Optical Resolution Limit Research Articles

Modern Methods of Fluorescence Nanoscopy in Biology

Optical microscopy has undergone significant changes in recent decades due to the breaking of the diffraction limit of optical resolution and the development of high-resolution imaging techniques, which are collectively known as fluorescence nanoscopy. These techniques allow researchers to observe biological structures and processes at a nanoscale level of detail, revealing previously hidden features and aiding in answering fundamental biological questions. Among the advanced methods of fluorescent nanoscopy are: STED (Stimulated Emission Depletion Microscopy), STORM (STochastic Optical Reconstruction Microscopy), PALM (Photo-activated Localization Microscopy), TIRF (Total Internal Reflection Fluorescence), SIM (Structured Illumination Microscopy), MINFLUX (Minimal Photon Fluxes), PAINT (Points Accumulation for Imaging in Nanoscale Topography) и RESOLFT (REversible Saturable Optical Fluorescence Transitions) and others. In addition, most of these methods make it possible to obtain volumetric (3D) images of the objects under study. In this review, we will look at the principles of these methods, their advantages and disadvantages, and their application in biological researches.

Super-resolved highly multiplexed immunofluorescence imaging for precise protein localization and podocyte ultrastructure.

Deep insights into the complex cellular and molecular changes occurring during (patho-)physiological conditions are essential for understanding the interactions and regulation of proteins. This understanding is crucial for research and diagnostics. However, the effectiveness of conventional immunofluorescence and light microscope, tools for visualizing the spatial distribution of cells or proteins, are limited both in resolution and multiplexity in complex tissues. This is mainly due to challenges such as the spectral overlap of fluorophore wavelengths, a limited range of antibody types, the inherent variability of samples and the optical resolution limit. The herein demonstrated combination of multiplex immunofluorescence imaging and super resolution microscopy offers a solution to these limitations by enabling the identification of different cell types and precise subcellular localization of proteins in tissue sections. In this study, we demonstrate the cyclic staining and de-staining of paraffin kidney sections, making it suitable for routine use and compatible with super-resolution microscopy for podocyte ultrastructural studies. We have further developed a computerized workflow for data processing which is accessible through available reagents and open-access code. As a proof of principle, we identified CDH2 as a marker for cellular lesions of sclerotic glomeruli in the nephrotoxic serum nephritis mouse model and cross-validated this finding with a human Nephroseq dataset indicating its translatability. In summary, our work represents an advance in multiplex imaging, which is crucial for understanding the localization of numerous proteins in a single FFPE kidney section and the compatibility with super-resolution microscopy to study ultrastructural changes of podocytes.

Overlay Mark Design Using Irregular Grating Patterns

Overlay refers to controlling the vertical alignment and misalignment between circuit patterns manufactured in the previous process and circuit patterns manufactured in the current process during the stacking process of circuit patterns in the semiconductor manufacturing process. A critical factor in ensuring precise overlay measurements is using an optimized overlay mark design. Many semiconductor manufacturers have put considerable effort into optimizing overlay measurement marks to reduce high costs. However, image-based overlay measurement encounters several challenges, including resolution degradation, image distortion, and noise introduction during digital data conversion. These issues can amplify the uncertainty of overlay measurements, impair optical resolution, and heighten the risk of mismeasurement. In this paper, we introduce irregular grating patterns designed to address the principal challenges faced by the commonly utilized Advanced Imaging Metrology (AIM) marks, namely large overlays and image distortion. Furthermore, we propose an overlay mark capable of precisely identifying overlays exceeding half a pitch and incorporating as much data as possible within the limits of optical resolution. This aims to improve overlay measurement performance. The experimental findings demonstrate that the proposed Pulsated Grating Target (PGT) mark enhances overall measurement uncertainty by approximately 17% compared to the AIM mark. The practical applicability of the PGT was validated through experiments, and we achieved enhanced precision and reliability in overlay measurements by resolving the issue of large overlays—the most significant challenge with grating patterns—and simultaneously enhancing mark performance.

