Computational resolution in single molecule localization - impact of noise level and emitter density.

Classical fluorescence microscopy is a powerful technique to image biological specimen under close-to-native conditions, but light diffraction limits its optical resolution to 200-300 nm-two orders of magnitude worse than the size of biomolecules. Assuming single fluorescent emitters, the final image of the optical system can be described by a convolution with the point spread function (PSF) smearing out details below the size of the PSF. In mathematical terms, fluorescence microscopy produces bandlimited space-continuous images that can be recovered from their spatial samples under the conditions of the classical Shannon-Nyquist theorem. During the past two decades, several single molecule localization techniques have been established and these allow for the determination of molecular positions with sub-pixel accuracy. Without noise, single emitter positions can be recovered precisely - no matter how close they are. We review recent work on the computational resolution limit with a sharp phase transition between two scenarios: 1) where emitters are well-separated with respect to the bandlimit and can be recovered up to the noise level and 2) closely distributed emitters which results in a strong noise amplification in the worst case. We close by discussing additional pitfalls using single molecule localization techniques based on structured illumination.

Microemitter-Based IR Spectroscopy and Imaging with Multilayer Graphene Thermal Emission.

IR analyses such as Fourier transform infrared spectroscopy (FTIR) are widely used in many fields; however, the performance of FTIR is limited by the slow speed (∼10 Hz), large footprint (∼ millimeter), and glass bulb structure of IR light sources. Herein, we present IR spectroscopy and imaging based on multilayer-graphene microemitters, which have distinct features: a planar structure, bright intensity, a small footprint (sub-µm2), and high modulation speed of >50 kHz. We developed an IR analysis system based on the multilayer-graphene microemitter and performed IR absorption spectroscopy. We show two-dimensional IR chemical imaging that visualizes the distribution of the chemical information. In addition, we present high-spatial-resolution IR imaging with a spatial resolution of ∼1 µm, far higher than the diffraction limit. The graphene-based IR spectroscopy and imaging can open new routes for IR applications in chemistry, material science, medicine, biology, electronics, and physics.

Seeing beyond the limit: A guide to choosing the right super-resolution microscopy technique.

Super-resolution microscopy has become an increasingly popular and robust tool across the life sciences to study minute cellular structures and processes. However, with the increasing number of available super-resolution techniques has come an increased complexity and burden of choice in planning imaging experiments. Choosing the right super-resolution technique to answer a given biological question is vital for understanding and interpreting biological relevance. This is an often-neglected and complex task that should take into account well-defined criteria (e.g., sample type, structure size, imaging requirements). Trade-offs in different imaging capabilities are inevitable; thus, many researchers still find it challenging to select the most suitable technique that will best answer their biological question. This review aims to provide an overview and clarify the concepts underlying the most commonly available super-resolution techniques as well as guide researchers through all aspects that should be considered before opting for a given technique.

λ/12 Super Resolution Achieved in Maskless Optical Projection Nanolithography for Efficient Cross-Scale Patterning.

The emerging demand for device miniaturization and integration prompts the patterning technique of micronano-cross-scale structures as an urgent desire. Lithography, as a sufficient patterning technique, has been playing an important role in achieving functional micronanoscale structures for decades. As a promising alternative, we have proposed and demonstrated the maskless optical projection nanolithography (MLOP-NL) technique for efficient cross-scale patterning. A minimum feature size of 32 nm, which is λ/12 super resolution breaking the optical diffraction limit, has been achieved by a single exposure. Furthermore, multiscale two-dimensional micronano-hybrid structures with the size over hundreds of micrometers and the precision at tens of nanometers have been fabricated by simply controlling the exposure conditions. The proposed MLOP-NL technique provides a powerful tool for achieving cross-scale patterning with both large-scale and precise configuration with high efficiency, which can be potentially used in the fabrication of multiscale integrated microsystems.

Inverse-phase composite zone plate providing deeper focus than the normal diffraction-limited depth of X-ray microbeams.

A novel type of zone plate (ZP), termed an inverse-phase composite ZP, is proposed to gain a deeper focus than the standard diffraction-limited depth of focus, with little reduction in spatial resolution. The structure is a combination of an inner ZP functioning as a conventional phase ZP and an outer ZP functioning with third-order diffraction with opposite phase to the inner ZP. Two-dimensional complex amplitude distributions neighboring the focal point were calculated using a wave-optical approach of diffraction integration with a monochromatic plane-wave illumination, where one dimension is the radial direction and the other dimension is the optical-axis direction. The depth of focus and the spatial resolution were examined as the main focusing properties. Two characteristic promising cases regarding the depth of focus were found: a pit-intensity focus with the deepest depth of focus, and a flat-intensity focus with deeper depth of focus than usual ZPs. It was found that twice the depth of focus could be expected with little reduction in the spatial resolution for 10 keV X-ray energy, tantalum zone material, 84 nm minimum fabrication zone width, and zone thickness of 2.645 µm. It was also found that the depth of focus and the spatial resolution were almost unchanged in the photon energy range from 8 to 12 keV. The inverse-phase composite ZP has high potential for use in analysis of practical thick samples in X-ray microbeam applications.

