Sub-diffraction Limited Resolution Research Articles

One-Shot Dual-Detection-Based Single-Molecule Super-Resolution Imaging Method for Real-Time Observation of Spatiotemporal Catalytic Activity Variations on the Plasmonic Gold Nanoparticle Surface.

Understanding the relationship between the surface properties of a single plasmonic nanoparticle and its catalytic performance is critical for developing highly efficient nanocatalysts. In this study, a one-shot dual-detection-based single-molecule super-resolution imaging method in the evanescent field was developed to observe real-time spatiotemporal catalytic activity on a single plasmonic gold nanoparticle (AuNP) surface. The scattering intensity of AuNPs and the fluorescence of resorufin molecules produced on the AuNP surface were obtained simultaneously to investigate the relationship between nanoparticles and catalytic reactions at a single-molecule level. Chemisorbed adsorbates (i.e., catalytic product and resorufin) changed the electron density of individual AuNPs throughout the catalytic cycle, resulting in the fluctuation of the scattering intensity of individual AuNPs, which was attributed to the electron transfer between reactant resazurin molecules and AuNPs. The increase in the electron density of individual AuNPs affected the catalytic reaction rate. Furthermore, sequential mapping of individual catalytic events at the subdiffraction limit resolution was completed for real-time surface dynamics and spatiotemporal activity variations on the single AuNP surface. The developed method can aid in understanding surface-property-dependent catalytic kinetics and facilitate the development of nanoparticle-based heterogeneous catalysts at subdiffraction limit resolution.

An entropy-controlled objective chip for reflective confocal microscopy with subdiffraction-limit resolution

Planar diffractive lenses (PDLs) with optimized but disordered structures can focus light beyond the diffraction limit. However, these disordered structures have inevitably destroyed wide-field imaging capability, limiting their applications in microscopy. Here, we introduce information entropy S to evaluate the disorder of an objective chip by using the probability of its structural deviation from standard Fresnel zone plates. Inspired by the theory of entropy change, we predict an equilibrium point {S}_{0}=0.5 to balance wide-field imaging (theoretically evaluated by the Strehl ratio) and subdiffraction-limit focusing. To verify this, a {NA}=0.9 objective chip with a record-long focal length of 1 mm is designed with S=0.535, which is the nearest to the equilibrium point among all reported PDLs. Consequently, our fabricated chip can focus light with subdiffraction-limit size of 0.44 λ and image fine details with spatial frequencies up to 4000 lp/mm experimentally. These unprecedented performances enable ultracompact reflective confocal microscopy for superresolution imaging.

The interwoven fibril-like structure of amyloid-beta plaques in mouse brain tissue visualized using super-resolution STED microscopy

BackgroundStandard neuropathologic analysis of Alzheimer’s brain relies on traditional fluorescence microscopy, which suffers from limited spatial resolution due to light diffraction. As a result, it fails to reveal intricate details of amyloid plaques. While electron microscopy (EM) offers higher resolution, its extensive sample preparation, involving fixation, dehydration, embedding, and sectioning, can introduce artifacts and distortions in the complex brain tissue. Moreover, EM lacks molecular specificity and has limited field of view and imaging depth.ResultsIn our study, we employed super-resolution Stimulated Emission Depletion (STED) microscopy in conjunction with the anti-human APP recombinant antibody 1C3 fluorescently labelled with DyLightTM633 (1C3-DyLight633). This combination allowed us to visualize amyloidogenic aggregates in vitro and in brain sections from a 17-month-old 3×Tg-AD mouse with sub-diffraction limited spatial resolution. Remarkably, we achieved a spatial resolution of 29 nm in vitro and 62 nm in brain tissue sections, surpassing the capabilities of conventional confocal microscopy by 5–10 times. Consequently, we could discern individual fibrils within plaques, an achievement previously only possible with EM.ConclusionsThe utilization of STED microscopy represents a groundbreaking advancement in the field, enabling researchers to delve into the characterization of local mechanisms that underlie Amyloid (Aβ) deposition into plaques and their subsequent clearance. This unprecedented level of detail is especially crucial for comprehending the etiology of Alzheimer’s disease and developing the next generation of anti-amyloid treatments. By facilitating the evaluation of drug candidates and non-pharmacological interventions aiming to reduce amyloid burden, STED microscopy emerges as an indispensable tool for driving scientific progress in Alzheimer’s research.

Compact, Fast Blinking Cd-Free Quantum Dots for Super-Resolution Fluorescence Imaging.