The collapse of a sonoluminescent cavitation bubble imaged with X-ray free-electron laser pulses

Single bubble sonoluminescence (SBSL) is the phenomenon of synchronous light emission due to the violent collapse of a single spherical bubble in a liquid, driven by an ultrasonic field. During the bubble collapse, matter inside the bubble reaches extreme conditions of several gigapascals and temperatures on the order of 10000 K, leading to picosecond flashes of visible light. To this day, details regarding the energy focusing mechanism rely on simulations due to the fast dynamics of the bubble collapse and spatial scales below the optical resolution limit. In this work we present phase-contrast holographic imaging with single x-ray free-electron laser (XFEL) pulses of a SBSL cavitation bubble in water. X-rays probe the electron density structure and by that provide a uniquely new view on the bubble interior and its collapse dynamics. The involved fast time-scales are accessed by sub-100 fs XFEL pulses and a custom synchronization scheme for the bubble oscillator. We find that during the whole oscillation cycle the bubble’s density profile can be well described by a simple step-like structure, with the radius R following the dynamics of the Gilmore model. The quantitatively measured internal density and width of the boundary layer exhibit a large variance. Smallest reconstructed bubble sizes reach down to R≃0.8μm , and are consistent with spherical symmetry. While we here achieved a spatial resolution of a few 100 nm, the visibility of the bubble and its internal structure is limited by the total x-ray phase shift which can be scaled with experimental parameters.

Size and density control of skyrmions with picometer CoFeB thickness variations—observation of zero-field skyrmions and skyrmion merging

The controlled formation and adjustment of size and density of magnetic skyrmions in Ta/CoFeB/MgO trilayers with low Dzyaloshinskii–Moriya interaction is demonstrated. Close to the out-of-plane to in-plane magnetic spin reorientation transition, we find that small energy contributions enable skyrmion formation in a narrow window of 20 pm in CoFeB thickness. Zero-field stable skyrmions are established with proper magnetic field initialization within a 10 pm CoFeB thickness range. Using magneto-optical imaging with quantitative image processing, variations in skyrmion distribution and diameter are analyzed quantitatively, the latter for sizes well below the optical resolution limit. We demonstrate the controlled merging of individual skyrmions. The overall demonstrated degree of comprehension of skyrmion control aids to the development of envisioned skyrmion based magnetic memory devices.

Domain Imaging in Periodic Submicron Wide Nanostructures by Digital Drift Correction in Kerr Microscopy

Magneto‐optical Kerr microscopy is a powerful method for imaging magnetic domains. Even though domain imaging below the diffractive resolution limit is possible, such investigations are getting increasingly complex with decreasing structure size due to the decreasing Kerr contrast. As magnetic domain images free of topographical artifacts are obtained by subtracting a reference image from the actual image, the corresponding challenges are additionally increased by unavoidable sample motion in the time interval between acquiring the two images. Software‐based drift corrections typically rely on a unique structure in the image's region of interest (ROI), recognized automatically or selected manually by the user. By digital image shifting, the ROI positions in the actual and reference images are aligned, and the sample motion is compensated. For magnetic domain imaging in periodically arranged micro‐ or nano‐objects, unique topographical features are not given, making the drift correction by ROIs difficult, often even impossible. Herein, a novel software‐based approach is presented for drift corrections to image domains with features close/below the optical resolution limit and for investigating periodically arranged micro‐ or nano‐objects without utilizing ROIs. High‐contrast images are obtained, enabling the characterization of periodically arranged 1D, 2D, and 3D magnetic objects with lateral dimensions below 100 nm.