Laser-slicing at a low-emittance storage ring.

Laser-slicing at a diffraction-limited storage ring light source in the soft X-ray region is investigated with theoretical and numerical modelling. It turns out that the slicing efficiency is favoured by the ultra-low beam emittance, and that slicing can be implemented without interference to the standard multi-bunch operation. Spatial and spectral separation of the sub-picosecond radiation pulse from a hundreds of picosecond-long background is achieved by virtue of 1:1 imaging of the radiation source. The spectral separation is enhanced when the radiator is a transverse gradient undulator. The proposed configuration applied to the Elettra 2.0 six-bend achromatic lattice envisages total slicing efficiency as high as 10-7, one order of magnitude larger than the demonstrated state-of-the-art, at the expense of pulse durations as long as 0.4 ps FWHM and average laser power as high as ∼40 W.

Sub-Wavelength Focusing in Inhomogeneous Media with a Metasurface Near Field Plate.

Overcoming the diffraction limit, which enables focusing much less than the wavelength, requires tailoring the evanescent spectrum of an aperture's field distribution. We model and simulate a corrugated near field plate, which can generate a sub-wavelength focus in inhomogeneous background media. All reactive coupling, between the metasurface near field plate and the focusing domain and among the corrugations in the metasurface, is taken into consideration with the finite element method, which we solve in combination with a constraint to generate a desired focus. Various geometries for the near field plate are considered and we demonstrate that the proposed method can effectively create a deeply sub-wavelength focus within a layered medium having properties resembling brain tissue. Such a device could find use as a detector of biological signals or for hyperthermic treatment near the skin surface.

One way only to synchrotron light sources upgrade?

The last decade has seen a renaissance of machine-physics studies and technological advancements that aim to upgrade at least 15 synchrotron light sources worldwide to diffraction-limited storage rings. This is expected to improve the average spectral brightness and transversally coherent fraction of photons by several orders of magnitude in the soft- and hard-X-ray wavelength range, at the expense of pulse durations longer than ∼80 ps FWHM. This paper discusses the compatibility of schemes for the generation of sub-picosecond photon-pulse durations in synchrotron light sources with standard multi-bunch user operation and, in particular, diffraction-limited electron optics design. The question of this compatibility is answered taking into consideration the storage ring beam energy and the constraint of existing synchrotrons' infrastructure. An alternative scheme for the upgrade of medium-energy synchrotron light sources to diffraction-limited storage rings and the simultaneous production of picosecond-long photon pulses in a high-gain free-electron laser scheme are illustrated.

The coherent radiation fraction of low-emittance synchrotrons.

In this work the coherence properties of the synchrotron radiation beam from an X-ray undulator in a fourth-generation storage ring are analyzed. A slightly focused X-ray beam is simulated using a wavefront propagation through a non-redundant array of slits and the mutual coherence function is directly obtained and compared with the Gaussian-Schell approximation. The numerical wave propagation and the approximate analytical approaches are shown to agree qualitatively, and it is also shown that, when the coherent fraction is selected by a finite aperture before the focusing element, even achromatic focusing systems like total reflection mirrors become slightly chromatic. This effect is also well accounted for in the Gaussian-Schell model. The wavefront propagation simulation through the non-redundant array was repeated with an imperfect mirror demonstrating that, although the wavefront is distorted, its coherent length is practically unchanged.

Super-resolution measurement of distance between transcription sites using RNA FISH with intronic probes.

Nascent transcripts being copied from specific human genes can be detected using RNA FISH (fluorescence in situ hybridization) with intronic probes, and the distance between two different nascent transcripts is often measured when studying structure-function relationships. Such distance measurements are limited by the resolution of the light microscope. Here we describe methods for measuring these distances in cultured cells with a precision of a few tens of nanometers, using equipment found in most laboratories (i.e., a wide-field fluorescence microscope equipped with a charged-coupled-device camera). Using images of pairs of transcripts that are often co-transcribed, we discuss how selection of cell type, design of FISH probes, image acquisition, and image processing affect the precision that can be achieved.