Quantum dots (QDs) can be used as fluorescent probes in single molecule localization microscopy to achieve subdiffraction limit resolution (super-resolution fluorescence imaging). However, the toxicity of Cd in the prototypical CdSe-based QDs can limit their use in biological applications. Furthermore, commercial CdSe QDs are usually modified with relatively thick shells of both inorganic and organic materials to render them in the 10-20 nm size range, which is relatively large for biological labels. In this report, we present compact (4-6 nm) CuInS2/ZnS (CIS/ZnS) and compare them to commercially sourced CdSe/ZnS QDs for their blinking behavior, localization precision and super-resolution imaging. Although commercial CdSe/ZnS QDs are brighter than the more compact Cd-free CIS/ZnS QD, both give comparable results of 4.5-5.0-fold improvement in imaging resolution over conventional TIRF imaging of actin filaments. This likely results from the fact that CIS/ZnS QDs show very short on-times and long off times which leads to less overlap in the point spread functions of emitting CIS/ZnS QD labels on the actin filaments at the same labeling density. These results demonstrate that CIS/ZnS QDs are an excellent candidate to complement and perhaps even replace the larger and more toxic CdSe-based QDs for robust single- molecule super-resolution imaging.

Wavelength-dependent three-dimensional single-molecule superlocalization imaging for yoctomole detection of thyroid-stimulating hormone on a quantum dot nanobiosensor

A wavelength-dependent three-dimensional (3D) superlocalization imaging method on gold nanoislands (GNIs) chip was developed as a supersensitive single-molecule thyroid-stimulating hormone (TSH) nanobiosensor. Scattered and fluorescent signals from gold nanoislands on the substrate and quantum dots (QDs) nanoprobes were simultaneously isolated and acquired within an evanescent field layer generated by total internal reflection (TIR) of incident light using a dual-view device. The 3D TIR fluorescence images of TSH-bound QDs on the GNIs were obtained using z-axis optical sectioning at 10 nm intervals before/after immunoreaction to identify the optimal conditions for detection. The localized centroid position of QD nanoprobes and GNI were distinguished at a subdiffraction limit resolution using 3D Gaussian fitting to the point spread function. The QD TSH nanobiosensor using wavelength-dependent 3D TIR fluorescence-based single-molecule localization microscopy (3D TIRF-SLM) imaging technique showed an excellent detection limit of 90 yoctomoles (∼54 molecules) and a wide linear dynamic range of 1.14 zmol/L − 100 pmol/L for TSH. The detection sensitivity was about 4.4 × 109 times higher than conventional enzyme-linked immunosorbent assay and could successfully quantify TSH in human serum. The wavelength-dependent 3D TIRF-SLM technique may emerge as a reliable platform for ultrahigh-sensitive nanobiosensors at the single-molecule level and early diagnosis with quantification of disease-related ultra-trace biomolecules.

Polarization-Resolved Electron Energy Gain Nanospectroscopy With Phase-Structured Electron Beams

Free-electron-based measurements in scanning transmission electron microscopes (STEMs) reveal valuable information on the broadband spectral responses of nanoscale systems with deeply subdiffraction limited spatial resolution. Leveraging recent advances in manipulating the spatial phase profile of the transverse electron wavefront, we theoretically describe interactions between the electron probe and optically stimulated nanophotonic targets in which the probe gains energy while simultaneously transitioning between transverse states with distinct phase profiles. Exploiting the selection rules governing such transitions, we propose phase-shaped electron energy gain nanospectroscopy for probing the 3D polarization-resolved response field of an optically excited target with nanoscale spatial resolution. Considering ongoing instrumental developments, polarized generalizations of STEM electron energy loss and gain measurements hold the potential to become powerful tools for fundamental studies of quantum materials and their interaction with nearby nanostructures supporting localized surface plasmon or phonon polaritons as well as for noninvasive imaging and nanoscale 3D field tomography.

Raman-based assay for quantifying how environmental changes affect bR activity

Bacteriorhodopsin (bR) is one of the best studied transport proteins, having been first purified in 1974 by Oesterhelt and Stoeckenius. Acting as a proton pump in halophilic archaea, bR forms crystalline patches in the cell membrane of about 100 nm in size. Despite decades of research into bR, the purpose of these crystalline patches is widely unknown. To determine what benefit patches offer the cell, a method is needed that can measure the activity of bR with sub-diffraction limited resolution.

