Osip Schwartz, Dan Oron
Breaking the diffraction limit in microscopy by utilizing quantum properties of light has been the goal of intense research in the recent years. We propose a quantum superresolution technique based on non-classical emission statistics of fluorescent markers, routinely used as contrast labels for bio-imaging. The technique can be readily implemented using standard fluorescence microscopy equipment.
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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 …
Excitonic Coupling and Photon Antibunching in Venus Yellow Fluorescent Protein Dimers: A Lindblad Master Equation Approach
本文利用 Lindblad 主方程框架模拟金星黄色荧光蛋白二聚体动力学，通过预测多飞秒退相干产生的明暗态混合，调和了强激子耦合与光子反聚束共存现象，且无需引入长寿命量子相干性。
One of the main characteristics of optical imaging systems is the spatial resolution, which is restricted by the diffraction limit to approximately half the wavelength of the incident light. Along with the recently developed classical super-resolution techniques, which aim at breaking the diffraction limit in classical systems, there is a class of quantum super-resolution techniques which leverage the non-classical nature of the optical signals radiated by quantum emitters, the so-called antibunching super-resolution microscopy. This approach can ensure a factor of $\sqrt{n}$ improvement in th…
We present a new technique for obtaining simultaneous multimodal quantitative phase and fluorescence microscopy of biological cells, providing both quantitative phase imaging and molecular specificity using a single camera. Our system is based on an interferometric multiplexing module, externally positioned at the exit of an optical microscope. In contrast to previous approaches, the presented technique allows conventional fluorescence imaging, rather than interferometric off-axis fluorescence imaging. We demonstrate the presented technique for imaging fluorescent beads and live biological cel…
We report a new way to slow down the spontaneous emission rate of excitons in the wetting layer (WL) through radiative field coupling between the exciton emissions and the dipole field of metal islands. As a result, a long-lifetime decay process is detected in the emission of InAs/GaAs single quantum dots (QDs). It is found that when the separation distance from WL layer (QD layer) to the metal islands is around 20 nm and the islands have an average size of approximately 50 nm, QD lifetime may change from approximately 1 to 160 ns. The corresponding second-order autocorrelation function g(2) (…
We propose the use of the subwavelength localization via adiabatic passage technique for fluorescence microscopy with nanoscale resolution in the far field. This technique uses a Λ-type medium coherently coupled to two laser pulses: the pump, with a node in its spatial profile, and the Stokes. The population of the Λ system is adiabatically transferred from one ground state to the other except at the node position, yielding a narrow population peak. This coherent localization allows fluorescence imaging with nanometer lateral resolution. We derive an analytical expression to asses the resoluti…
Calibration-free single-frame super-resolution fluorescence microscopy
该研究提出一种深度学习方法，无需校准即可从单帧衍射极限相机重建超分辨率荧光图像，可实现7倍分辨率提升，泛化能力强，适用于标准宽场装置的快速密集或光敏感样本成像。
Single gold nanoparticles can act as nanoantennas for enhancing the fluorescence of emitters in their near-fields. Here we present experimental and theoretical studies of scanning antenna-based fluorescence microscopy as a function of the diameter of the gold nanoparticle. We examine the interplay between fluorescence enhancement and spatial resolution and discuss the requirements for deciphering single molecules in a dense sample. Resolutions better than 20 nm and fluorescence enhancement up to 30 times are demonstrated experimentally. By accounting for the tip shaft and the sample interface …
In this letter, the leakage radiation microscopy (LRM) is extended into characterization of planar waveguide modes (WMs) rather than surface plasmon polaritons (SPPs) taking advantages of the coupling between WMs and fluorescence emission. Propagation constants of different WMs allowed in the same planar waveguide can be simultaneously and rapidly derived from the Fourier plane image of fluorescence based LRM. Numerical simulations are also carried out to calculate propagation constants of these modes, which are consistent with experimental results. Our experiments provide a simple but high ef…
Fifth Harmonic and Five-Photon Excitation Fluorescence Multiphoton Microscopy
本研究将五阶非线性光学过程应用于多光子显微技术，通过五次谐波和5光子激发荧光实现更高分辨率和更深穿透深度，适用于生物、材料、地质及半导体等领域。
Fluorescence microscopy is a powerful tool for imaging biological samples with molecular specificity. In contrast, phase microscopy provides label-free measurement of the sample's refractive index (RI), which is an intrinsic optical property that quantitatively relates to cell morphology, mass, and stiffness. Conventional imaging techniques measure either the labeled fluorescence (functional) information or the label-free RI (structural) information, though it may be valuable to have both. For example, biological tissues have heterogeneous RI distributions, causing sample-induced scattering th…
