Abstract
Live cells are three-dimensional environments where biological molecules move to find their targets and accomplish their functions. However, up to now, most single molecule investigations have been limited to bi-dimensional studies owing to the complexity of 3d-tracking techniques. Here, we present a novel method for three-dimensional localization of single nano-emitters based on automatic recognition of out-of-focus diffraction patterns. Our technique can be applied to track the movements of single molecules in living cells using a conventional epifluorescence microscope. We first demonstrate three-dimensional localization of fluorescent nanobeads over 4 microns depth with accuracy below 2 nm in vitro. Remarkably, we also establish three-dimensional tracking of Quantum Dots, overcoming their anisotropic emission, by adopting a ligation strategy that allows rotational freedom of the emitter combined with proper pattern recognition. We localize commercially available Quantum Dots in living cells with accuracy better than 7 nm over 2 microns depth. We validate our technique by tracking the three-dimensional movements of single protein-conjugated Quantum Dots in living cell. Moreover, we find that important localization errors can occur in off-focus imaging when improperly calibrated and we give indications to avoid them. Finally, we share a Matlab script that allows readily application of our technique by other laboratories.
Highlights
Live cells are three-dimensional environments where biological molecules move to find their targets and accomplish their functions
Wide-field fluorescence detection is obtained through a dichroic mirror and an emission filter, an EMCCD camera (Andor Ixon X3), and two laser sources for fluorescence excitation (532 nm for fluorescent beads and 488 nm for Quantum Dots (QDs) excitation, see supplementary materials for a detailed description of the setup)
As reported previously[51], we observed that the number of rings and their diameter increased as we moved the focal plane deeper inside the sample (Fig. 1a)
Summary
Live cells are three-dimensional environments where biological molecules move to find their targets and accomplish their functions. To address the need for accurate localization in all three dimensions, many techniques have been recently proposed for accurate axial position determination of single molecules Among these techniques, on one hand, bifocal imaging[15] has been demonstrated to reach few nanometres accuracy using fluorescent beads, but limited to about half micron depth, whereas multiplane imaging techniques[16,17,18,19,20] can span thicker volumes but at the expense of the axial localization precision. A different approach that does not rely on the changes of the PSF shape with the axial position is adopted in Parallax[29] Through this technique nanometre localization in all three coordinates was achieved for fluorescent beads over 1 μ m range. Without an automated procedure to measure the radius of the diffraction pattern, this method is time-consuming making it difficult to be applied
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