Quantifying intracellular trafficking of silica-coated magnetic nanoparticles in live single cells by site-specific direct stochastic optical reconstruction microscopy

BackgroundNanoparticles have been used for biomedical applications, including drug delivery, diagnosis, and imaging based on their unique properties derived from small size and large surface-to-volume ratio. However, concerns regarding unexpected toxicity due to the localization of nanoparticles in the cells are growing. Herein, we quantified the number of cell-internalized nanoparticles and monitored their cellular localization, which are critical factors for biomedical applications of nanoparticles.MethodsThis study investigates the intracellular trafficking of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)] in various live single cells, such as HEK293, NIH3T3, and RAW 264.7 cells, using site-specific direct stochastic optical reconstruction microscopy (dSTORM). The time-dependent subdiffraction-limit spatial resolution of the dSTORM method allowed intracellular site-specific quantification and tracking of MNPs@SiO2(RITC).ResultsThe MNPs@SiO2(RITC) were observed to be highly internalized in RAW 264.7 cells, compared to the HEK293 and NIH3T3 cells undergoing single-particle analysis. In addition, MNPs@SiO2(RITC) were internalized within the nuclei of RAW 264.7 and HEK293 cells but were not detected in the nuclei of NIH3T3 cells. Moreover, because of the treatment of the MNPs@SiO2(RITC), more micronuclei were detected in RAW 264.7 cells than in other cells.ConclusionThe sensitive and quantitative evaluations of MNPs@SiO2(RITC) at specific sites in three different cells using a combination of dSTORM, transcriptomics, and molecular biology were performed. These findings highlight the quantitative differences in the uptake efficiency of MNPs@SiO2(RITC) and ultra-sensitivity, varying according to the cell types as ascertained by subdiffraction-limit super-resolution microscopy.Graphical

Development of a New Approach for Low-Laser-Power Super-Resolution Fluorescence Imaging.

The development of super-resolution fluorescence microscopy over the past decade has drastically improved the resolution of light microscopy to ∼10 nm. Stochastic optical reconstruction microscopy (STORM) can be used to achieve subdiffraction-limit resolution by sequentially imaging and localizing individual fluorophores. In principle, the super-resolution of STORM can be obtained by high-accuracy localization of photoswitchable fluorophores, which require fast photoswitching and bright fluorescence intensity from a single emitter. It is known that the switching rate of photoswitchable fluorophores depends on the laser power─a high laser power being required for the enhancement of imaging resolution. However, high laser power is usually harmful to biological specimens and limits the imaging time because of its photobleaching effects and high phototoxicity. In this study, we attempted to overcome this problem by improving the STORM resolution at a lower laser power. Through the quantitative analysis of the photoswitching behavior of single fluorophores under different laser power conditions, we developed a new approach to achieve super-resolution fluorescence images at a laser power 10 times lower than had previously been reported. This approach is expected to play an increasingly significant role in super-resolution imaging of power-sensitive samples.

Pupil function design for multifocal confocal, STED, and isoSTED microscopy.

Point scanning super-resolution microscopy techniques such as stimulated emission depletion (STED) microscopy are powerful tools to observe biological samples at sub-diffraction limited resolution in three dimensions. However, scanning the sample with only a single beam limits the imaging speed in these microscopes. Here, we propose a concept to increase this speed by introducing highly flexible multifocal illumination and detection. We introduce phase patterns in the objectives’ pupil planes to create arrays of foci in the sample plane with negligible loss of laser power. High uniformity of these foci’s intensities is achieved by iteratively applying a weighted Gerchberg–Saxton phase retrieval algorithm. We characterize the performance of this iterative approach numerically and present simulation results that demonstrate the high quality of the focus arrays for future implementations in laser-scanning STED and isoSTED microscopes. The same approach can also be applied in diffraction-limited confocal laser scanning microscopy.

Cubic spline-based depth-dependent localization of mitochondria-endoplasmic reticulum contacts by three-dimensional light-sheet super-resolution microscopy.

