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arXiv:2504.04611 · Nature Communications

A Bayesian approach towards atomically-precise localization in fluorescence microscopy

Yuqin Duan, Qiushi Gu, Hanfeng Wang, Yong Hu, Kevin C. Chen, Matthew E. Trusheim, Dirk Englund

Published Oct 21, 2025• 4 citations
arXivHTMLPDF

Abstract

Super-resolution microscopy has revolutionized the imaging of complex physical and biological systems by surpassing the Abbe diffraction limit. Recent advancements, particularly in single-molecule localization microscopy, have pushed localization below nanometer precision, by applying prior knowledge of correlated fluorescence emission from single emitters. However, achieving a refinement from 1 nm to 1 Ångström demands a hundred-fold increase in collected photon signal. This quadratic resource scaling imposes a fundamental barrier in single-molecule localization microscopy, where the intense photon collection is challenged by photo-bleaching, prolonged integration times, and inherent practical constraints. Here, we break this limit by harnessing the periodic nature of the atomic lattice structure. Applying this discrete grid imaging technique (DIGIT) in a quantum emitter system, we observe an exponential collapse of localization uncertainty once surpassing the host crystal's atomic lattice constant. We further applied DIGIT to a large-scale quantum emitter array, enabling parallel positioning of each emitter through wide-field imaging. Collectively, these advancements establish DIGIT as a competitive tool for achieving unprecedented, precise measurements, ultimately paving the way to direct optical resolution of crystal and atomic features within quantum and biological systems.

