High-Density Single Particle Tracking on the Plasma Membrane using High-Speed Hyperspectral Imaging

Abstract

We have developed a high-speed, line-scanning hyperspectral microscope (HSM) capable of acquiring 30 frames per second with 128 spectral channels per spatial pixel. The HSM allows simultaneous single particle tracking (SPT) of up to eight spectrally distinct quantum dots (QDs), improving the useable labeling density in SPT by nearly an order of magnitude as compared to single-color SPT. In addition, HSM imaging allows the spectral uniqueness of QDs within the same spectral family to be used for resolving ambiguities in trajectory reconstruction.We describe our physical instrument as well as the required analysis for hyperspectral SPT of multi-color QDs. Our analysis extends multi-emitter fitting previously developed in our lab for accurate localization of single fluorophores at higher density [Huang, F. et al., Biomed Opt Express 2011;2(5):1377] to three dimensions (x, y, lambda). Using a finite pixel 3D Gaussian estimate for their combined point spread function and spectral features, simultaneous spatial localization and spectral identification of QDs is achieved. The algorithm is implemented in CUDA to take advantage of parallel processing of GPUs. Trajectories are constructed with spatial and spectral localization information using a cost matrix approach for global optimization. Finally, squared displacements from single particle trajectories are used to build viscosity maps of the plasma membrane.We demonstrate high-density SPT by performing viscosity mapping of the plasma membrane, which provides important spatial and temporal information about local membrane effects on receptor mobility. We have applied HSM viscosity mapping to investigate the influence of the membrane environment on QD-IgE bound to FceRI. Changes in mobility and confinement of FceRI due to receptor crosslinking (receptor aggregation and signal initiation), latrunculin-beta treatment (induces actin depolymerization), and PMA treatment (induces actin polymerization) are examined.