Nanoscale localization of point emitters is critical to several methods in optical fluorescence microscopy, including single-molecule super-resolution imaging and tracking. While the precision of the localization procedure has been the topic of extensive study, localization accuracy has been less emphasized, in part due to the challenge of producing an experimental sample containing unperturbed point emitters at known three-dimensional positions in a relevant geometry. We report a new experimental system which reproduces a widely-adopted geometry in high-numerical aperture localization microscopy, in which molecules are situated in an aqueous medium above a glass coverslip imaged with an oil-immersion objective. We demonstrate a calibration procedure that enables measurement of the depth-dependent point spread function (PSF) for open aperture imaging as well as imaging with engineered PSFs with index mismatch. We reveal the complicated, depth-varying behavior of the focal plane position in this system and discuss the axial localization biases incurred by common approximations of this behavior. We compare our results to theoretical calculations.
We demonstrate super-resolution imaging of single rare-earth emitting centers, namely, trivalent cerium, in yttrium aluminum garnet (YAG) crystals by means of stimulated emission depletion (STED) microscopy. The achieved all-optical resolution is $\approx$ 80nm. Similar results were obtained on H3 color centers in diamond with resolution of $\approx$ 60nm. In both cases, STED resolution is improving slower than the inverse square-root of the depletion beam intensity. This is caused by excited state absorption (ESA) and interaction of the emitter with non-fluorescing crystal defects in its near…
Wide-field fluorescence microscopy, while much faster than confocal microscopy, suffers from a lack of optical sectioning and poor axial resolution. 3D structured illumination microscopy (SIM) has been demonstrated to provide optical sectioning and to double the achievable resolution both laterally and axially, but even with this the axial resolution is still worse than the lateral resolution of unmodified wide-field detection. Interferometric schemes using two high numerical aperture objectives, such as 4Pi confocal and I5S microscopy, have improved the axial resolution beyond that of the lat…
Single-emitter microscopy has emerged as a promising method of imaging nanostructures with nanoscale resolution. This technique uses the centroid position of an emitters far-field radiation pattern to infer its position to a precision that is far below the diffraction limit. However, nanostructures composed of high-dielectric materials such as noble metals can distort the far-field radiation pattern. Nanoparticles also exhibit a more complex range of distortions, because in addition to introducing a high dielectric surface, they also act as efficient scatterers. Thus, the distortion effects of…
We report an experimental technique to map and exploit the local density of optical states of arbitrary planar nano-photonic structures. The method relies on positioning a spontaneous emitter attached to a scanning probe deterministically and reversibly with respect to its photonic environment while measuring its lifetime. We demonstrate the method by imaging the enhancement of the local density of optical states around metal nanowires. By nano-positioning, the decay rate of a pointlike source of fluorescence can be reversibly and repeatedly changed by a factor of two by coupling it to the gui…
We present a new imaging method for pump-probe microscopy that explores non-collinear excitation. This method (crossed-beam pump-probe microscopy, or CBPM) can significantly improve the axial resolution when imaging through low-NA lenses, providing an alternative way for depth resolved, large field-of-view imaging. We performed a proof-of-concept demonstration, characterized CBPM's resolution using different imaging lenses, and measured an enhanced axial resolution for certain types of low-NA lenses.
