Anal. Chem. 2008, 80, 9830–9834
Confocal, Three-Dimensional Tracking of Individual Quantum Dots in High-Background Environments Nathan P. Wells, Guillaume A. Lessard, and James H. Werner* Center for Integrated Nanotechnologies (MPA-CINT), Los Alamos National Laboratory, Los Alamos, New Mexico 87545 We demonstrate a custom confocal fluorescence-microscope that is capable of tracking individual quantum dots undergoing three-dimensional Brownian motion (diffusion coefficient ∼ 0.5 µm2/s) in environments with a signalto-background ratio as low as 2:1, significantly worse than observed in a typical cellular environment. By utilizing a pulsed excitation source and time-correlated single photon counting, the time-resolved photon stream can be used to determine changes in the emission lifetime as a function of position and positively identify single quantum dots via photon-pair correlations. These results indicate that this microscope will be capable of following protein and RNA transport throughout the full three-dimensional volume of a live cell for durations up to 15 s. Single-molecule detection, particularly via laser induced fluorescence, has matured over the last two decades to a point that in vivo assays are routinely accessible.1-4 Of particular interest to biologists and biophysicists are in vivo observations of proteins, RNA, DNA, and viruses performing biological functions.3,5-7 To this extent, observing single-molecule fluorescence via camera based wide field microscopy has been used to study biological events in two-dimensions (2D), particularly membrane dynamics,4,5,8-10 and in at least one instance has been extended to three-dimensions (3D) with limited (e1 µm) depth of field.11 While camera based techniques can have very good 2D localization accuracy (
