Multicolor 3D Single Particle Tracking using Spectrally Displaced Localization

Single particle tracking (SPT) techniques such as sptPALM, uPAINT, and quantum dot tracking have given unprecedented insight into molecular dynamics in living cells. They allow monitoring the behavior of individual proteins in the plasma membrane as well as their molecular interaction with scaffold proteins at millisecond temporal resolution and high spatial resolution (u003c30 nm) by fitting the point spread function (PSF) of the individual emitter. While these SPT methods have been extended to study the temporal dynamics and co-organization of multiple proteins, conventional experimental setups used to perform multicolor imaging are typically limited to two simultaneous wavelengths. Increasing the number of colors requires additional filters for specific fluorescent tags and is usually performed at the expense of a loss of spatial or temporal resolution. This limits the minimum diffusion coefficient that can be measured, thereby degrading the ability to differentiate between molecular diffusion regimes like immobilization and confined diffusion.By employing a dual collection objective imaging configuration compatible with live cell imaging, we will present a single molecule tracking technique that allows for simultaneous 3D single particle tracking of multiple distinct species without compromising the spatio-temporal resolution. A dispersive element introduced into the second optical path induces a spectrally-dependent displacement, which is used to separate numerous fluorescent species of single emitters based on their emission spectra. We will present the systemu0027s spectral separation capabilities in a quantitative manner and validate the technique in fixed samples. We will conclude by demonstrating the ability to simultaneously track several fluorescing species in live cells.