Intensity correlation microscopy (ICM), which is prominently known through antibunching microscopy or super-resolution optical fluctuation imaging (SOFI), provides superresolution through a correlation analysis of antibunching of independent quantum emitters or temporal fluctuations of blinking fluorophores. For correlation order $m$ the PSF in the signal is effectively taken to the $m$th power, and is thus directly shrunk by the factor $\sqrt{m}$. Combined with deconvolution a close to linear resolution improvement of factor $m$ can be obtained. Yet, analysis of high correlation orders is cha…
Fluorescence imaging is the most widely used method for unveiling the molecular composition of biological specimens. However, the weak optical emission of fluorescent probes and the tradeoff between imaging speed and sensitivity is problematic for acquiring blur-free images of fast phenomena, such as sub-millisecond biochemical dynamics in live cells and tissues, and cells flowing at high speed. We report a solution that achieves real-time pixel readout rates one order of magnitude faster than a modern electron multiplier charge coupled device (EMCCD) - the gold standard in high-speed fluoresc…
Fluorescence microscopy is indispensable in nanoscience and biological sciences. The versatility of labeling target structures with fluorescent dyes permits to visualize structure and function at a subcellular resolution with a wide field of view. Due to the diffraction limit, conventional optical microscopes are limited to resolving structures larger than 200 nm. The resolution can be enhanced by near-field and far-field super-resolution microscopy methods. Near-field methods typically have a limited field of view and far-field methods are limited by the involved conventional optics. Here, we…
We introduce the basic elements of a spatio-angular theory of fluorescence microscopy, providing a unified framework for analyzing systems that image single fluorescent dipoles and ensembles of overlapping dipoles that label biological molecules. We model an aplanatic microscope imaging an ensemble of in-focus fluorescent dipoles as a linear Hilbert-space operator, and we show that the operator takes a particularly convenient form when expressed in a basis of complex exponentials and spherical harmonics$-$a form we call the dipole spatio-angular transfer function. We discuss the implications o…
We suggest a new multimodal imaging technique for quantitatively measuring the integral (thickness-average) refractive index of the nuclei of live biological cells in suspension. For this aim, we combined quantitative phase microscopy with simultaneous 2-D fluorescence microscopy. We used 2-D fluorescence microscopy to localize the nucleus inside the quantitative phase map of the cell, as well as for measuring the nucleus radii. As verified offline by both 3-D confocal fluorescence microscopy and by 2-D fluorescence microscopy while rotating the cells during flow, the nucleus of cells in suspe…
Fluorescence microscopy is widely used in biological imaging, however scattering from tissues strongly limits its applicability to a shallow depth. In this work we adapt a methodology inspired from stellar speckle interferometry, and exploit the optical memory effect to enable fluorescence microscopy through a turbid layer. We demonstrate efficient reconstruction of micrometer-size fluorescent objects behind a scattering medium in epi-microscopy, and study the specificities of this imaging modality (magnification, field of view, resolution) as compared to traditional microscopy. Using a modifi…
Super-resolution fluorescence microscopy is an important tool in biomedical research for its ability to discern features smaller than the diffraction limit. However, due to its difficult implementation and high cost, the universal application of super-resolution microscopy is not feasible. In this paper, we propose and demonstrate a new kind of super-resolution fluorescence microscopy that can be easily implemented and requires neither additional hardware nor complex post-processing. The microscopy is based on the principle of stepwise optical saturation (SOS), where $M$ steps of raw fluoresce…
Dynamic Spectral fluorescence microscopy via Event-based & CMOS image-sensor fusion
本文提出融合事件传感器与CMOS的宽场荧光显微镜，结合衍射光学编码和深度神经网络，实现微秒级时间分辨与纳米级光谱区分，验证了超快动态光谱成像能力。
A fluorescence correlation spectroscopy (FCS) system based on two independent measurement volumes is presented. The optical setup and data acquisition hardware are detailed, as well as a complete protocol to control the location, size and shape of the measurement volumes. A method that allows to monitor independently the excitation and collection efficiency distribution is proposed. Finally, a few examples of measurements that exploit the two spots in static and/or scanning schemes, are reported.