The design of single-molecule photoswitchable emitters was the first milestone toward the advent of single-molecule localization microscopy that sets a new paradigm in the field of optical imaging. Several photoswitchable emitters have been developed but they all fluoresce in the visible or far-red ranges, missing the desirable near-infrared window where biological tissues are most transparent. Moreover, photocontrol of individual emitters in the near-infrared would be highly desirable for elementary optical molecular switches or information storage elements since most communication data trans…
Multifocus microscopy enables recording of entire volumes in a single camera exposure. In dense samples, multifocus microscopy is severely hampered by background haze. Here, we introduce a scalable multifocus method that incorporates optical sectioning and offers axial superresolution capabilities. In our method, a dithered oblique light-sheet scans the sample volume during a single exposure, while generated fluorescence is linearised onto the camera with a multifocus optical element. A synchronised rolling shutter readout realised optical sectioning. We describe the technique theoretically an…
Fast, volumetric imaging over large scales has been a long-standing goal in biological microscopy. Scanning techniques such as fluorescence confocal microscopy can acquire 2D images at high resolution and high speed, but extending the acquisition to multiple planes at different depths requires an axial scanning mechanism that drastically reduces the acquisition speed. To address this challenge, we report an augmented variant of confocal microscopy where the key innovation consists to use a series of reflecting pinholes axially distributed in the detection plane, each one probing a different de…
Accurate distance measurement in 3D confocal microscopy is important for quantitative analysis, volume visualization and image restoration. However, axial distances can be distorted by both the point spread function and by a refractive-index mismatch between the sample and immersion liquid, which are difficult to separate. Additionally, accurate calibration of the axial distances in confocal microscopy remains cumbersome, although several high-end methods exist. In this paper we present two methods to calibrate axial distances in 3D confocal microscopy that are both accurate and easily impleme…
We investigate a nonlinear localization microscopy method based on Rabi oscillations of single emitters. We demonstrate the fundamental working principle of this new technique using a cryogenic far-field experiment in which subwavelength features smaller than $λ$/10 are obtained. Using Monte Carlo simulations, we show the superior localization accuracy of this method under realistic conditions and a potential for higher acquisition speed or a lower number of required photons as compared to conventional linear schemes. The method can be adapted to other emitters than molecules and allows for th…
Deterministically integrating single solid-state quantum emitters with photonic nanostructures serves as a key enabling resource in the context of photonic quantum technology. Due to the random spatial location of many widely-used solid-state quantum emitters, a number of positoning approaches for locating the quantum emitters before nanofabrication have been explored in the last decade. Here, we review the working principles of several nanoscale positioning methods and the most recent progress in this field, covering techniques including atomic force microscopy, scanning electron microscopy, …
Fast confocal imaging was achieved by combining remote focusing with differential spinning disk optical sectioning to rapidly acquire images of live samples at cellular resolution. Axial and lateral full width half maxima less than 5 um and 490 nm respectively are demonstrated over 130 um axial range with a 256 x 128 um field of view. A water-index calibration slide was used to achieve an alignment that minimises image volume distortion. Application to live biological samples was demonstrated by acquiring image volumes over a 24 um axial range at 1 volume/s, allowing for the detection of calci…
Super-resolution localization microscopy is based on determining the positions of individual fluorescent markers in a sample. The major challenge in reaching an ever higher localization precision lies in the limited number of collected photons from single emitters. To tackle this issue, it has been shown that one can exploit the increased photostability at low temperatures, reaching localization precisions in the sub-nanometer range. Another crucial ingredient of single-molecule super-resolution imaging is the ability to activate individual emitter within a diffraction-limited spot. Here, we r…
Three-dimensional direct laser writing has become a well established, versatile, widespread, and even readily commercially available "workhorse" of nano- and micro-technology. However, its lateral and axial spatial resolution is inherently governed by Abbe's diffraction limitation - analogous to optical microscopy. In microscopy, stimulated-emission-depletion approaches have lately circumvented Abbe's barrier and lateral resolutions down to 5.6 nm using visible light have been achieved. In this paper, after very briefly reviewing our previous efforts with respect to translating this success in…