The contact distance between mitochondria (Mito) and endoplasmic reticulum (ER) has received considerable attention owing to their crucial function in maintaining lipid and calcium homeostasis. Herein, cubic spline algorithm-based depth-dependent fluorescence-free three-dimensional light-sheet super-resolution microscopy (3D LSRM) with dual-wavelength illumination sources was investigated to study the distance of Mito-ER contacts in various live cells. To detect wavelength-dependent scattering, 12 nm gold nanoparticles (AuNPs) and 20 nm silver nanoparticles (AgNPs) as fluorescence-free nanoprobes were conjugated with Mito and ER. The cubic spline algorithm-based method showed improved localization precision in lateral and axial directions compared with that for previously used least squares and least cubic algorithms. The cubic spline-based depth-dependent localization was applied to the spatial localization of nanoprobes in super-resolution images, in which the average distance of Mito and ER was 22.4 nm in HeLa cells, 22.2 nm in RAW264.7 macrophage cells, 21.9 nm in AGS cells, 21.4 nm in HT29 cells, and 21.3 nm in HEK293 cells. The distances were ∼12% larger than those previously determined by electron microscopy, which demonstrated that this method was accessible and reliable for studying the intracellular structures of various live cells at the subdiffraction limit resolution.

Smart Scattering Scanning Near-Field Optical Microscopy

Scattering scanning near-field optical microscopy (s-SNOM) provides spectroscopic imaging from molecular to quantum materials with few nanometer deep subdiffraction limited spatial resolution. Howe...

Chiral Structured Illumination Microscopy

We propose a chiral imaging modality based on optical chirality engineering, fluorescence-detected circular dichroism and structured illumination microscopy. In this method, the optical chirality of the illumination is structured and the circular dichroism dependent fluorescence is detected. With image reconstruction, the spatial distribution of chiral domains can be obtained at sub-diffraction limited resolution. We theoretically demonstrate this method and discuss the feasibility using an optical chirality engineering approach based on far-field optics.

Long working distance high resolution reflective sample imaging via structured embedded speckle illumination

Imaging beyond the diffraction limit at longer working distances using enhanced microscopic configurations has always been a challenge for biological and engineering samples. Even though multiple techniques have been widely used for sub-diffraction limit resolution imaging, the achievable resolution was relying on the use of objective lenses with a high numerical aperture (NA). In the case of engineering samples, in addition to sustaining higher resolutions at large working distances, improving the signal-to-noise ratio (SNR) is also critical. In this context, we propose and demonstrate a concept for high-resolution imaging at large working distances, termed as structured illumination embedded speckle microscopy. An imaging resolution of ~ 310 ± 5 nm was achieved with a microscope objective (0.55 NA; 50X) having 11 mm long working distance using a Siemen's star as the test sample. The demonstrated microscopy is therefore envisaged for engineering applications that demands high-resolution, high SNR imaging at long working distances.

Transcription and translation contribute to gene locus relocation to the nucleoid periphery in E. coli

Transcription by RNA polymerase (RNAP) is coupled with translation in bacteria. Here, we observe the dynamics of transcription and subcellular localization of a specific gene locus (encoding a non-membrane protein) in living E. coli cells at subdiffraction-limit resolution. The movement of the gene locus to the nucleoid periphery correlates with transcription, driven by either E. coli RNAP or T7 RNAP, and the effect is potentiated by translation.

Deep plasmonic direct writing lithography with ENZ metamaterials and nanoantenna

In this article, we demonstrate a specially designed resonant metamaterial with epsilon-near-zero (ENZ) and nanoantenna to enhance the exposure depth in plasmonic direct writing lithography more than 10 times. The ENZ metamaterial composed of a Ag/Si3N4 multilayer thin film, converts the evanescent field generated by the bowtie aperture nanoantenna to propagating waves with low divergence and high collimation. Deep sub-diffraction limited resolution of less than 65 nm (λ/7) with exposure depth greater than 100 nm is achieved. This work brings plasmonic direct writing lithography one step closer to practical applications.

Nanoscopy on-a-chip: super-resolution imaging on the millimeter scale.

Optical nanoscopy techniques can image intracellular structures with high specificity at sub-diffraction limited resolution, bridging the resolution gap between optical microscopy and electron microscopy. So far conventional nanoscopy lacks the ability to generate high throughput data, as the imaged region is small. Photonic chip-based nanoscopy has demonstrated the potential for imaging large areas, but at a lateral resolution of 130 nm. However, all the existing super-resolution methods provide a resolution of 100 nm or better. In this work, chip-based nanoscopy is demonstrated with a resolution of 75 nm over an extraordinarily large area of 0.5 mm × 0.5 mm, using a low magnification and high N.A. objective lens. Furthermore, the performance of chip-based nanoscopy is benchmarked by studying the localization precision and illumination homogeneity for different waveguide widths. The advent of large field-of-view chip-based nanoscopy opens up new routes in diagnostics where high throughput is needed for the detection of non-diffuse disease, or rare events such as the early detection of cancer.