Computational Cannula Microscopy (CCM) is a high-resolution widefield fluorescence imaging approach deep inside tissue, which is minimally invasive. Rather than using conventional lenses, a surgical cannula acts as a lightpipe for both excitation and fluorescence emission, where computational methods are used for image visualization. Here, we enhance CCM with artificial neural networks to enable 3D imaging of cultured neurons and fluorescent beads, the latter inside a volumetric phantom. We experimentally demonstrate transverse resolution of ~6um, field of view ~200um and axial sectioning of ~…
Fluorescence lifetime imaging microscopy (FLIM) is an important technique to understand the chemical micro-environment in cells and tissues since it provides additional contrast compared to conventional fluorescence imaging. When two fluorophores within a diffraction limit are excited, the resulting emission leads to non-linear spatial distortion and localization effects in intensity (magnitude) and lifetime (phase) components. To address this issue, in this work, we provide a theoretical model for convolution in FLIM to describe how the resulting behavior differs from conventional fluorescenc…
Asynchronous-spectral fusion fluorescence microscopy for microsecond-scale behavioral dynamics
本文提出一种融合异步事件流与衍射编码CMOS测量的荧光显微镜架构，实现微秒级时间分辨率与光谱分辨的解耦，支持10万帧/秒的光谱追踪。
We introduce a full field fluorescence imaging technique with axial confinement of about 100 nm at the sample/substrate interface. Contrary to standard surface imaging techniques, this confinement is obtained through emission filtering. This technique is based on supercritical emission selectivity. It can be implemented on any epifluorescence microscope with a commercial high numerical aperture objective and offers a real time surface imaging capability. This technique is of particular interest for live cell membrane and adhesion studies. Using HEK cells, we show that one can observe simultane…
A statistical resolution measure of fluorescence microscopy with finite photons
本文提出有限光子下荧光显微镜分辨能力的统计度量方法，结合衍射极限与光子统计特性，利用Cramer-Rao下界量化周期结构可分辨性，为现代荧光显微技术确立了含噪声的实际分辨率理论度量。
Light tailored by multimode fiber for multiphoton fluorescence microscopy
本研究提出将高斯光束注入石英渐变折射率多模光纤生成仅含方位角不变导模的光束，该极简低成本方法有效提升了双光子显微镜的轴向分辨率。
Fluorescence Microscopy: a statistics-optics perspective
本文从统计与光学交叉视角，综述了荧光显微成像中光与荧光团特性对图像特征的影响，并介绍了相应的概率建模工具，旨在促进定量显微图像解释。
The fluorescence microscope is one of the most important tools in modern clinical diagnosis and biological science. However, its expense, size and limited field-of-view (FOV) are becoming bottlenecks in key applications such as large-scale phenotyping and low-resource-setting diagnostics. Here we report a low-cost, compact chip-scale fluorescence-imaging platform, termed the Fluorescence Talbot Microscopy (FTM), which utilizes the Talbot self-imaging effect to enable efficient fluorescence imaging over a large and directly-scalable FOV. The FTM prototype has a resolution of 1.2 microns and an …
Sample Motion for Structured Illumination Fluorescence Microscopy
本研究针对结构照明显微镜需多照明模式的问题，提出利用单散斑照明和样品固有运动，从运动中拍摄的荧光图像联合估计运动与超分辨图像的方法，并通过仿真与实验验证了可行性。
Photobleaching is a major limitation of superresolution Stimulated Depletion Emission (STED) microscopy. Fast scanning has long been considered an effective means to reduce photobleaching in fluorescence microscopy, but a careful quantitative study of this issue is missing. In this paper, we show that the photobleaching rate in STED microscopy is slowed down and fluorescence yield is enhanced by scanning with high linear speed, enabled by the large field of view in our custom-built resonant-scanning STED microscope. The effect of scanning speed on photobleaching and fluorescence yield is more …
Optical Diffraction Tomography Meets Fluorescence Localization Microscopy
本文将单分子定位显微数据重新建模为无相位光学衍射层析系统，并提出联合优化框架，仅从SMLM帧中同时估计折射率图和荧光分子位置。
Nonlinear structured illumination microscopy (nSIM) is an effective approach for super-resolution wide-field fluorescence microscopy with a theoretically unlimited resolution. In nSIM, carefully designed, highly-contrasted illumination patterns are combined with the saturation of an optical transition to enable sub-diffraction imaging. While the technique proved useful for two-dimensional imaging, extending it to three-dimensions (3D) is challenging due to the fading/fatigue of organic fluorophores under intense cycling conditions. Here, we present a compressed sensing approach that allows for…
