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Proceedings of SPIE, the International Society for Optical Engineering/Proceedings of SPIE

Fluorescence antibunching microscopy

Osip Schwartz, Jonathan M. Levitt, Dan Oron

Published Jan 6, 2012• 3 citations
Publisher

Abstract

Utilizing quantum properties of light to break the diffraction limit has been the goal of intense research in the recent years. This paper is a progress report on a study aimed at experimentally demonstrating a superresolution microscopy technique enabled by photon antibunching, a non-classical emission statistics feature exhibited by most emitters used as fluorescent markers. We find that photon antibunching gives rise to correlations that encode high spatial frequency information on the distribution of fluorescent emitters. Detecting these correlations using photon counting instrumentation in a standard fluorescence microscope setting allows for three-dimensional superresolution imaging of fluorophore stained samples. The technique provides a quantum alternative to the established superresolution tools.

DOI
10.1117/12.907807

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Utilizing quantum properties of light to break the diffraction limit has been the goal of intense research in the recent years. This paper is a progress report on a study aimed at experimentally demonstrating a superresolution microscopy technique enabled by photon antibunching, a non-classical emission statistics feature exhibited by most emitters used as fluorescent markers. We find that photon antibunching gives rise to correlations that encode high spatial frequency information on the distribution of fluorescent emitters. Detecting these correlations using photon counting instrumentation in a standard fluorescence microscope setting allows for three-dimensional superresolution imaging of fluorophore stained samples. The technique provides a quantum alternative to the established superresolution tools.
The optical diffraction limit 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. Here, we demonstrate a quantum superresolution imaging method taking advantage of nonclassical light naturally produced in fluorescence microscopy due to photon antibunching, a fundamentally quantum phenomenon inhibiting simultaneous emission of multiple photons. Using a photon counting digital camera, we detect antibunching-induced second and third order intensity correlations and perform subdiffraction limited quantum imaging in a standard wide-field fluorescence microscope.
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.
We propose to use intensity correlation microscopy in combination with structured illumination to image quantum emitters that exhibit antibunching with a spatial resolution reaching far beyond the Rayleigh limit. Combining intensity measurements and intensity auto correlations up to order mmm creates an effective PSF with FWHM shrunk by the factor m\sqrt{m}m​. Structured Illumination microscopy on the other hand introduces a resolution improvement of factor 2 by use of the principle of moiré fringes. Here, we show that for linear low-intensity excitation and linear optical detection the simultaneous use of both techniques leads to an in theory unlimited resolution power with the improvement scaling favorably as m+mm + \sqrt{m}m+m​ in dependence of the correlation order mmm. Hence, yielding this technique to be of interest in microscopy for imaging a variety of samples including biological ones. We present the underlying theory and simulations demonstrating the highly increased spatial superresolution, and point out requirements for an experimental implementation.