Optical-resolution Microscopy Research Articles

3D micro-devices for enhancing the lateral resolution in optical microscopy

3D micro-devices for enhancing the lateral resolution in optical microscopy

High throughput spectrally resolved super-resolution fluorescence microscopy with improved photon usage.

Single-molecule localization microscopy (SMLM), a type of super-resolution fluorescence microscopy, has become a strong technique in the toolbox of chemists, biologists, physicists, and engineers in recent years for its unique ability to resolve characteristic features at the nanoscopic level. It drastically improves the resolution of optical microscopes beyond the diffraction limit, with which previously unresolvable structures can now be studied. Spectrally resolved super-resolution fluorescence microscopy via multiplexing of different fluorophores is one of the greatest advancements among SMLM techniques. However, current spectrally resolved SMLM (SR-SMLM) methodologies present low spatial resolution due to loss of photons, low throughput due to spectral interferences, or require complex optical systems. Here, we overcome these drawbacks by developing a SR-SMLM methodology using a color glass filter. It enables high throughput and improved photon usage for hyperspectral imaging at the nanoscopic level. Our methodology can readily distinguish fluorophores of close spectral emission and achieves sub-10 nm localization and sub-5 nm spectral precisions.

Heptatic liquid quasi-crystals by colloidal lithographic pre-assembly

HypothesisWe hypothesize that pre-assembled lithographic Brownian seven-fold quasi-crystals (QCs) of colloidal tiles at high densities can exhibit a heptatic liquid quasi-crystal (LQC) phase upon release; such heptatic LQCs can undergo heterogeneous dynamics at different length scales, reflecting the underlying symmetry, corrugation, and hierarchy of local sets of tiles. ExperimentsWe design, fabricate, and release a seven-fold QC composed of three differently shaped rhombic tiles using the method of lithographically pre-assembled monolayers (litho-PAMs). High resolution optical microscopy enables spatio-temporal particle tracking of Brownian fluctuations of many tiles in a large area over a long time. We develop an edge-proximity tessellation method for analyzing nearest neighboring particles that can be applied to assemblies and dense systems of complex shapes. FindingsA fluctuating heptatic LQC phase is identified at high tile area fractions. Heterogenous dynamics and order at different length scales indicate diverse, hierarchical motif structures. We show that certain motifs can collectively rotate without any cage breaking, leading to alterations of the local tile-structure reminiscent of phason-flips in atomic QCs; this rotation causes a slow decline in the system's spatial order. We anticipate that edge-proximity tessellation will help elucidate phase transitions of other systems made of diverse building blocks having significant geometrical complexity at multiple length scales.

Utilization of Curvelet Transform in Reconstructing Cellular Images for Undersampled Optical-resolution Photoacoustic Microscopy.

We address the problem of limited temporal resolution in optical-resolution microscopy (OR-PAM) for cellular imaging by undersampling and reconstruction. A curvelet transform method in a compressed sensing framework (CS-CVT) was devised to specifically reconstruct the boundary and separability of cells object in an image. The performance of CS-CVT approach was justified by comparisons with the natural neighbor interpolation (NNI) followed by smoothing filters on various imaging objects. In addition, a full-raster scanned image was provided as reference. In terms of structure, CS-CVT produces cellular images with smoother boundary but less aberration. We found the strength of CS-CVT in recovering high frequency that is important in representing sharp edges which often missing in typical smoothing filter. In a noisy environment, CS-CVT was less affected by the noise compared to NNI with smoothing filter. Furthermore, CS-CVT could attenuate noise beyond the full raster scanned image. By considering the finest structure in the cellular image, CS-CVT was performing well with minimum range of undersampling around 5% to 15%. In practice, this undersampling were easily translates into 8- to 4-fold faster OR-PAM imaging. In summary, our approach improves temporal resolution of OR-PAM without significant trade-off in image quality.