Experimental Demonstration of Demagnifying Hyperlens.

The emergence of optical metamaterials opens new opportunities for spatial pattern compression from the micro- to nanoscale. By exploiting strongly anisotropic optical properties of engineered nanostructures, we realize the first experimental demonstration of demagnifying hyperlens enabling optical patterning below the diffraction limit. We show that the diffraction-limited features on a mask can be demagnified to form the subwavelength patterns on the photoresist using visible light.

High Quantum Efficiency Nanopillar Photodiodes Overcoming the Diffraction Limit of Light.

InAs1-xSbx nanowires have recently attracted interest for infrared sensing applications due to the small bandgap and high thermal conductivity. However, previous reports on nanowire-based infrared sensors required low operating temperatures in order to mitigate the high dark current and have shown poor sensitivities resulting from reduced light coupling efficiency beyond the diffraction limit. Here, InAsSb nanopillar photodiodes with high quantum efficiency are achieved by partially coating the nanopillar with metal that excites localized surface plasmon resonances, leading to quantum efficiencies of ∼29% at 2390 nm. These high quantum efficiency nanopillar photodiodes, with 180 nm diameters and 1000 nm heights, allow operation at temperatures as high as 220 K and exhibit a detection wavelength up to 3000 nm, well beyond the diffraction limit. The InAsSb nanopillars are grown on low cost GaAs (111)B substrates using an InAs buffer layer, making our device architecture a promising path toward low-cost infrared focal plane arrays with high operating temperature.

Plasmonic superlensing in doped GaAs.

We demonstrate a semiconductor based broadband near-field superlens in the mid-infrared regime. Here, the Drude response of a highly doped n-GaAs layer induces a resonant enhancement of evanescent waves accompanied by a significantly improved spatial resolution at radiation wavelengths around λ = 20 µm, adjustable by changing the doping concentration. In our experiments, gold stripes below the GaAs superlens are imaged with a λ/6 subwavelength resolution by an apertureless near-field optical microscope utilizing infrared radiation from a free-electron laser. The resonant behavior of the observed superlensing effect is in excellent agreement with simulations based on the Drude-Lorentz model. Our results demonstrate a rather simple superlens implementation for infrared nanospectroscopy.

Nanoscopy with Focused Light (Nobel Lecture).

A picture is worth a thousand words-This doesn't only apply to everyday life but also to the natural sciences. It is, therefore, probably not by chance that the historical beginning of modern natural sciences very much coincides with the invention of light microscopy. S. W. Hell shows in his Nobel Lecture that the diffraction resolution barrier has been overcome by using molecular state transitions (e.g. on/off) to make nearby molecules transiently discernible.

A Lossless Sink Based on Complex Frequency Excitations.

The creation of a sink in a lossless wave-bearing medium is achieved using complex frequency signals-harmonic excitations that exponentially grow in time. The wave sink, where incident waves are confined to a point, has attracted interest for imaging and sensing since it may lead to arbitrarily small hotspots that surpass the diffraction limit. However, most methods of creating sinks require careful tuning, such as by impedance matching the sink to free space through the inclusion of loss, which imposes constraints on emerging applications. An alternative method, proposed here, relies on complex frequency excitations, bypassing the need to modify the scattering system by instead shaping the input signal. Eigenvalue zeros derived from a scattering formalism extended to the complex frequency plane reveal operating conditions that induce complete energy trapping under steady-state conditions in a framework generally applicable to 2D and 3D media. To support the developed theory, an experiment is performed where a sink is realized using elastic waves on a plate with a circular cutout. These findings may lead to imaging and sensing applications relying on subwavelength focal points and nonlinear wave generation due to the high amplitudes achieved over short timescales.

Super-resolution STED imaging in the inner and outer whole-mount mouse retina.

Since its invention, super-resolution microscopy has become a popular tool for advanced imaging of biological structures, allowing visualisation of subcellular structures at a spatial scale below the diffraction limit. Thus, it is not surprising that recently, different super-resolution techniques are being applied in neuroscience, e.g. to resolve the clustering of neurotransmitter receptors and protein complex composition in presynaptic terminals. Still, the vast majority of these experiments were carried out either in cell cultures or very thin tissue sections, while there are only a few examples of super-resolution imaging in deeper layers (30 - 50 µm) of biological samples. In that context, the mammalian whole-mount retina has rarely been studied with super-resolution microscopy. Here, we aimed at establishing a stimulated-emission-depletion (STED) microscopy protocol for imaging whole-mount retina. To this end, we developed sample preparation including horizontal slicing of retinal tissue, an immunolabeling protocol with STED-compatible fluorophores and optimised the image acquisition settings. We labelled subcellular structures in somata, dendrites, and axons of retinal ganglion cells in the inner mouse retina. By measuring the full width at half maximum of the thinnest filamentous structures in our preparation, we achieved a resolution enhancement of two or higher compared to conventional confocal images. When combined with horizontal slicing of the retina, these settings allowed visualisation of putative GABAergic horizontal cell synapses in the outer retina. Taken together, we successfully established a STED protocol for reliable super-resolution imaging in the whole-mount mouse retina at depths between 30 and 50 µm, which enables investigating, for instance, protein complex composition and cytoskeletal ultrastructure at retinal synapses in health and disease.

