Accurately measuring single fluorescent particles is often a challenge because their Brownian motion makes it difficult to keep them in focus for prolonged periods.As a result, investigators are forced to gather data from many samples to generate reliable statistics or to immobilize the particle with a gel, which might influence its dynamics and thus distort the results.The fluorescence from a quantum dot sample with the feedback tracking turned on. By using tracking, the researchers achieved a significant increase in observation time. Images courtesy of Kevin McHale.Researchers led by Kevin McHale at California Institute of Technology in Pasadena report that they have developed a method that keeps molecules with a fluorescent tag in sharp focus while allowing their 3-D motion to be tracked in real time and enabling variations in their fluorescent lifetimes to be detected.During experiments with the technique, the researchers tracked the Brownian motion of 20-nm CdSe/ZnS quantum dots in water that was not treated with viscous agents to slow the movement. A 532-nm Melles Griot diode laser was the excitation source. The 3-D rotation of the beam in the quantum dot sample was a critical part of the technique.The researchers achieved 2-D tracking along the X-Y axes by deflecting two laser beams in a circular orbit with acousto-optic modulators. The third dimension, which was the most difficult part, was achieved by switching the power of the two beams, which created a spot that moved back and forth on the Z-axis. The technique allowed for a clean signal and for 3-D tracking of the fast-moving particles.The three-dimensional motion of a quantum dot spans tens of micrometers along each axis. The plot shows the projection of the quantum dot’s motion onto the X-Y, X-Z and Y-Z planes as shadows in those planes. The beginning and ending coordinates of the trajectory are marked by the green and red dots, respectively. The black line shows a coarse version of the trajectory, to make the motion easier to discern.The laser beam was focused by a Carl Zeiss 1.2-NA water-immersion lens into the 25-μl liquid sample. The lens was a particularly important component because it transmitted the tracking laser beam into the sample with very little aberration, allowing good position estimates throughout the deep samples. Fluorescence was then measured by a pair of PerkinElmer photon-counting avalanche photodiodes, which are sensitive to single photons and have very high time resolution. A pair of Stanford Research Systems’ digital signal processing lock-in amplifiers demodulated the fluorescence signal, and a computer recorded the photon arrival times on a GuideTech time interval analyzer with 500-ps timing resolution.The researchers said that, in addition to tracking the movement of the quantum dots, the technique allowed measurements of photon antibunching — the minimum time delay between photons — on single fluorophores. Although this has been done with individual fluorophores attached to a glass surface (which could influence the results), it has not been done in a free solution. Photon correlations as fast as ~10 ns were resolved, a timescale that could be useful in studying certain proteins that have processes that are as fast or faster. McHale said that the tracking technique has been used successfully on large nucleic acid molecules tagged, in this case, with an organic dye instead of quantum dots. The team’s goal is to track proteins to determine how they behave in solution in terms of intramolecular motion and intermolecular interactions. Such information eventually could lead to insights into why certain proteins fail to interact and the diseases that result.Nano Letters, November 2007, pp. 3535-3539.