Traditional photon localization microscopy analyses only the spatial distributions of photons emitted by individual molecules to reconstruct super-resolution optical images. Unfortunately, however, the highly valuable spectroscopic information from these photons have been overlooked. Here we report a spectroscopic photon localization microscopy that is capable of capturing the inherent spectroscopic signatures of photons from individual stochastic radiation events. Spectroscopic photon localization microscopy achieved higher spatial resolution than traditional photon localization microscopy through spectral discrimination to identify the photons emitted from individual molecules. As a result, we resolved two fluorescent molecules, which were 15 nm apart, with the corresponding spatial resolution of 10 nm-a four-fold improvement over photon localization microscopy. Using spectroscopic photon localization microscopy, we further demonstrated simultaneous multi-colour super-resolution imaging of microtubules and mitochondria in COS-7 cells and showed that background autofluorescence can be identified through its distinct emission spectra.
Traditional photon localization microscopy analyses only the spatial distributions of photons emitted by individual molecules to reconstruct super-resolution optical images. Unfortunately, however, the highly valuable spectroscopic information from these photons have been overlooked. Here we report a spectroscopic photon localization microscopy that is capable of capturing the inherent spectroscopic signatures of photons from individual stochastic radiation events. Spectroscopic photon localization microscopy achieved higher spatial resolution than traditional photon localization microscopy through spectral discrimination to identify the photons emitted from individual molecules. As a result, we resolved two fluorescent molecules, which were 15 nm apart, with the corresponding spatial resolution of 10 nm-a four-fold improvement over photon localization microscopy. Using spectroscopic photon localization microscopy, we further demonstrated simultaneous multi-colour super-resolution imaging of microtubules and mitochondria in COS-7 cells and showed that background autofluorescence can be identified through its distinct emission spectra.
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.
Super-resolution microscopy is crucial for imaging sub-wavelength biological structures. However, most techniques rely on nonlinear saturation or stochastic switching of emitters, limiting imaging speed and increasing phototoxicity. Here, we achieve deep super-resolution without employing saturation or stochastic dynamics, instead using a form of spatial mode demultiplexing. By shaping the point-spread function of the emitted light, our Structured Detection Microscope (SDM) redistributes information away from high shot-noise regions of the image, enhancing sensitivity to sub-diffraction emitter separations in two-dimensions and without mode-sorting optics. Implementing SDM within a high-numerical aperture total internal reflection fluorescence microscope, we demonstrate imaging of fluorophores attached to DNA nanorulers with separations as small as 50 nm at resolutions surpassing 40 nm - fivefold below the diffraction limit. This shows that spatial mode demultiplexing can achieve far sub-wavelength resolution and is applicable to biologically relevant samples. By enabling super-resolution biomolecular imaging without emitter saturation and stochasticity, our work opens the door to better understanding biological structure, function and dynamics.
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.
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.
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.
Properties of quantum light represent a tool for overcoming limits of classical optics. Several experiments have demonstrated this advantage ranging from quantum enhanced imaging to quantum illumination. In this work, experimental demonstration of quantum-enhanced resolution in confocal fluorescence microscopy will be presented. This is achieved by exploiting the non-classical photon statistics of fluorescence emission of single nitrogen-vacancy (NV) color centers in diamond. By developing a general model of super-resolution based on the direct sampling of the kth-order autocorrelation function of the photoluminescence signal, we show the possibility to resolve, in principle, arbitrarily close emitting centers. Finally, possible applications of NV-based fluorescent nanodiamonds in biosensing and future developments will be presented.