Phase-sensitive lock-in detection for high-contrast mid-infrared photothermal imaging with sub-diffraction limited resolution.

Imaging of the phase output of a lock-in amplifier in mid-infrared photothermal vibrational microscopy is demonstrated for the first time in combination with nonlinear demodulation. In general, thermal blurring and heat transport phenomena contribute to the resolution and sensitivity of mid-infrared photothermal imaging. For heterogeneous samples with multiple absorbing features, if imaged in a spectral regime of comparable absorption with their embedding medium, it is demonstrated that differentiation with high contrast is achieved in complementary imaging of the phase signal obtained from a lock-in amplifier compared to standard imaging of the photothermal amplitude signal. Specifically, by investigating the relative contribution of the out-of-phase lock-in signal, information based on changes in the rate of heat transport can be extracted, and inhomogeneities in the thermal diffusion properties across the sample plane can be mapped with high sensitivity and sub-diffraction limited resolution. Under these imaging conditions, wavenumber regimes can be identified in which the thermal diffusion contributions are minimized and an enhancement of the spatial resolution beyond the diffraction limited spot size of the probe beam in the corresponding phase images is achieved. By combining relative diffusive phase imaging with nonlinear demodulation at the second harmonic, it is demonstrated that 1-μm-size melamine beads embedded in a thin layer of 4-octyl-4'-cyanobiphenyl (8CB) liquid crystal can be detected with a 1.3-μm spatial full-width at half-maximum (FWHM) resolution. Thus, imaging with a resolving power that exceeds the probe diffraction limited spot size by a factor of 2.5 is presented, which paves the route towards super-resolution, label-free imaging in the mid-infrared.

Photonic Nanojet Sub-Diffraction Nano-Fabrication With <italic>in situ</italic> Super-Resolution Imaging

In this paper, we report a system that uses a microsphere to induce a photonic nanojet to directly write on a sample surface with sub-diffraction limit resolution, while simultaneously observing the writing processing in situ . Because of diffraction limit, sub-wavelength laser processing resolution has been difficult to achieve. Recently, we have shown that a microsphere-induced photonics nanojet could be used to create an optical light spot size beyond the diffraction limits, i.e., the diameter of the light spot could be reduced to −200 nm or smaller. This capability could allow a laser (with significant reduction in input power) to pattern nano-scale structures on various substrate materials through the use of microspheres. Many researchers have attempted to use this photonic nanojet (PNJ) based technique to process sub-diffraction limit patterns on all types of materials. However, applying this method to process features at precise locations is very difficult, because the technique requires that a sample being “cut” by the PNJ be observed by a common optical microscope, which cannot resolve features smaller than the optical diffraction limit. This disadvantage limits the applicability of this novel method to nano-scale processing; for example, in trimming resistors in an integrated circuit chip, many circuit components on the chip are much smaller than 200 nm, and not being able to see features beyond the diffraction limit will inadvertently destroy many components while PNJ is used to trim resistive elements. We will show in this paper that processing resolution better than 200 nm is achievable using our laser-based photonic nanojet method, whereas a simultaneous imaging resolution of less than λ /2 could be obtained. Therefore, the method presented in this paper has potential applications in processing sub-diffraction features and bio-specimens in real time with optical information feedback.

Apparent self-heating of individual upconverting nanoparticle thermometers

Individual luminescent nanoparticles enable thermometry with sub-diffraction limited spatial resolution, but potential self-heating effects from high single-particle excitation intensities remain largely uninvestigated because thermal models predict negligible self-heating. Here, we report that the common “ratiometric” thermometry signal of individual NaYF4:Yb3+,Er3+ nanoparticles unexpectedly increases with excitation intensity, implying a temperature rise over 50 K if interpreted as thermal. Luminescence lifetime thermometry, which we demonstrate for the first time using individual NaYF4:Yb3+,Er3+ nanoparticles, indicates a similar temperature rise. To resolve this apparent contradiction between model and experiment, we systematically vary the nanoparticle’s thermal environment: the substrate thermal conductivity, nanoparticle-substrate contact resistance, and nanoparticle size. The apparent self-heating remains unchanged, demonstrating that this effect is an artifact, not a real temperature rise. Using rate equation modeling, we show that this artifact results from increased radiative and non-radiative relaxation from higher-lying Er3+ energy levels. This study has important implications for single-particle thermometry.