A standard paradigm of localization microscopy involves extension from two to three dimensions by engineering information into emitter images, and approximation of errors resulting from the field dependence of optical aberrations. We invert this standard paradigm, introducing the concept of fully exploiting the latent information of intrinsic aberrations by comprehensive calibration of an ordinary microscope, enabling accurate localization of single emitters in three dimensions throughout an ultrawide and deep field. To complete the extraction of spatial information from microscale bodies rang…
We present a method that can simultaneously locate positions of overlapped multi-emitters at the theoretical-limit precision. We derive a set of simple equations whose solution gives the maximum likelihood estimator of multi-emitter positions. We compare the performance of our simultaneous localization analysis with the conventional single-molecule analysis for simulated images and show that our method can improve the time-resolution of superresolution microscopy an order of magnitude. In particular, we derive the information-theoretical bound on time resolution of localization-based superreso…
Volumetric imaging of samples using fluorescence microscopy plays an important role in various fields including physical, medical and life sciences. Here we report a deep learning-based volumetric image inference framework that uses 2D images that are sparsely captured by a standard wide-field fluorescence microscope at arbitrary axial positions within the sample volume. Through a recurrent convolutional neural network, which we term as Recurrent-MZ, 2D fluorescence information from a few axial planes within the sample is explicitly incorporated to digitally reconstruct the sample volume over …
Localization microscopy is an imaging technique in which the positions of individual nanoscale point emitters (e.g. fluorescent molecules) are determined at high precision from their images. This is the key ingredient in single/multiple-particle-tracking and several super-resolution microscopy approaches. Localization in three-dimensions (3D) can be performed by modifying the image that a point-source creates on the camera, namely, the point-spread function (PSF). The PSF is engineered using additional optical elements to vary distinctively with the depth of the point-source. However, localizi…
Deep learning has become an extremely effective tool for image classification and image restoration problems. Here, we apply deep learning to microscopy, and demonstrate how neural networks can exploit the chromatic dependence of the point-spread function to classify the colors of single emitters imaged on a grayscale camera. While existing single-molecule methods for spectral classification require additional optical elements in the emission path, e.g. spectral filters, prisms, or phase masks, our neural net correctly identifies static as well as mobile emitters with high efficiency using a s…
We present an ultra-fast, precise, parameter-free method, which we term Deep-STORM, for obtaining super-resolution images from stochastically-blinking emitters, such as fluorescent molecules used for localization microscopy. Deep-STORM uses a deep convolutional neural network that can be trained on simulated data or experimental measurements, both of which are demonstrated. The method achieves state-of-the-art resolution under challenging signal-to-noise conditions and high emitter densities, and is significantly faster than existing approaches. Additionally, no prior information on the shape …
We discuss theoretically the concept of spatial resolution in near-field scanning optical microscopy (NSOM) in light of a recent work [Opt. Express 17 (2009) 19969] which reported on the achievement of active tips made of a single ultrasmall fluorescent nanodiamond grafted onto the apex of a substrate tip and on their validation in NSOM imaging. Since fluorescent nanodiamonds tend to decrease steadily in size, we assimilate a nanodiamond-based tip to a point-like single photon source and compare its ultimate resolution with that offered by standard metal-coated aperture NSOM tips. We demonstra…
We propose a straightforward sample-based technique to calibrate the axial detection in 3D single molecule localization microscopy (SMLM). Using microspheres coated with fluorescent molecules, the calibration curves of PSF-shaping- or intensity-based measurements can be obtained for any required depth range from a few hundreds of nanometers to several tens of microns. This experimental method takes into account the effect of the spherical aberration without requiring computational correction.
Their intrinsic properties render single quantum systems as ideal tools for quantum enhanced sensing and microscopy. As an additional benefit, their size is typically on an atomic scale which enables sensing with very high spatial resolution. Here, we report on utilizing a single nitrogen vacancy center in nanodiamond for performing three-dimensional scanning-probe fluorescence lifetime imaging microscopy. By measuring changes of the single emitter's lifetime information on the local density of optical states is acquired at the nanoscale. This technique to gather information on the local densi…
Traceability to the International System of Units (SI) is fundamental to measurement accuracy and reliability. In this study, we demonstrate subnanometer traceability of localization microscopy, establishing a metrological foundation for the maturation and application of super-resolution methods. To do so, we create a master standard by measuring the positions of submicrometer apertures in an array by traceable atomic-force microscopy. We perform correlative measurements of this master standard by optical microscopy, calibrating scale factor and correcting aberration effects. We introduce the …