Multiphoton microscopy has enabled us to image cellular dynamics in vivo. However, the excitation wavelength for imaging with commercially available lasers is mostly limited between 650-1040 nm. Here we develop a femtosecond fiber laser system that produces $\sim$150 fs pulses at 1.8 $μ$m. Our system starts from an erbium-doped silica fiber laser, and its wavelength is converted to 1.8 $μ$m using a Raman shift fiber. The 1.8 $μ$m pulses are amplified with a two-stage Tm:ZBLAN fiber amplifier. The final pulse energy is $\sim$1 $μ$J, sufficient for in vivo imaging.
Fluorescence microscopy has revolutionized biomedical research over the past three decades. Its high molecular specificity and unrivaled single molecule level sensitivity have enabled breakthroughs in a variety of research fields. For in vivo applications, its major limitation is the superficial imaging depth as random scattering in biological tissues causes exponential attenuation of the ballistic component of a light wave. Here we present fluorescence microscopy beyond the ballistic regime by combining single cycle pulsed ultrasound modulation and digital optical phase conjugation. We demons…
The efficient optical tool for elimination of the phase and amplitude distortions produced by imperfectness of the optical elements in microscope lightpath considered. This robust procedure described by simple theoretical model proved to be successful to repair the noisy images with noise to signal ratio close to 100. It is shown that Van Cittert - Zernike theorem provides adequate description of imperfect microscope operating under both coherent and incoherent illumination conditions and having turbid media in object and intermediate planes.
Phantom eye devices are designed to mimic the optical properties of the human eye; however, the retinal structures are typically emulated with synthetic tissues. Thus, it is currently impossible to exploit phantom eye devices for fluorescence microscopy experiments on cultures, or ex-vivo tissues, although these ophthalmic measurements would be of importance for many applications such as the testing of retinal biomarkers. Here we report and describe the development of a phantom eye designed to host biological samples, such as retinal cultures differentiated from human induced pluripotent stem …
Improving Richardson--Lucy Deconvolution with Diffusion Priors for Fluorescence Microscopy
针对荧光显微镜反卷积的ill-posed问题，提出将基于分数的扩散先验集成到Richardson-Lucy迭代框架中，在保持泊松数据一致性的同时抑制噪声放大，改善低光子计数下弱丝状和点状结构的重建。
Superresolution fluorescence microscopy techniques beat the diffraction limit, enabling ultra-high resolution imaging in biological physics and nanoscience. In all cases that have been studied experimentally, the resolution scales inversely with the square root of some parameter that measures the number of photons used. However, this ubiquitous limit arises from very distinct mechanisms in different approaches, raising the question of whether it is a fundamental limit that cannot be exceeded, or merely a coincidence of the techniques studied thus far. We demonstrate that, under very general as…
Computational TIRF enables optical sectioning beyond the evanescent field for widefield fluorescence microscopy
计算TIRF通过深度学习实现宽场荧光显微镜超越衰减场的光学分层，无需光学修改即可生成TIRF-like图像，并在密集标记样本中实现单帧和体积分层重建。
Noninvasive megapixel fluorescence microscopy through scattering layers by a virtual reflection-matrix
本文提出利用少量宽场荧光图像构建虚拟反射矩阵，经矩阵散射补偿算法实现散射层后非侵入式百万像素荧光成像，无需空间光调制器，计算高效。
The mathematical theory of compressed sensing (CS) asserts that one can acquire signals from measurements whose rate is much lower than the total bandwidth. Whereas the CS theory is now well developed, challenges concerning hardware implementations of CS-based acquisition devices---especially in optics---have only started being addressed. This paper presents an implementation of compressive sensing in fluorescence microscopy and its applications to biomedical imaging. Our CS microscope combines a dynamic structured wide-field illumination and a fast and sensitive single-point fluorescence dete…