Modelling strong lenses from wide-field ground-based observations in KiDS and GAMA

ABSTRACT Despite the success of galaxy-scale strong gravitational lens studies with Hubble-quality imaging, a number of well-studied strong lenses remains small. As a result, robust comparisons of the lens models to theoretical predictions are difficult. This motivates our application of automated Bayesian lens modelling methods to observations from public data releases of overlapping large ground-based imaging and spectroscopic surveys: Kilo-Degree Survey (KiDS) and Galaxy and Mass Assembly (GAMA), respectively. We use the open-source lens modelling software pyautolens to perform our analysis. We demonstrate the feasibility of strong lens modelling with large-survey data at lower resolution as a complementary avenue to studies that utilize more time-consuming and expensive observations of individual lenses at higher resolution. We discuss advantages and challenges, with special consideration given to determining background source redshifts from single-aperture spectra and to disentangling foreground lens and background source light. High uncertainties in the best-fitting parameters for the models due to the limits of optical resolution in ground-based observatories and the small sample size can be improved with future study. We give broadly applicable recommendations for future efforts, and with proper application, this approach could yield measurements in the quantities needed for robust statistical inference.

Influence of effusion cooling air on the CO production in the primary zone of a pressurized single sector model gas turbine combustor

In this study, the influence of effusion cooling air on the CO production in the primary zone of a pressurized single sector model gas turbine combustor is investigated in a parametric study using a combination of laser-based diagnostics. Three species (OH, CO, CH2O) are probed using laser-induced fluorescence (LIF). In principle, this combination of probed species allows to derive the forward reaction rate of CO oxidation and the local heat release rate from the LIF signals. Based on simulations and measurements in a laminar flame, we show that the limited optical resolution of the intensified cameras severely limits the applicability of the method for pressurized flames. However, by employing a conditioning strategy, mainly using CH2O-LIF as a marker for low temperature chemistry, it was shown that effusion cooling air affects the structure of the premixed flame at large distances from the effusion cooled liner. At higher blowing ratios, samples measured in the primary zone during the CO production phase exhibit a higher CO concentration at a given level of OH concentration. Most likely, this is caused by the strong non-linear sensitivity of OH concentration to temperature, shifting the local equilibrium towards higher CO concentrations, when fractions of the effusion cooling air are recirculated towards the inlet nozzle. In the exhaust phase, dilution effects seem to dominate, as a higher effusion cooling air mass flow systematically lowers both measured OH and CO signals.

Automated 3D segmentation of guard cells enables volumetric analysis of stomatal biomechanics

Automating the three-dimensional (3D) segmentation of stomatal guard cells and other confocal microscopy data is extremely challenging due to hardware limitations, hard-to-localize regions, and limited optical resolution. We present a memory-efficient, attention-based, one-stage segmentation neural network for 3D images of stomatal guard cells. Our model is trained end to end and achieved expert-level accuracy while leveraging only eight human-labeled volume images. As a proof of concept, we applied our model to 3D confocal data from a cell ablation experiment that tests the "polar stiffening" model of stomatal biomechanics. The resulting data allow us to refine this polar stiffening model. This work presents a comprehensive, automated, computer-based volumetric analysis of fluorescent guard cell images. We anticipate that our model will allow biologists to rapidly test cell mechanics and dynamics and help them identify plants that more efficiently use water, a major limiting factor in global agricultural production and an area of critical concern during climate change.

Diamond-Based Nanoscale Quantum Relaxometry for Sensing Free Radical Production in Cells.

Diamond magnetometry makes use of fluorescent defects in diamonds to convert magnetic resonance signals into fluorescence. Because optical photons can be detected much more sensitively, this technique currently holds several sensitivity world records for room temperature magnetic measurements. It is orders of magnitude more sensitive than conventional magnetic resonance imaging (MRI) for detecting magnetic resonances. Here, the use of diamond magnetometry to detect free radical production in single living cells with nanometer resolution is experimentally demonstrated. This measuring system is first optimized and calibrated with chemicals at known concentrations. These measurements serve as benchmarks for future experiments. While conventional MRI typically has millimeter resolution, measurements are performed on individual cells to detect nitric oxide signaling at the nanoscale, within 10-20 nm from the internalized particles localized with a diffraction limited optical resolution. This level of detail is inaccessible to the state-of-the-art techniques. Nitric oxide is detected and the dynamics of its production and inhibition in the intra- and extracellular environment are followed.

