Abstract
Nanoscale particle tracking in three dimensions is crucial to directly observe dynamics of molecules and nanoparticles in living cells. Here we present a three-dimensional particle tracking method based on temporally focused two-photon excitation. Multiple particles are imaged at 30 frames/s in volume up to 180 × 180 × 100 µm3. The spatial localization precision can reach 50 nm. We demonstrate its capability of tracking fast swimming microbes at speed of ~200 µm/s. Two-photon dual-color tracking is achieved by simultaneously exciting two kinds of fluorescent beads at 800 nm to demonstrate its potential in molecular interaction studies. Our method provides a simple wide-field fluorescence imaging approach for deep multiple-particle tracking.
Highlights
In biological applications particle tracking is an essential method on investigating a wide range of molecular behaviors and interactions in living cells, such as protein movement and interactions, RNA transport [1,2,3,4]
The temporal focusing two-photon microscope is first characterized with fluorescent nanospheres (100 nm)
The point spread function (PSF) (Fig. 2(a)) of one single bead shows full width at half maximum (FWHM) of 0.5 μm (Fig. 2(a)), which is at the diffraction limit
Summary
In biological applications particle tracking is an essential method on investigating a wide range of molecular behaviors and interactions in living cells, such as protein movement and interactions, RNA transport [1,2,3,4]. Based on super resolution imaging modalities such as photoactivated localization microscopy (PALM) [11] and stochastic optical reconstruction microscopy (STORM) [12], point spread function (PSF) engineering approach encodes the z position information into the shapes of the microscope’s PSF, such as astigmatism, double helix, Airy function, saddle-point and Tetrapod PSFs [13,14,15,16,17,18]. This approach can track nanoscale emitters over axial range up to 20 μm. This approach has achieved Ångström accuracy [23] and over 100 μm range in axial dimension [24]
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