Zhaxylyk A. Kudyshev, Demid Sychev, Zachariah Martin, Simeon I. Bogdanov, Xiaohui Xu, Alexander V. Kildishev, Alexandra Boltasseva, Vladimir M. Shalaev
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 the spatial resolution by measuring the n-th order autocorrelation function. The main bottleneck of the antibunching super-resolution microscopy is the time-consuming acquisition of multi-photon event histograms. We present a machine learning-assisted approach for the realization of rapid antibunching super-resolution imaging and demonstrate 12 times speed-up compared to conventional, fitting-based autocorrelation measurements. The developed framework paves the way to the practical realization of scalable quantum super-resolution imaging devices that can be compatible with various types of quantum emitters.
Correlative light-electron microscopy (CLEM) unifies the versatility of light microscopy (LM) with the high resolution of electron microscopy (EM), allowing one to zoom into the complex organization of cells. Most CLEM techniques use ultrathin sections, and thus lack the 3D-EM structural information, and focusing on a very restricted field of view. Here, we introduce photonic chip assisted CLEM, enabling multi-modal total internal reflection fluorescence (TIRF) microscopy over large field of view and high precision localization of the target area of interest within EM. The chip-based direct st…
We demonstrate stimulated emission depletion (STED) microscopy with 20 nm gold nanospheres coated by fluorescent silica. Compared with previous demonstrations of STED with a hybrid plasmonic fluorescent label, the current implementation offers a substantially smaller label and a better resolution improvement of up to 2.5-fold beyond the diffraction limit of confocal microscopy. This is achieved at approximately 2 times lower intensity than conventional STED based on dyes alone, and in an aqueous environment, demonstrating the relevance to bio-imaging. Finally, we also show, for the first time …
Microsphere-assisted imaging emerged as a surprisingly simple way of achieving optical super-resolution imaging. In this work, we use movable PDMS thin films with embedded high-index barium titanate glass microspheres a sample scanning capability was developed, thus removing the main limitation of this technology based on its small field-of-view.
Structured Illumination Microscopy (SIM) is an imaging technique for achieving both super-resolution (SR) and optical sectioning (OS) in wide-field microscopy. It consists in illuminating the sample with periodic patterns at different orientations and positions. The resulting images are then processed to reconstruct the observed object with SR and/or OS. In this work, we present BOSSA-SIM, a general-purpose SIM reconstruction method, applicable to moving objects such as encountered in in vivo retinal imaging, that enables SR and OS jointly in a fully unsupervised Bayesian framework. By modelin…
Two-Photon Microscopy has become an invaluable tool for biological and medical research, providing high sensitivity, molecular specificity, inherent three-dimensional sub-cellular resolution and deep tissue penetration. In terms of imaging speeds, however, mechanical scanners still limit the acquisition rates to typically 10-100 frames per second. Here we present a high-speed non-linear microscope achieving kilohertz frame rates by employing pulse-modulated, rapidly wavelength-swept lasers and inertia-free beam steering through angular dispersion. In combination with a high bandwidth, single-p…
The emergence of far-field super-resolution microscopy has rejuvenated the possibility for nanoscale imaging. Approaches to far-field super-resolution that utilize point scanning often depends on spatially reducing the size of the focused spot. However, the focused spot always achieves high resolution at the expense of extremely low light efficiency for the probing mainlobe and high-intensity sidelobes, which limits the applications in nanoscale imaging and might cause misinterpretation of samples. Here we report a sharp probing spot with the diffraction efficiency of 3.76% at the resolution o…
We extend image scanning microscopy to second harmonic generation (SHG) by extracting the complex field amplitude of the second-harmonic beam. While the theory behind coherent image scanning microscopy (ISM) is known, an experimental demonstration wasn't yet established. The main reason is that the naive intensity-reassignment procedure cannot be used for coherent scattering as the point spread function is now defined for the field amplitude rather than for the intensity. We use an inline interferometer to demonstrate super-resolved phase-sensitive SHG microscopy by applying the ISM reassignme…
Super-resolution light microscopy overcomes the physical barriers due to light diffraction, allowing for the observation of otherwise indistinguishable subcellular entities. However, the specific acquisition conditions required by state-of-the-art super-resolution methods to achieve adequate spatio-temporal resolution are often very challenging. Exploiting molecules fluctuations allows good spatio-temporal resolution live-cell imaging by means of common microscopes and conventional fluorescent dyes. In this work, we present the method COL0RME for COvariance-based $\ell_0$ super-Resolution Micr…
In recent years several far-field microscopy techniques have been developed which manage to overcome the diffraction limit of resolution. A unifying classification scheme for them is clearly desirable. We argue that existing schemes based on the information capacity of the optical system can not easily be extended to cover e.g., STED microscopy or techniques based on single molecule imaging. We suggest a classification based on a reconstruction of the Abbe limit.
