High spatial and temporal resolution using upconversion nanoparticles and femtosecond pulsed laser in single particle tracking

Recent advanced biophysical techniques allow us to monitor the cellular dynamics of biologically important molecules in real time. Bright, stable fluorophores are needed to accomplish this: photoblinking and photobleaching occurring in organic fluorophores and qdots make them an ill-suited option. In this study, we employed upconversion nanoparticles (UCNPs) since they exhibit no photobleaching or photoblinking. Another advantage of using UCNPs is that these particles absorb IR light (980 nm) and emit visible light (560 nm and 640 nm), which sig-nificantly eliminates background noise caused by autofluorescence. Moreover, excitation of UCNPs can occur using a CW-laser because they can be excited by wide-field illumination rather than requiring confocal illumination. Although a CW-laser would have been capable of exciting UCNPs, we were able to maximize the photon density and resulting number of photons emitted from UCNPs by employing a femto-second laser. Using a femto-second laser, we achieved 2.4 nm single-molecule localization accuracy with an exposure time of 2 ms. The UCNP particles and femto-second laser allowed us to stably monitor the molecular motors, kinesin and dynein, in cells.