Nanoscale and ultrafast in situ techniques to probe plasmon photocatalysis

Plasmonic photocatalysis uses the light-induced resonant oscillation of free electrons in a metal nanoparticle to concentrate optical energy for driving chemical reactions. By altering the joint electronic structure of the catalyst and reactants, plasmonic catalysis enables reaction pathways with improved selectivity, activity, and catalyst stability. However, designing an optimal catalyst still requires a fundamental understanding of the underlying plasmonic mechanisms at the spatial scales of single particles, at the temporal scales of electron transfer, and in conditions analogous to those under which real reactions will operate. Thus, in this review, we provide an overview of several of the available and developing nanoscale and ultrafast experimental approaches, emphasizing those that can be performed in situ. Specifically, we discuss high spatial resolution optical, tip-based, and electron microscopy techniques; high temporal resolution optical and x-ray techniques; and emerging ultrafast optical, x-ray, tip-based, and electron microscopy techniques that simultaneously achieve high spatial and temporal resolution. Ab initio and classical continuum theoretical models play an essential role in guiding and interpreting experimental exploration, and thus, these are also reviewed and several notable theoretical insights are discussed.

Ultrastructural skin alterations of healthy subjects with anti-desmoglein 1 antibodies in endemic areas to pemphigus foliaceus: A case series

BackgroundEndemic pemphigus foliaceus and endemic pemphigus vulgaris are autoimmune dermatologic disorders endemic to the Peruvian Amazon. ObjectiveTo determine the ultrastructural skin alterations of three healthy subjects with anti DSG-1 antibodies in areas endemic to pemphigus foliaceus and pemphigus vulgaris in the Peruvian Amazon. Patients and methodsCase series carried out from data of three clinically healthy subjects positive to anti DSG-1 antibodies, from Peru. This study consists of a sub-analysis of data gathered in a previous study. ResultsUltrastructural results are presented from the skin biopsies of three clinically healthy patients positive to anti-desmoglein 1 (DSG-1) antibodies. High Resolution Optical Microscopy (HROM) showed the absence of acantholysis. Transmission Electron Microscopy (TEM) showed the widening of intercellular space between keratinocytes, the presence of vacuoles in intercellular space with granular material and cytoplasmic vacuolization, loss of desmosome structure, loss of normal distribution among tonofilaments and lateral separation among cells in the stratum basale. ConclusionAccording to our results, healthy subjects that present anti-desmoglein 1 antibodies can develop ultrastructural alterations that are visible through transmission electron microscopy but not through conventional optical microscopy.

Experimental and numerical polarization analysis of the 3D transfer behavior in microsphere-assisted interferometry for 1D phase gratings

Enhancing the lateral resolution in optical microscopy and interferometry is of great interest in recent research. In order to laterally resolve structures including feature dimensions below the Abbe resolution limit, microspheres in the optical near-field of the specimen are shown to locally improve the resolution of the imaging system. Experimental and simulated results following this approach are obtained by a high NA Linnik interferometer and analyzed in this contribution. They show the reconstructed surface of a 1D phase grating below the resolution limit. For further understanding of the transfer characteristics, measured interference data are compared with FEM (finite element method) based simulations with respect to the polarization dependency of the relevant image information for 1D phase gratings. Therefore, the implemented Koehler illumination as well as the experimental setup utilize polarized light.

Amphipathic peptide–phospholipid nanofibers: Kinetics of fiber formation and molecular transfer between assemblies

Understanding the kinetics of nano-assembly formation is important to elucidate the biological processes involved and develop novel nanomaterials with biological functions. In the present study, we report the kinetic mechanisms of nanofiber formation from a mixture of phospholipids and the amphipathic peptide 18A[A11C], carrying cysteine substitution of the apolipoprotein A-I-derived peptide 18A at residue 11. 18A[A11C] with acetylated N-terminus and amidated C-terminus can associate with phosphatidylcholine to form fibrous aggregates at neutral pH and lipid-to-peptide molar ratio of ∼1, although the reaction pathways of self-assembly remain unclear. Here, the peptide was added to giant 1-palmitoyl-2-oleoyl phosphatidylcholine vesicles to monitor nanofiber formation under fluorescence microscopy. The peptide initially solubilized the lipid vesicles into particles smaller than the resolution of optical microscope, and fibrous aggregates appeared subsequently. Transmission electron microscopy and dynamic light scattering analyses revealed that the vesicle-solubilized particles were spherical or circular, measuring ∼10–20 nm in diameter. The rate of nanofiber formation of 18A with 1,2-dipalmitoyl phosphatidylcholine from the particles was proportional to the square of lipid–peptide concentration in the system, suggesting that the association of particles, accompanied by conformational changes, was the rate-limiting step. Moreover, molecules in the nanofibers could be transferred between aggregates faster than those in the lipid vesicles. These findings provide useful information for the development and control of nano-assembling structures using peptides and phospholipids.