We demonstrate a new versatile method for 3D super-resolution microscopy by using a deformable mirror to shape the point spread function of our microscope in a continuous and controllable way. We apply this for 3D STORM imaging of microtubules.
We demonstrate an optical far-field super-resolution microscopy using array of nitrogen vacancy centers in bulk diamond as near-field optical probes. The local optical field, which transmits through the nanostructures on the diamond surface, is measured by detecting the charge state conversion of nitrogen vacancy center. And the locating of nitrogen vacancy center with spatial resolution of 6.1 nm is realized with the charge state depletion nanoscopy. The nanostructures on the surface of diamond are then imaged with resolution below optical diffraction limit. The results offer an approach to b…
Super-resolution optical microscopy is a rapidly evolving scientific field dedicated to imaging sub-wavelength sized objects, leaving its mark in multiple branches of biology and technology. While several super-resolution optical microscopy methods have become a common tool in life science imaging, new methods, supported by cutting-edge technology, continue to emerge. One rather recent addition to the super-resolution toolbox, image scanning microscopy (ISM), achieves an up to twofold lateral resolution enhancement in a robust and straightforward manner. To further enhance ISM's resolution in …
I explain what is, what is not, and what is only sort of superresolution microscopy. I discuss optical resolution, first in terms of diffraction theory, then in terms of linear systems theory, and finally in terms of techniques that use prior information, nonlinearity, and other tricks to improve performance. The discussion reveals two classes of superresolution: Pseudo superresolution techniques improve images up to the diffraction limit but not much beyond. True superresolution techniques allow substantial, useful improvements beyond the diffraction limit. The two classes are distinguished b…
Super-resolution microscopy in the visible is an established powerful tool in several disciplines. In the infrared (IR) spectral range, however, no comparable schemes have been demonstrated to date. In this work, we experimentally demonstrate super-resolution microscopy in the IR range ($λ_{IR}\approx 10-12\,μ$m) using IR-visible sum-frequency generation. We operate our microscope in a wide-field scheme and image localized surface phonon polaritons in 4H-SiC nanostructures as a proof-of-concept. With this technique, we demonstrate an enhanced spatial resolution of $\simλ_{IR}/9$, enabling to r…
Structured illumination microscopy (SIM) has emerged as an essential technique for 3D and live-cell super-resolution imaging. However, to date, there has not been a dedicated workshop or journal issue covering the various aspects of SIM, from bespoke hardware and software development and the use of commercial instruments to biological applications. This special issue aims to recap recent developments as well as outline future trends. In addition to SIM, we cover related topics such as complementary super-resolution microscopy techniques, computational imaging, visualisation and image processin…
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…
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…
Stimulated emission depletion (STED) microscopy has become a powerful imaging and localized excitation method beating the diffraction barrier for improved lateral spatial resolution in cellular imaging, lithography, etc. Due to specimen-induced aberrations and scattering distortion, it has been a great challenge for STED to maintain consistent lateral resolution deeply inside the specimens. Here we report on a deep imaging STED microscopy by using Gaussian beam for excitation and hollow Bessel beam for depletion (GB-STED). The proposed scheme shows the improved imaging depth up to ~155μm in so…
Hierarchical polymer structures such as pNIPAM microgels have been extensively studied for their ability to undergo significant structural and physical transformations that can be controlled by external stimuli such as temperature, pH or solvent composition. However, direct three-dimensional visualization of individual particles in situ have so far been hindered by insufficient resolution, with optical microscopy, or contrast, with electron microscopy. In recent years superresolution microscopy techniques have emerged that in principle can provide nanoscopic optical resolution. Here we report …
Quadriwave lateral shearing interferometry (QLSI) is a quantitative phase imaging technique based on the use of a diffraction grating placed in front of a camera. This grating creates a wire-mesh-like image, called an interferogram, that is postprocessed to retrieve both the intensity and phase profiles of an incoming light beam. Invented in the 90s, QLSI has been used in numerous applications, e.g., laser beam characterization, lens metrology, topography measurements, adaptive optics, or gas jet metrology. More recently, the technique has been implemented on optical microscopes to characteriz…