Super-Resolution Reconstruction of Cytoskeleton Image Based on A-Net Deep Learning Network.

To date, live-cell imaging at the nanometer scale remains challenging. Even though super-resolution microscopy methods have enabled visualization of sub-cellular structures below the optical resolution limit, the spatial resolution is still far from enough for the structural reconstruction of biomolecules in vivo (i.e., ~24 nm thickness of microtubule fiber). In this study, a deep learning network named A-net was developed and shows that the resolution of cytoskeleton images captured by a confocal microscope can be significantly improved by combining the A-net deep learning network with the DWDC algorithm based on a degradation model. Utilizing the DWDC algorithm to construct new datasets and taking advantage of A-net neural network’s features (i.e., considerably fewer layers and relatively small dataset), the noise and flocculent structures which originally interfere with the cellular structure in the raw image are significantly removed, with the spatial resolution improved by a factor of 10. The investigation shows a universal approach for exacting structural details of biomolecules, cells and organs from low-resolution images.

Super-resolution laser probing of integrated circuits using algorithmic methods

Laser probing remains invaluable to the semiconductor industry for isolating and diagnosing defects in silicon transistors in integrated circuits during electrical stress tests. However, continuous device miniaturization below the 20 nm technology node has crammed multiple transistors within the focal spot of the laser beam, resulting in signal crosstalk, poor beam positioning accuracy and degraded fault isolation capabilities. The challenge is analogous to focusing attention to a single speaker in a crowd despite the multiple simultaneous conversations in the background. Through algorithms introduced in this patented work, consisting of cross-correlations, clustering, and our previously developed combinational logic analysis, we achieved beam positioning accuracy to better than 10 nm, extracted electrooptic waveforms from a node of a group of transistors (~18 times beyond the optical resolution limit), and applied this to isolate and identify an actual fault on a defective device. While problems associated with probing with shorter wavelength lasers continue to be addressed, our approach enhances and enables the continued probing of ICs using sub-bandgap photon energies without hardware modification to existing technology at semiconductor technology nodes below 10 nm.

Resolution formulas and near-resolution-limit images of a periodic structure

Abbe’s resolution formulas for a grating imaged under on-axis, off-axis, and incoherent illuminations are obtained with the help of the line-spread function. A rule-of-thumb relationship applicable to the extreme ultraviolet mask for patterning a grating near the resolution limit of the imaging optics is given.

Revealing fundamentals of charge extraction in photovoltaic devices through potentiostatic photoluminescence imaging

Revealing fundamentals of charge extraction in photovoltaic devices through potentiostatic photoluminescence imaging

High-speed X-ray ptychographic tomography

X-ray ptychography is a coherent scanning imaging technique widely used at synchrotron facilities for producing quantitative phase images beyond the resolution limit of conventional x-ray optics. The scanning nature of the technique introduces an inherent overhead to the collection at every scan position and limits the acquisition time of each 2D projection. The overhead associated with motion can be minimised with a continuous-scanning approach. Here we present an acquisition architecture based on continuous-scanning and up-triggering which allows to record ptychographic datasets at up to 9 kHz. We demonstrate the method by applying it to record 2D scans at up to 273 µm2/s and 3D scans of a (20 µm)3 volume in less than three hours. We discuss the current limitations and the outlook toward the development of sub-second 2D acquisition and minutes-long 3D ptychographic tomograms.