Scan-less full-field fluorescence-lifetime dual-comb microscopy using two-dimensional spectral mapping and frequency multiplexing of dual-optical-comb beats
本文提出一种基于双光梳拍频二维光谱映射和频率复用的无扫描全场荧光寿命显微成像方法，无需机械扫描即可实现高定量性的荧光幅度与寿命双模态成像。
Super-resolution Live-cell Fluorescence Lifetime Imaging
该研究将超分辨率结构光照明显微镜与电光荧光寿命成像结合，提出SIMFLIM技术，实现156 nm分辨率的高帧率活细胞理化参数监测，可用于多重成像、环境传感等场景。
Fluorescence Lifetime Imaging Microscopy Analysis of Isolated Melanosomes
本研究首次利用FLIM技术分析分离的黑素体，发现黑色素在长时间激光照射下会发生光致结构变化，寿命在几分钟至一小时内显著改变。
We investigate the properties of a single-view fluorescence microscope in a 4$f$ geometry when imaging fluorescent dipoles without using the monopole or scalar approximations. We show that this imaging system has a spatio-angular band limit, and we exploit the band limit to perform efficient simulations. Notably, we show that information about the out-of-plane orientation of ensembles of in-focus fluorophores is recorded by paraxial fluorescence microscopes. Additionally, we show that the monopole approximation may cause biased estimates of fluorophore concentrations, but these biases are smal…
Mid-infrared photothermal microscopy is a new chemical imaging technology in which a visible beam senses the photothermal effect induced by a pulsed infrared laser. This technology provides infrared spectroscopic information at sub-micron spatial resolution and enables infrared spectroscopy and imaging of living cells and organisms. Yet, current mid-infrared photothermal imaging sensitivity suffers from a weak dependance of scattering on temperature and the image quality is vulnerable to the speckles caused by scattering. Here, we present a novel version of mid-infrared photothermal microscopy…
Non-degenerate 2-photon excitation (ND-2PE) of a fluorophore with two laser beams of different photon energies offers an independent degree of freedom in tuning of the photon flux for each beam. This feature takes advantage of the infrared wavelengths used in 3-photon microscopy to achieve an increased penetration depth, while preserving a relatively high degenerate 2-photon excitation (D-2PE) cross section, exceeding that achievable with 3-photon excitation. Here, using spatially and temporally aligned Ti:Sapphire laser and optical parametric oscillator beams operating at near infrared (NIR) …
Three-dimensional (3D) fluorescence microscopy in general requires axial scanning to capture images of a sample at different planes. Here we demonstrate that a deep convolutional neural network can be trained to virtually refocus a 2D fluorescence image onto user-defined 3D surfaces within the sample volume. With this data-driven computational microscopy framework, we imaged the neuron activity of a Caenorhabditis elegans worm in 3D using a time-sequence of fluorescence images acquired at a single focal plane, digitally increasing the depth-of-field of the microscope by 20-fold without any axi…
Fluholoscopy. Compact and Simple Platform Combining Fluorescence and Holographic Microscopy
本研究提出一种名为Fluholoscopy的紧凑型简易共路双模显微平台，采用单一波长同时激发荧光与提供相干照明，经带通滤光片分光后由双相机同步实现单快照荧光与定量相位成像，并通过静态和动态样本完成验证。
We report a novel extension to structured illumination (SI) microscopy that utilizes the Talbot self-imaging effect to generate a SI pattern on a sample with field-of-view (FOV) and resolution unconstrained by the numerical aperture (NA) of the microscope objective. This enables high-resolution SI pattern generation (up to the free-space propagation limit) across large FOVs, which in turn allows SI microscopy to be efficiently adapted for high-throughput imaging. We demonstrated this concept using a simple prototype microscope to image a large FOV (450 x 350 um) with a diffraction-limited fluo…
Super-resolution capacity of variance-based stochastic fluorescence microscopy
本文从数学上刻画了随机散斑照明图像方差中的样品信息，证明当散斑相关长度与观测点扩散函数宽度一致时可获得两倍分辨率提升，并比较了RIM与SOFI两类基于方差的方法的差异。