Momentum microscopy of ultrafast electron emission from a strongly driven optical nanoantenna
Carl von Ossietzky Universität Oldenburg（奥尔登堡卡尔·冯·奥西茨基大学）；ELI ALPS, The Extreme Light Infrastructure ERIC（ELI ALPS，极端光基础设施ERIC）；Leibniz Institute of Photonic Technology（莱布尼兹光子技术研究所）；Universität Würzburg（维尔茨堡大学）；Friedrich-Schiller-Universität Jena（耶拿弗里德里希·席勒大学）；HUN-REN Wigner Research Center for Physics（匈牙利科学院Wigner物理研究中心）
Coherence properties and wavelength of light sources are indispensable for optical coherence microscopy/tomography as they greatly influence the signal to noise ratio, axial resolution, and penetration depth of the system. In the present letter, we investigated the longitudinal spatial coherence properties of the pseudo-thermal light source (PTS) as a function of spot size at the diffuser plane, which is controlled by translating microscope objective lens towards or away from the diffuser plane. The axial resolution of PTS is found to be maximum ~ 13 microns for the beam spot size of 3.5 mm at…
We address the issue of the second-order coherence of single surface plasmons launched by a quantum source of light into extended gold films. The quantum source of light is made of a scanning fluorescent nanodiamond hosting five nitrogen-vacancy (NV) color centers. By using a specially designed microscopy that combines near-field optics with far-field leakage-radiation microscopy in the Fourier space and adapted spatial filtering, we find that the quantum statistics of the initial source of light is preserved after conversion to surface plasmons and propagation along the polycrystalline gold f…
An interferometric fluorescent microscope and a novel theoretic image reconstruction approach were developed and used to obtain super-resolution images of live biological samples and to enable dynamic real time tracking. The tracking utilizes the information stored in the interference pattern of both the illuminating incoherent light and the emitted light. By periodically shifting the interferometer phase and a phase retrieval algorithm we obtain information that allow localization with sub-2 nm axial resolution at 5 Hz.
We present a new two-snapshot structured light illumination (SLI) reconstruction algorithm for fast image acquisition. The new algorithm, which only requires two mutually π phase-shifted raw structured images, is implemented on a custom-built temporal focusing fluorescence microscope (TFFM) to enhance its axial resolution via a digital micromirror device (DMD). First, the orientation of the modulated sinusoidal fringe patterns is automatically identified via spatial frequency vector detection. Subsequently, the modulated in-focal-plane images are obtained via rotation and subtraction. Lastly, …
Fluorescent observation of cells generally suffers from the limited axial resolution due to the elongated point spread function of the microscope optics. Consequently, three-dimensional imaging results in axial resolution being several times worse than the transversal. The optical solutions to this problem usually require complicated optics and extreme spatial stability. A straightforward way to eliminate anisotropic resolution is to fuse images recorded from multiple viewing directions achieved mostly by the mechanical rotation of the entire sample. In the presented approach, multiview imagin…
Over the last decade, single-molecule optical microscopy has become the gold-standard approach to decipher complex molecular processes in cellular environments. [1-3] Single-molecule fluorescence microscopy has several advantages such as ease of application, high sensitivity, low invasiveness and versatility due the large number of available fluorescent probes. It bears however some drawbacks related to the poor photostability of organic dye molecules [4] and auto-fluorescent proteins [5-7] and and to the relatively large size of semiconductor nanoparticles in the context of live cell applicat…
We experimentally demonstrate precision addressing of single quantum emitters by combined optical microscopy and spin resonance techniques. To this end we utilize nitrogen-vacancy (NV) color centers in diamond confined within a few ten nanometers as individually resolvable quantum systems. By developing a stochastic optical reconstruction microscopy (STORM) technique for NV centers we are able to simultaneously perform sub diffraction-limit imaging and optically detected spin resonance (ODMR) measurements on NV spins. This allows the assignment of spin resonance spectra to individual NV center…
Light sheet fluorescence microscopy provides optical sectioning and is widely used in volumetric imaging of large specimens. However, the axial resolution and the lateral Field of View (FoV) of the system, defined by the light sheet, typically limit each other due to the spatial band product of the excitation objective. Here, we develop a simple multi-focus scheme to extend the FoV, where a Gaussian light sheet can be focused at three or more consecutive positions. Axially overlapped multiple light sheets significantly enlarge the FoV with improved uniformity and negligible loss in axial resol…
