Time-Resolved Three Dimensional Tracking of Individual GFP Molecules in Solution at Biologically Relevant Transport Rates

Abstract

Many important cellular functions such as intracellular signaling and protein trafficking involve three dimensional molecular motion. Monitoring these processes at the single molecule level requires the ability to track individual fluorescent emitters in high background environments at biologically relevant diffusion rates, and over several microns in all three dimensions. We have previously reported a custom confocal 3D tracking fluorescence microscope design capable of tracking single quantum dots (QDs) over several microns in X, Y, and Z in high background environments (Anal. Chem., 2008, 80, 9830-9834), and followed QD labeled antibodies bound to membrane receptors on live cells (Proc. of SPIE, 2009, 7185, 71850Z). While QDs are useful for obtaining long trajectories, their use as biological labels is generally limited to receptors that can be externally labeled or to permeabilized cells. GFP labeling circumvents these limitations, and as such, is the fluorescent label of choice for many intracellular fluorescence microscopy experiments. While CCD-based methods for 2D tracking of individual GFPs in live cells are well established, 3D tracking of single GFP labeled intracellular proteins in live cells will represent a tremendous technical achievement. Towards this end, here we show our 3D tracking methods can follow the Brownian diffusion of single GFPs in glycerol-water mixtures at rates comparable to intracellular protein traffic (D ∼ 1 um2/s). Trajectory durations are limited by GFP photostability, with some trajectories lasting for seconds. Unlike CCD-based tracking schemes, the arrival time of each photon is recorded with ∼400 picoseconds accuracy, enabling time-resolved spectroscopy to be performed on the molecules tracked. In particular, we demonstrate fluorescence correlation spectroscopy (FCS) of individual GFPs, with these FCS curves being obtained as we follow individual molecules moving through 3 dimensional space.