In this paper we review the applicability of autofluorescent proteins for single-molecule imaging in biology. The photophysical characteristics of several mutants of the Green Fluorescent Protein (GFP) and those of DsRed are compared and critically discussed for their use in cellular biology. The alternative use of two-photon excitation at the single-molecule level or Fluorescence Correlation Spectroscopy is envisaged for the study of individual autofluorescent proteins. Single-molecule experiments performed in live cells using eGFP and preferably eYFP fusion proteins are reviewed. Finally, th…
Optical phase-space functions describe spatial and angular information simultaneously; examples include light fields in ray optics and Wigner functions in wave optics. Measurement of phase-space enables digital refocusing, aberration removal and 3D reconstruction. High-resolution capture of 4D phase-space datasets is, however, challenging. Previous scanning approaches are slow, light inefficient and do not achieve diffraction-limited resolution. Here, we propose a multiplexed method that solves these problems. We use a spatial light modulator (SLM) in the pupil plane of a microscope in order t…
Learned Single-Pixel Fluorescence Microscopy
本文提出自监督学习的自编码器以联合优化单像素荧光显微成像的测量矩阵与重建网络，在真实多光谱和强度数据上实现百倍提速、更高画质及低成本多光谱成像。
FRET-based approaches are a unique tool for sensing the immediate surroundings and interactions of (bio)molecules. FRET imaging and FLIM (Fluorescence Lifetime Imaging Microscopy) enable the visualization of the spatial distribution of molecular interactions and functional states. However, conventional FLIM and FRET imaging provide average information over an ensemble of molecules within a diffraction-limited volume, which limits the spatial information, accuracy, and dynamic range of the observed signals. Here, we demonstrate an approach to obtain super-resolved FRET imaging based on single-m…
Optical fluorescence imaging is capable of measuring both the spatial and rotational dynamics of single molecules. However, unavoidable measurement noise will result in inaccurate estimates of rotational dynamics, causing a molecule to appear to be more rotationally constrained than it actually is. We report a mathematical framework to compute the fundamental limit of accuracy in measuring the rotational mobility of dipole-like emitters. By applying our framework to both in-plane and three-dimensional methods, we provide a means to choose the optimal orientation-measurement technique based on …
Measuring the lifetime of fluorescent emitters by time-correlated single photon counting (TCSPC) is a routine procedure in many research areas spanning from nanophotonics to biology. The precision of such measurement depends on the number of detected photons but also on the various sources of noise arising from the measurement process. Using Fisher information theory, we calculate the lower bound on the precision of lifetime estimations for mono-exponential and bi-exponential distributions. We analyse the dependence of the lifetime estimation precision on experimentally relevant parameters, in…
Fluorescence detection, either involving propagating or near-field emission, is widely being used in spectroscopy, sensing and microscopy. Total internal reflection fluorescence (TIRF) confines fluorescence excitation by an evanescent (near-) field and it is a popular contrast generator for surface-selective fluorescence assays. Its emission equivalent, supercritical angle fluorescence (SAF) is comparably less established although it achieves a similar optical sectioning as does TIRF. SAF emerges when a fluorescing molecule is located very close to an interface and its near-field emission coup…
Depth-dependent resolution quantification in 3D fluorescence microscopy
本研究提出一种量化3D荧光显微镜中随深度变化分辨率的方法，在果蝇大脑验证，量化双光子相对共聚焦的成像提升，可客观测量激光功率调优的效果，适用于跨系统比较及指导生物实验设计。
We demonstrate sub-wavelength sectioning on biological samples with a conventional confocal microscope. This optical sectioning is achieved by the phenomenon of supercritical angle fuorescence, wherein only a fluorophore next to the interface of a refractive index discontinuity can emit propagating components of radiation into the so-called forbidden angles. The simplicity of this technique allows it to be integrated with a high numerical aperture confocal scanning microscope by only a simple modification on the detection channel. Confocal-SAF microscopy would be a powerful tool to achieve hig…
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