Quantitative dSTORM superresolution microscopy

AbstractLocalization based superresolution technique provides the highest spatial resolution in optical microscopy. The final image is formed by the precise localization of individual fluorescent dyes, therefore the quantification of the collected data requires special protocols, algorithms and validation processes. The effects of labelling density and structured background on the final image quality were studied theoretically using the TestSTORM simulator. It was shown that system parameters affect the morphology of the final reconstructed image in different ways and the accuracy of the imaging can be determined. Although theoretical studies help in the optimization procedure, the quantification of experimental data raises additional issues, since the ground truth data is unknown. Localization precision, linker length, sample drift and labelling density are the major factors that make quantitative data analysis difficult. Two examples (geometrical evaluation of sarcomere structures and counting the γH2AX molecules in DNA damage induced repair foci) have been presented to demonstrate the efficiency of quantitative evaluation experimentally.

Calcite-Assisted Localization and Kinetics (CLocK) Microscopy.

Localization-based super-resolution imaging techniques have improved the spatial resolution of optical microscopy well below the diffraction limit, yet encoding additional information into super-resolved images, such as anisotropy and orientation, remains a challenge. Here we introduce calcite-assisted localization and kinetics (CLocK) microscopy, a multiparameter super-resolution imaging technique easily integrated into any existing optical microscope setup at low cost and with straightforward analysis. By placing a rotating calcite crystal in the infinity space of an optical microscope, CLocK microscopy provides immediate polarization and orientation information while maintaining the ability to localize an emitter/scatterer with <10 nm resolution. Further, kinetic information an order of magnitude shorter than the integration time of the camera is encoded in the unique point spread function of a CLocK image, allowing for new mechanistic insight into dynamic processes such as single-nanoparticle dissolution and single-molecule surface-enhanced Raman scattering.

Correlative nano-imaging of metals and proteins in primary neurons by synchrotron X-ray fluorescence and STED super resolution microscopy: Experimental validation

BackgroundIt is becoming increasingly clear that biological metals such as iron, copper or zinc are involved in synaptic functions, and in particular in the mechanisms of synaptogenesis and subsequent plasticity. Understanding the role of metals on synaptic functions is a difficult challenge due to the very low concentration of these elements in neurons and to the submicrometer size of synaptic compartments. New methodTo address this challenge we have developed a correlative nano-imaging approach combining metal and protein detection. First, stimulated emission depletion (STED) microscopy, a super resolution optical microscopy technique, is applied to locate fluorescently labeled proteins. Then, synchrotron radiation induced X-ray fluorescence (SXRF) is performed on the same regions of interest, e.g. synaptic compartments. ResultsWe present here the principle scheme that allows this correlative nano-imaging and its experimental validation. We applied this correlative nano-imaging to the study of the physiological distribution of metals in synaptic compartments of primary rat hippocampal neurons. We thus compared the nanometric distribution of metals with that of synaptic proteins, such as PSD95 or cytoskeleton proteins. Comparison with existing method(s)Compared to correlative imaging approaches currently used to characterize synaptic structures, such as electron microscopy correlated with optical fluorescence, our approach allows for ultra-sensitive detection of trace metals using highly focused synchrotron radiation beams. ConclusionWe provide proof-of-principle for correlative imaging of metals and proteins at the synaptic scale and discuss the present limitations and future developments in this area.

