Abstract
In this work, we present a 3D single-particle tracking system that can apply tailored sampling patterns to selectively extract photons that yield the most information for particle localization. We demonstrate that off-center sampling at locations predicted by Fisher information utilizes photons most efficiently. When performing localization in a single dimension, optimized off-center sampling patterns gave doubled precision compared to uniform sampling. A ~20% increase in precision compared to uniform sampling can be achieved when a similar off-center pattern is used in 3D localization. Here, we systematically investigated the photon efficiency of different emission patterns in a diffraction-limited system and achieved higher precision than uniform sampling. The ability to maximize information from the limited number of photons demonstrated here is critical for particle tracking applications in biological samples, where photons may be limited.
Highlights
Single-particle tracking (SPT) [1] has led to numerous advances in unveiling sophisticated intracellular biophysical events, including diffusion of membrane proteins [2], transportation of intracellular vesicles [3], and viral internalization events [4,5]
Hou et by al. [12], a focused laser spot is guided sion of achieving isotropicby information-efficient sampling in alltothree an electro-optic deflector (EOD)
This study showed that implementing information-efficient laser scanning patterns led to dramatic improvements in precision in all three dimensions
Summary
Single-particle tracking (SPT) [1] has led to numerous advances in unveiling sophisticated intracellular biophysical events, including diffusion of membrane proteins [2], transportation of intracellular vesicles [3], and viral internalization events [4,5]. While the use of artificial particles [8] can produce a higher flux of photons and improve localization precision, conventional organic fluorophores and fluorescent proteins remain essential in biophysical studies These probes can only yield a finite number of photons before undergoing irreversible photobleaching, so it is crucial to maximize the information available from this limited number of photons. An under-explored avenue for increasing precision is adjusting the excitation pattern around the emitter to get beyond the localization limit described above This type of advance is only possible if the particle position is known a priori, at least to some degree of certainty. This theory indicated that the optimized sampling pattern is non-uniform, and the particle position should not be directly sampled, but did not provide experimental support Both of these works suggest that non-uniform illumination shows promise for improved localization with a limited number of photons.
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