Super-resolved far-field microscopy has emerged as a powerful tool for investigating the structure of objects with resolution well below the diffraction limit of light. Nearly all super-resolution imaging techniques reported to date rely on real energy states of probe molecules to circumvent the diffraction limit, preventing super-resolved imaging of contrast mechanisms that occur via virtual energy states such as harmonic generation (HG). Here we report a super-resolution technique based on SPatIal Frequency modulated Imaging (SPIFI) that permits super-resolved nonlinear microscopy with any c…
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 present a novel experimental setup in which magnetic and optical tweezers are combined for torque and force transduction onto single filamentous molecules in a transverse configuration to allow simultaneous mechanical measurement and manipulation. Previously we have developed a super-resolution imaging module which in conjunction with advanced imaging techniques such as Blinking assisted Localisation Microscopy (BaLM) achieves localisation precision of single fluorescent dye molecules bound to DNA of ~30 nm along the contour of the molecule; our work here describes developments in producing…
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…
Super-resolution microscopy is providing unprecedented insights into biology by resolving details much below the diffraction limit. State-of-the-art Single Molecule Localization Microscopy (SMLM) techniques for super-resolution are restricted by long acquisition and computational times, or the need of special fluorophores or chemical environments. Here, we propose a novel statistical super-resolution technique of wide-field fluorescence microscopy called MUltiple SIgnal Classification ALgorithm (MUSICAL) which has several advantages over SMLM techniques. MUSICAL provides resolution down to at …
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 $m$ creates an effective PSF with FWHM shrunk by the factor $\sqrt{m}$. 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 simulta…
We present experimental demonstration of tilt-mirror assisted transmission structured illumination microscopy (tSIM) that offers a large field of view super resolution imaging. An assembly of custom-designed tilt-mirrors are employed as the illumination module where the sample is excited with the interference of two beams reflected from the opposite pair of mirror facets. Tunable frequency structured patterns are generated by changing the mirror-tilt angle and the hexagonal-symmetric arrangement is considered for the isotropic resolution in three orientations. Utilizing high numerical aperture…
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.
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 principles of quantum optics have yielded a plethora of ideas to surpass the classical limitations of sensitivity and resolution in optical microscopy. While some ideas have been applied in proof-of-principle experiments, imaging a biological sample has remained challenging mainly due to the inherently weak signal measured and the fragility of quantum states of light. In principle, however, these quantum protocols can add new information without sacrificing the classical information and can therefore enhance the capabilities of existing super-resolution techniques. Image scanning microscop…
Sub-diffraction resolution, gentle sample illumination, and the possibility to image in multiple colors make Structured Illumination Microscopy (SIM) an imaging technique which is particularly well suited for live cell observations. Here, we present Machine learning Assisted Interferometric-SIM (MAI-SIM), an easy-to-implement method for high speed SIM imaging in multiple colors. The instrument is based on an interferometer design in which illumination patterns are generated, rotated, and stepped in phase through movement of a single galvanometric mirror element. The design is robust, flexible,…
Although several optical techniques have been recently developed in order to overcome the resolution limit in microscopy, the imaging of sub-wavelength features is still a real challenge. In practise, super-resolution techniques remain difficult to build or are photo-toxic for the biological samples. However, microsphere-assisted microscopy has recently made super-resolution imaging accessible to scientists (e.g. optical metrologists, engineers and biologists). This paper presents an easy-to-implement optical setup to perform full-field and contactless super-resolution measurements of nanostru…