Improved resolution Fourier ptychography scheme using oil‐filled dome‐shaped LED array

Utilising angular-varied illumination in Fourier Ptychography, the resolution of optical microscopes can be increased and the phase of the sample can also be recovered while maintaining a wide field-of-view. In this work, an Fourier Ptychography microscopy imaging setup is demonstrated using a 10X 0.28 NA objective lens and a dome-shaped light-emitting diode array illuminator. By increasing the refractive index of the medium between the sample and light sources using oil filled dome, a synthetic system numerical aperture of 0.68 is achieved using only 37 light-emitting diodes. The measured resolution of 345 nm at a wavelength of 530 nm closely matches the theoretical prediction. Furthermore, the results of the oil-medium illumination are compared to free-space illumination. The oil immersion on the illumination side effectively enhances the illumination numerical aperture as compared to air and reduces the need for far off-axis light-emitting diodes. The result is high-resolution Fourier Ptychography imaging with relatively fewer light-emitting diodes providing both short capture and reconstruction times.

Ultralow Laser Power Three-Dimensional Superresolution Microscopy Based on Digitally Enhanced STED.

The resolution of optical microscopes is limited by the optical diffraction limit; in particular, the axial resolution is much lower than the lateral resolution, which hinders the clear distinction of the three-dimensional (3D) structure of cells. Although stimulated emission depletion (STED) superresolution microscopy can break through the optical diffraction limit to achieve 3D superresolution imaging, traditional 3D STED requires high depletion laser power to acquire high-resolution images, which can cause irreversible light damage to biological samples and probes. Therefore, we developed an ultralow laser power 3D STED superresolution imaging method. On the basis of this method, we obtained lateral and axial resolutions of 71 nm and 144 nm, respectively, in fixed cells with 0.65 mW depletion laser power. This method will have broad application prospects in 3D superresolution imaging of living cells.

Determining the internal orientation, degree of ordering, and volume of elongated nanocavities by NMR: Application to studies of plant stem

This study investigates the fibril nanostructure of fresh celery samples by modeling the anisotropic behavior of the transverse relaxation time (T2) in nuclear magnetic resonance (NMR). Experimental results are interpreted within the framework of a previously developed theory, which was successfully used to model the nanostructures of several biological tissues as a set of water filled nanocavities, hence explaining the anisotropy the T2 relaxation time in vivo. An important feature of this theory is to determine the degree of orientational ordering of the nanocavities, their characteristic volume, and their average direction with respect to the macroscopic sample. Results exhibit good agreement between theory and experimental data, which are, moreover, supported by optical microscopic resolution. The quantitative NMR approach presented herein can be potentially used to determine the internal ordering of biological tissues noninvasively.

Modulated illumination localization microscopy-enabled sub-10 nm resolution

Optical microscopy is an essential tool for exploring the structures and activities of cells and tissues. To break the limit of resolution caused by diffraction, researchers have made continuous advances and innovations to improve the resolution of optical microscopy since the 1990s. These contributions, however, still make sub-10[Formula: see text]nm imaging an obstacle. Here, we name a series of technologies as modulated illumination localization microscopy (MILM), which makes ultra-high-resolution imaging practical. Besides, we review the recent progress since 2017 when MINFLUX was proposed and became the inspiration and foundation for the follow-up development of MILM. This review divides MILM into two types: point-scanning and wide-field. The schematics, principles and future research directions of MILM are discussed elaborately.

Angular birefringence phase matching behaviour of Lithium Triborate (LBO) crystal for second harmonic generation in broad wavelength range

Second Harmonic Generation (SHG) is a nonlinear optical process in which the frequency of the incident radiation gets doubled. There are various types of applications of SHG such as ultra-short pulse measurement, high resolution optical microscopy, characterization of the structure of 2D materials and its physical properties like the thickness of interlayer stacking, stacking angle between different layers, the grain boundary etc. It has also been used to determine the structure of Langmuir-Blodgett films of low symmetry. For SHG, various commercial nonlinear optical (NLO) crystals are generally used. Lithium Triborate (LBO) is one of the best NLO material among them due to its wide transparency range (160–2600 nm), high damage threshold (19 GW/cm2 @1064 nm, 1 ns) and high second order nonlinear coefficient. It has also the ability to establish birefringence phase matching condition for efficient SHG for different wavelengths. The crystal can be used for generation for SHG in broad wavelength range through angular phase matching method. For this, in this work detail investigation has been made to find out the optimum wavelength range that can be used for efficient SHG. This has been done by finding the variation of phase matching angle, second order nonlinearity etc. with respect to wavelength using the SNLO software. Both Type I and Type II phase matching has been considered for various planes of the crystal.