Application of ImPASS to translated images produces image resolution below the optical diffraction resolution limit

The image resolution produced by a lens/camera system is limited by the digital sampling frequency of the sensor and the diffraction limit as imposed by the front aperture diameter of the optics. A previous study using Image Phase Alignment Super-Sampling (ImPASS) demonstrated that Fourier phase information from a sequence of slightly displaced images can be used to achieve image resolution beyond the digital sampling frequency. In continuation of that work, this study applies ImPASS to sequences of slightly displaced empirical images for a range of aperture settings. The frames are up-sampled, aligned, and combined into a single frame. Application of Self-Deconvolving Data Restoration Algorithm (SeDDaRA) deconvolution reveals features with higher resolution. The slanted edge technique is applied to the processed images to establish the angular resolution of the system as a function of the effective f-number. When compared to Abbe’s resolution criteria, the measurements reveal that this super-sampling method produces image resolution that subceeds the diffraction limit of the lens/camera system.

Optimal transfer functions for bandwidth-limited imaging

One of the fundamental limits of classical optical microscopy is the diffraction limit of optical resolution. It results from the finite bandwidth of the optical transfer function (or OTF) of an optical microscope, which restricts the maximum spatial frequencies that are transmitted by a microscope. However, given the frequency support of the OTF, which is fully determined by the used optical hardware, an open and unsolved question is what is the optimal amplitude and phase distribution of spatial frequencies across this support that delivers the ``sharpest'' possible image. In this paper, we will answer this question and present a general rule how to find the optimal OTF for any given imaging system. We discuss our result in the context of optical microscopy, by considering in particular the cases of wide-field microscopy, confocal image scanning microscopy (ISM), 4pi microscopy, and structured illumination microscopy (SIM). Our results are important for finding optimal deconvolution algorithms for microscopy images, and we demonstrate this experimentally on the example of ISM. They can also serve as a guideline for designing optical systems that deliver best possible images, and can be easily generalized to nonoptical imaging such as telescopic imaging, ultrasound imaging, or magnetic resonance imaging.

Super-resolution microscopy: a brief history and new avenues.

Super-resolution microscopy (SRM) is a fast-developing field that encompasses fluorescence imaging techniques with the capability to resolve objects below the classical diffraction limit of optical resolution. Acknowledged with the Nobel prize in 2014, numerous SRM methods have meanwhile evolved and are being widely applied in biomedical research, all with specific strengths and shortcomings. While some techniques are capable of nanometre-scale molecular resolution, others are geared towards volumetric three-dimensional multi-colour or fast live-cell imaging. In this editorial review, we pick on the latest trends in the field. We start with a brief historical overview of both conceptual and commercial developments. Next, we highlight important parameters for imaging successfully with a particular super-resolution modality. Finally, we discuss the importance of reproducibility and quality control and the significance of open-source tools in microscopy.This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 2)'.

Pushing the limits of optical resolution in the study of the tiniest fossil arthropods

ABSTRACT Studies of minute fossils trapped in amber shed light on the chronology of species and allow us to track evolutionary, ecological and biospheric changes. However, the resolution of commonly used methods for imaging amber fossils is limited, and the quality of preservation is not the only reason for this limitation. We have used super-resolution confocal laser microscopy (Airyscan) to study fossil mites (Acari) from the Eocene and Cretaceous amber (35–100 Ma). Here we report our technical approaches and methodological findings, including the selection of mounting medium, clearing of specimens, the effects of hydration of fossils in amber and the advantages of super-resolution confocal microscopy in obtaining images with sub-micron detail. Our results show that modern microscopy, in combination with specific non-destructive methods of amber preparation, can facilitate achieving image resolutions that were previously hardly feasible in fossils.

Super-Resolution Imaging of Plasmodesmata Using 3D Structured Illumination Microscopy.

Plasmodesmata (PD) have a diameter of around 30-50nm which is well below the 200nm limit of optical resolution, making analysis by light microscopy difficult and resolving internal structures of the PD such as the desmotubule impossible. Modern super-resolution methods such as 3D structured illumination microscopy (3D-SIM) can increase the lateral and axial resolution and work well on fixed, sectioned material. However, imaging in live plant cells requires careful optimization. Here we present a method to image PD using 3D-SIM in live BY2 cells.