Limitations of fluorescence for particle tracking

Fluorescent dyes offer a limited photon budget (@fo2020Tracking and Analyzing the Brownian Motion of Nano-objects Inside Hollow Core Fibers): the fluorescence lifetime limits the maximum rate at which photons can be emitted, and photobleaching limits the total number of photons that can be emitted overall. Therefore, there is a tradeoff between amount of photons per second and overall length of the experiment. In the case of nanoparticle tracking, this affects the accuracy of the localization and the number of frames over which a particle can be tracked through fluorescence (see: limitations of nanoparticle tracking analysis).