Super-resolution imaging of micro- and nanoplastics using confocal Raman with Gaussian surface fitting and deconvolution.

Confocal Raman imaging can directly identify and visualise microplastics and even nanoplastics. However, due to diffraction, the excitation laser spot has a size, which defines the image resolution. Consequently, it is difficult to image nanoplastic that is smaller than the diffraction limit. Within the laser spot, fortunately, the excitation energy density behaves an axially transcended distribution, or a 2D Gaussian distribution. By mapping the emission intensity of Raman signal, the imaged nanoplastic pattern is axially transcended as well and can be fitted as a 2D Gaussian surface via deconvolution, to re-construct the Raman image. The image re-construction can intentionally and selectively pick up the weak signal of nanoplastics, average the background noise/the variation of the Raman intensity, smoothen the image surface and re-focus the mapped pattern towards signal enhancement. Using this approach, along with nanoplastics models with known size for validation, real samples are also tested to image microplastics and nanoplastics released from the bushfire-burned face masks and water tanks. Even the bushfire-deviated surface group can be visualised as well, to monitor the different degrees of burning by visualising micro- and nanoplastics. Overall, this approach can effectively image regular shape of micro- and nanoplastics, capture nanoplastics smaller than the diffraction limit, and realise super-resolution imaging via confocal Raman.

High- and Super-Resolution Imaging of Cell-Cell Interfaces.

Physical interfaces mediate interactions between multiple types of cells. Despite the importance of such interfaces to the cells' function, their high-resolution optical imaging has been typically limited due to poor alignment of the interfaces relative to the optical plane of imaging. Here, we present a simple and robust method to align cell-cell interfaces in parallel to the coverslip by adhering the interacting cells to two opposing coverslips and bringing them into contact in a controlled and stable fashion. We demonstrate aberration-free high-resolution imaging of interfaces between live T cells and antigen-presenting cells, known as immune synapses, as an outstanding example. Imaging methods may include multiple diffraction-limited and super-resolution microscopy techniques (e.g., bright-field, confocal, STED, and dSTORM). Thus, our simple and widely compatible approach allows imaging with high- and super-resolution the intricate structure and molecular organization within a variety of cell-cell interfaces.

Roadmap on Label-Free Super-Resolution Imaging.

Label-free super-resolution (LFSR) imaging relies on light-scattering processes in nanoscale objects without a need for fluorescent (FL) staining required in super-resolved FL microscopy. The objectives of this Roadmap are to present a comprehensive vision of the developments, the state-of-the-art in this field, and to discuss the resolution boundaries and hurdles which need to be overcome to break the classical diffraction limit of the LFSR imaging. The scope of this Roadmap spans from the advanced interference detection techniques, where the diffraction-limited lateral resolution is combined with unsurpassed axial and temporal resolution, to techniques with true lateral super-resolution capability which are based on understanding resolution as an information science problem, on using novel structured illumination, near-field scanning, and nonlinear optics approaches, and on designing superlenses based on nanoplasmonics, metamaterials, transformation optics, and microsphere-assisted approaches. To this end, this Roadmap brings under the same umbrella researchers from the physics and biomedical optics communities in which such studies have often been developing separately. The ultimate intent of this paper is to create a vision for the current and future developments of LFSR imaging based on its physical mechanisms and to create a great opening for the series of articles in this field.

Speed Microscopy: High-Speed Single Molecule Tracking and Mapping of Nucleocytoplasmic Transport.

The nuclear pore complex (NPC) functions as a gateway through which molecules translocate into and out of the nucleus. Understanding the transport dynamics of these transiting molecules and how they interact with the NPC has great potentials in the discovery of clinical targets. Single-molecule microscopy techniques are powerful tools to provide sub-diffraction limit information about the dynamic and structural details of nucleocytoplasmic transport. Here we detail single-point edge-excitation subdiffraction (SPEED) microscopy, a high-speed superresolution microscopy technique designed to track and map proteins and RNAs as they cross native NPCs.