, slowing the particle and, therefore, affecting measurements. Other factors that affect sizing and counting include surface charges of the particles being studied, the surface properties of the substrate, the ionic and buffer strength of the solution, and the properties of any surfactants that might be used. The investigators noted, however, that it was not critical to eliminate these and other factors as long as only the particle’s trajectories in the center of the droplet were used — a condition that they satisfied by “parking” the laser probe volume in the center of the droplet.According to Chiu, the investigators will use the droplet technique to study synapses from other animals as well as mitochondria and other subcellular organelles.“We want to integrate this capability with other droplet microfluidic techniques we developed as well as with other single-molecule separation and analysis techniques that we have been working on,” he said.Analytical Chemistry, ASAP Edition, March 26, 2008, doi: 10.1021/ac8000385.