Characterization of the thickness of the Tear Film Lipid Layer in Meibomian Gland Dysfunction using high resolution optical microscopy

PurposeTo evaluate the thickness of the tear film lipid layer (TFLL) in meibomian gland dysfunction (MGD) using a high-resolution optical microscope. MethodsThe Ocular Surface Disease Index (OSDI) and meibum grade score (MGS) were used to classify 190 subjects into four groups: normal (OSDI<13 and MGS<10), mixed (OSDI≥13 and MGS<10), asymptomatic MGD (OSDI<13 and MGS≥10), and MGD (OSDI≥13 and MGS≥10). The high-resolution optical microscope was used to capture TFLL images in vivo. The histograms of TFLL thickness were analyzed and curve-fitted using probability density functions (PDFs). ResultsThere were three obvious peaks in the distributions of TFLL across the groups. From the curve-fitting process, the main outcomes are displayed according to each Gaussian function with the position of peak (μ) and the summed percentage within the range of standard deviation (σ). The normal group had distribution as follows: 33.3 ± 0.005 nm, 26%; 53.9 ± 0.019 nm, 40%; 79.4 ± 0.064 nm, 12%. The mixed group had a distribution as follows: 33.8 ± 0.004 nm, 32%; 53.1 ± 0.115 nm, 21%; 71.7 ± 0.232 nm, 27%. The asymptomatic MGD group had a distribution as follows: 33.5 ± 0.004 nm, 20%; 49.2 ± 0.041 nm, 25%; 62.9 ± 0.063 nm, 47%. The MGD group had a distribution as follows: 34.3 ± 0.004 nm, 34%; 53.7 ± 0.022 nm, 28%; 74.9 ± 0.060 nm, 16%. ConclusionsThe MGD and mixed groups had the largest percentages of TFLL thicknesses fall within the thinnest modes (peak 34.3 and 33.8 nm, respectively). These data show that measures of central tendency (e.g., averages, medians) do not fully appreciate the variable distributions of TFLL across disease spectra.

Aberration measurement and correction on a large field of view in fluorescence microscopy.

The aberrations induced by the sample and/or by the sample holder limit the resolution of optical microscopes. Wavefront correction can be achieved using a deformable mirror with wavefront sensorless optimization algorithms but, despite the complexity of these systems, the level of correction is often limited to a small area in the field of view of the microscope. In this work, we present a plug and play module for aberration measurement and correction. The wavefront correction is performed through direct wavefront reconstruction using the spinning-pupil aberration measurement and controlling a deformable lens in closed loop. The lens corrects the aberrations in the center of the field of view, leaving residual aberrations at the margins, that are removed by anisoplanatic deconvolution. We present experimental results obtained in fluorescence microscopy, with a wide field and a light sheet fluorescence microscope. These results indicate that detection and correction over the full field of view can be achieved with a compact transmissive module placed in the detection path of the fluorescence microscope.

Image quality improvement for optical imaging interferometric microscopy.

Imaging interferometric microscopy (IIM) is an optical microscopy resolution enhancement technique involving combining multiple sub-images to increase resolution. Several image reconstruction challenges can degrade the image quality including the frequency, phase deviations between sub-images, and maintenance of a uniform frequency response across the entire space. This work proposes methods to address these issues. The methods are first compared in simulation using a Manhattan structure of 260-nm critical dimension with 2-µm-pitch calibration grating on the sides. The proposed correction methods are then applied to the experimental results and found to be effective in improving the image quality of IIM.

A minimal subwavelength focal spot for the energy flux

It is shown theoretically and numerically that circularly and linearly polarized incident beams produce at the tight focus identical circularly symmetric distributions of an on-axis energy flux. It is also shown that the on-axis energy fluxes from radially and azimuthally polarized optical vortices with unit topological charge are equal to each other. An optical vortex with azimuthal polarization is found to generate the minimum focal spot measured for the intensity (all other parameters being equal). Slightly larger (by a fraction of a percent) is the spot size calculated for the energy flux for the circularly and linearly polarized light. The spot size in terms of intensity is of importance in light-matter interaction, whereas the spot size in terms of energy flux affects the resolution in optical microscopy.