DOI
10.1038/s41467-025-64083-w

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Super-resolution microscopy has revolutionized the imaging of complex physical and biological systems by surpassing the Abbe diffraction limit. Recent advancements, particularly in single-molecule localization microscopy, have pushed localization below nanometer precision, by applying prior knowledge of correlated fluorescence emission from single emitters. However, achieving a refinement from 1 nm to 1 Ångström demands a hundred-fold increase in collected photon signal. This quadratic resource scaling imposes a fundamental barrier in single-molecule localization microscopy, where the intense photon collection is challenged by photo-bleaching, prolonged integration times, and inherent practical constraints. Here, we break this limit by harnessing the periodic nature of the atomic lattice structure. Applying this discrete grid imaging technique (DIGIT) in a quantum emitter system, we observe an exponential collapse of localization uncertainty once surpassing the host crystal's atomic lattice constant. We further applied DIGIT to a large-scale quantum emitter array, enabling parallel positioning of each emitter through wide-field imaging. Collectively, these advancements establish DIGIT as a competitive tool for achieving unprecedented, precise measurements, ultimately paving the way to direct optical resolution of crystal and atomic features within quantum and biological systems.
We report on image processing techniques and experimental procedures to determine the lattice-site positions of single atoms in an optical lattice with high reliability, even for limited acquisition time or optical resolution. Determining the positions of atoms beyond the diffraction limit relies on parametric deconvolution in close analogy to methods employed in super-resolution microscopy. We develop a deconvolution method that makes effective use of the prior knowledge of the optical transfer function, noise properties, and discreteness of the optical lattice. We show that accurate knowledge of the image formation process enables a dramatic improvement on the localization reliability. This allows us to demonstrate super-resolution of the atoms' position in closely packed ensembles where the separation between particles cannot be directly optically resolved. Furthermore, we demonstrate experimental methods to precisely reconstruct the point spread function with sub-pixel resolution from fluorescence images of single atoms, and we give a mathematical foundation thereof. We also discuss discretized image sampling in pixel detectors and provide a quantitative model of noise sources in electron multiplying CCD cameras. The techniques developed here are not only beneficial to neutral atom experiments, but could also be employed to improve the localization precision of trapped ions for ultra precise force sensing.
The fundamental understanding of biological pathways requires minimally invasive nanoscopic optical resolution imaging. Many approaches to high-resolution imaging rely on localization of single emitters, such as fluorescent molecules or quantum dots. Additionally, the exact determination of the number of such emitters in an imaging volume is essential for a number of applications; however, in standard intensity-based microscopy it is not possible to determine the number of individual emitters within a diffraction limited spot without initial knowledge of system parameters. Here we explore how quantum measurements of the emitted photons using photon number resolving detectors can be used to address this challenging task. In the proposed new approach, the problem of counting emitters reduces to the task of determining differences between the emitted photon distribution and the Poisson limit. We show that quantum measurements of the number of photons emitted from an ensemble of emitters enable the determination of both the number of emitters and the probability of emission. This method can be applied for any type of single-photon emitters. The scaling laws of this new approach are presented by the Cramer-Rao Lower Bounds, and this technique has great potential in quantum optical imaging with nanoscopic resolution.
Quantum simulations with ultracold atoms typically create atomic wavefunctions with structures at optical length scales, where direct imaging suffers from the diffraction limit. In analogy to advances in optical microscopy for biological applications, we use a non-linear atomic response to surpass the diffraction limit. Exploiting quantum interference, we demonstrate imaging with super-resolution of λ/50 and excellent temporal resolution of 500 ns. We characterize our microscope's performance by measuring the ensemble averaged probability density of atoms within the unit cells of an optical lattice, and observe the dynamics of atoms excited into motion. This approach can be readily applied to image any atomic or molecular system, as long as it hosts a three-level system.
Far-field optical microscopy beyond the Abbe diffraction limit, making use of nonlinear excitation (e.g. STED), or temporal fluctuations in fluorescence (PALM, STORM, SOFI) is already a reality. In contrast, overcoming the diffraction limit using non-classical properties of light is very difficult to achieve due to the fragility of quantum states of light. Here, we experimentally demonstrate superresolution microscopy based on quantum properties of light naturally emitted by fluorophores used as markers in fluorescence microscopy. Our approach is based on photon antibunching, the tendency of fluorophores to emit photons one by one rather than in bursts. Although a distinctively quantum phenomenon, antibunching is readily observed in most common fluorophores even at room temperature. This nonclassical resource can be utilized directly to enhance the imaging resolution, since the non-classical far-field intensity correlations induced by antibunching carry high spatial frequency information on the spatial distribution of emitters. Detecting photon statistics simultaneously in the entire field of view, we were able to detect non-classical correlations of the second and third order, and reconstructed images with resolution significantly beyond the diffraction limit. Alternatively, we demonstrate the utilization of antibunching for augmenting the capabilities of localization-based superresolution imaging in the presence of multiple emitters, using a novel detector comprised of an array of single photon detectors connected to a densely packed fiber bundle. These features allow us to enhance the spatial and temporal resolution with which multiple emitters can be imaged compared with other techniques that rely on CCD cameras.
The optical diffraction limit, formulated by Abbe 140 years ago, imposes a bound on imaging resolution in classical optics. Over the last twenty years, many theoretical schemes have been presented for overcoming the diffraction barrier in optical imaging using quantum properties of light. An experimental realization of sub-diffraction limited quantum imaging has, however, remained elusive. Here, we demonstrate a quantum imaging method taking advantage of non-classical light naturally produced in fluorescence microscopy due to photon antibunching, a fundamentally quantum phenomenon prohibiting simultaneous emission of multiple photons. Using a photon counting digital camera, we detect antibunching-induced second and third order intensity correlations and perform sub-diffraction limited quantum imaging in a standard wide-field fluorescence microscope.
The classical optical diffraction limit can be overcome by exploiting the quantum properties of light in several theoretical studies; however, they mostly rely on an entangled light source. Recent experiments have demonstrated that quantum properties are preserved in many fluorophores, which makes it possible to add a new dimension of information for super-resolution fluorescence imaging. Here, we developed a statistical quantum coherence model for fluorescence emitters and proposed a new super-resolution method using fluorescence quantum coherence in fluorescence microscopy. In this study, by exploiting a single-photon avalanche detector (SPAD) array with a time-correlated single-photon-counting technique to perform spatial-temporal photon statistics of fluorescence coherence, the subdiffraction-limited spatial separation of emitters is obtained from the determined coherence. We numerically demonstrate an example of two-photon interference from two common fluorophores using an achievable experimental procedure. Our model provides a bridge between the macroscopic partial coherence theory and the microscopic dephasing and spectral diffusion mechanics of emitters. By fully taking advantage of the spatial-temporal fluctuations of the emitted photons as well as coherence, our quantum-enhanced imaging method has the significant potential to improve the resolution of fluorescence microscopy even when the detected signals are weak.