We theorize the surface step characterization by reflected incoherent-light differential interference microscopy with consideration of the optical diffraction effect. With the integration of localization analysis, we develop a quantitative differential interference optical system, by which we demonstrate that the axial resolution of measuring surface height variation is sensitive to the shear distance between the two spatially differentiated beams. We fabricate three nanometer-size steps by photolithography, and successfully characterize their 1D height variations with 0.13 nm Hz^(-1/2) axial …
How far a particle moves along the optical axis in a holographic optical trap is not simply dictated by the programmed motion of the trap, but rather depends on an interplay of the trap's changing shape and the particle's material properties. For the particular case of colloidal spheres in optical tweezers, holographic video microscopy reveals that trapped particles tend to move farther along the axial direction than the traps that are moving them and that different kinds of particles move by different amounts. These surprising and sizeable variations in axial placement can be explained by a d…
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 Defocusing Microscopy (DM), a bright-field optical microscopy technique able to perform total 3D imaging of transparent objects. By total 3D imaging we mean the determination of the actual shapes of the upper and lower surfaces of a phase object. We propose a new methodology using DM and apply it to red blood cells subject to different osmolality conditions: hypotonic, isotonic and hypertonic solutions. For each situation the shape of the upper and lower cell surface-membranes (lipid bilayer/cytoskeleton) are completely recovered, displaying the deformation of RBCs surfaces due to a…
Multiphoton microscopy (MPM) has gained enormous popularity over the years for its capacity to provide high resolution images from deep within scattering samples1. However, MPM is generally based on single-point laser-focus scanning, which is intrinsically slow. While imaging speeds as fast as video rate have become routine for 2D planar imaging, such speeds have so far been unattainable for 3D volumetric imaging without severely compromising microscope performance. We demonstrate here 3D volumetric (multiplane) imaging at the same speed as 2D planar (single plane) imaging, with minimal compro…
Fast 3D super-resolution imaging is essential for decoding rapidly occurring biological processes. Encoding single molecules to their respective planes enable simultaneous multi-plane super-resolution volume imaging. This saves the data-acquisition time and as a consequence reduce radiation-dose that lead to photobleaching and other undesirable photochemical reactions. Detection and subsequent identification of the locus of individual molecule (both on the focal plane and off-focal planes) holds the key. Experimentally, this is achieved by accurate calibration of system PSF size and its natura…
The rise of interest in the study of quantum emitters has recently prompted many research groups to construct their own confocal epifluorescence microscopy/spectroscopy instruments. The low light levels typically involved in quantum emitter measurements makes it critically important to account for any potential sources of background fluorescence by components used within such setups. In this report, we show that emission originating from various microscope objectives can possess elusive sharp linewidths that could potentially be mistaken for quantum emission. Impurities present in many glasses…
In this paper, we show that it is possible to overcome one of the fundamental limitations of super-resolution microscopy techniques: the necessity to be in an \emph{optically homogeneous} environment. Using recent modal approximation results we show as a proof of concept that it is possible to recover the position of a single point-like emitter in a \emph{known resonant environment} from far-field measurements with a precision two orders of magnitude below the classical Rayleigh limit. The procedure does not involve solving any partial differential equation, is computationally light (optimisat…
Institute of Micro- and Nanotechnologies, Technische Universität Ilmenau（微纳技术研究所，埃尔朗根-纽伦堡工业大学）；Institute of Thermodynamics and Fluid Mechanics, Technische Universität Ilmenau（热力学与流体力学研究所，埃尔朗根-纽伦堡工业大学）
Optical cavities can enhance and control light-matter interactions. This has recently been extended to the nanoscale, and with single emitter strong coupling regime even at room temperature using plasmonic nano-cavities with deep sub-diffraction-limited mode volumes. However, with emitters in static nano-cavities, this limits the ability to tune coupling strength or to couple different emitters to the same cavity. Here, we present tip-enhanced strong coupling (TESC) spectroscopy, imaging, and control. Based on a nano-cavity formed between a scanning plasmonic antenna-tip and the substrate, by …
Single molecule localization microscopy has the potential to resolve structural details of biological samples at the nanometer length scale. However, to fully exploit the resolution it is crucial to account for the anisotropic emission characteristics of fluorescence dipole emitters. In case of slight residual defocus, localization estimates may well be biased by tens of nanometers. We show here that astigmatic imaging in combination with information about the dipole orientation allows to extract the position of the dipole emitters without localization bias and down to a precision of ~1nm, the…
