Super-resolution Optical Microscopy Research Articles

Microscopy Methods for Imaging MIF and Its Interaction Partners.

Fluorescence microscopy has become a powerful tool to investigate proteins in their natural environment. Well-established techniques like widefield and confocal fluorescence microscopy have commonly been used for decades to visualize biomolecules in single cells and tissue sections. Live cell microscopy allows for the investigation of biomolecular trafficking, and other specialized techniques, such as proximity ligation assays (PLA) and fluorescence lifetime imaging microscopy (FLIM), can be used to study interactions between biomolecules of interest. Finally, with the most recent rise of optical super-resolution microscopy, we can investigate target biomolecules in situ with unprecedented detail on the nanometer scale. Here, we discuss various optical microscopy techniques that have successfully been used to image MIF. We highlight applications, advantages, and limitations of each technique. The techniques described here can easily be adapted to investigate other target proteins, their localization, interaction partners, and mechanisms of action.

Rapid single-wavelength lightsheet localization microscopy for clarified tissue

Optical super-resolution microscopy allows nanoscale imaging of protein molecules in intact biological tissues. However, it is still challenging to perform large volume super-resolution imaging for entire animal organs. Here we develop a single-wavelength Bessel lightsheet method, optimized for refractive-index matching with clarified specimens to overcome the aberrations encountered in imaging thick tissues. Using spontaneous blinking fluorophores to label proteins of interest, we resolve the morphology of most, if not all, dopaminergic neurons in the whole adult brain (3.64 × 107 µm3) of Drosophila melanogaster at the nanometer scale with high imaging speed (436 µm3 per second) for localization. Quantitative single-molecule localization reveals the subcellular distribution of a monoamine transporter protein in the axons of a single, identified serotonergic Dorsal Paired Medial (DPM) neuron. Large datasets are obtained from imaging one brain per day to provide a robust statistical analysis of these imaging data.

Superresolution Multifunctional Sensing with the Nitrogen-Vacancy Center in Diamond

High spatial resolution and low perturbation are required for mapping the electromagnetic field at the nanoscale, to provide crucial information about local light-matter interaction. Using optical superresolution microscopy for detection via a nitrogen-vacancy center in diamond, this work demonstrates far-field noninvasive sensing of the local electromagnetic field with high accuracy and a spatial resolution of approximately 50 nm. As the nitrogen-vacancy center is sensitive to various physical quantities, a multifunctional far-field quantum nanoscope is anticipated.

Photonic nanojet beam shaping by illumination polarization engineering

Photonic Nanojets (PNJs) have attracted considerable research attention in the fields of super-resolution optical microscopy, nano-photolithography, and single molecule sensors because of their sub-wavelength near-field focusing properties. In this paper, we thoroughly studied how the polarizations and amplitude profiles of the incident beams affect the shape, size, and location of the PNJs generated from the illuminated microspheres. Numerical results showed that the PNJs generated by microspheres were strongly modulated by the polarizations and amplitude profiles of the illumination beams. Therefore, PNJs can be engineered according to the requirements of a specific application by designing the polarizations and amplitude profiles of the illumination light. Various fascinating properties of the PNJs generated with different illumination schemes were demonstrated and their implications for potential applications were discussed as well.

Subsynaptic Domains in Super-Resolution Microscopy: The Treachery of Images.

The application of super-resolution optical microscopy to investigating synaptic structures has revealed a highly heterogeneous and variable intra-synaptic organization. Dense subsynaptic protein assemblies named subsynaptic domains or SSDs have been proposed as structural units that regulate the efficacy of neuronal transmission. However, an in-depth characterization of SSDs has been hampered by technical limitations of super-resolution microscopy of synapses, namely the stochasticity of the signals during the imaging procedures and the variability of the synaptic structures. Here, we synthetize the available evidence for the existence of SSDs at central synapses, as well as the possible functional relevance of SSDs. In particular, we discuss the possible regulation of co-transmission at mixed inhibitory synapses as a consequence of the subsynaptic distribution of glycine receptors (GlyRs) and GABAA receptors (GABAARs).LAY ABSTRACTSuper-resolution imaging strategies bypass the resolution limit of conventional optical microscopy and have given new insights into the distribution of proteins at synapses in the central nervous system. Neurotransmitter receptors and scaffold proteins appear to occupy specialized locations within synapses that we refer to as subsynaptic domains or SSDs. Interestingly, these SSDs are highly dynamic and their formation seems to be related to the remodeling of synapses during synaptic plasticity. It was also shown that SSDs of pre-and post-synaptic proteins are aligned in so-called nanocolumns, highlighting the role of SSDs in the regulation of synaptic transmission. Despite recent advances, however, the detection of SSDs with super-resolution microscopy remains difficult due to the inherent technical limitations of these approaches that are discussed in this review article.

Fine mapping titin's C-zone: Matching cardiac myosin-binding protein C stripes with titin's super-repeats

Titin is largely comprised of serially-linked immunoglobulin (Ig) and fibronectin type-III (Fn3) domains. Many of these domains are arranged in an 11 domain super-repeat pattern that is repeated 11 times, forming the so-named titin C-zone in the A-band region of the sarcomere. Each super-repeat is thought to provide binding sites for thick filament proteins, such as cMyBP-C (cardiac myosin-binding protein C). However, it remains to be established which of titin's 11 C-zone super-repeats anchor cMyBP-C as titin contains 11 super-repeats and cMyBP-C is found in 9 stripes only. To study the layout of titin's C-zone in relation to MyBP-C, immunolabeling studies were performed on mouse skinned myocardium with antibodies to titin and cMyBP-C, using both immuno-electron microscopy and super-resolution optical microscopy. Results indicate that cMyBP-C locates near the interface between titin's C-zone super-repeats. Studies on a mouse model in which two of titin's C-zone repeats have been genetically deleted support that the first Ig domain of a super-repeat is important for anchoring cMyBP-C but also Fn3 domains located at the end of the preceding repeat. Furthermore, not all super-repeat interfaces are equal as the interface between super-repeat 1 and 2 (close to titin's D-zone) does not contain cMyBP-C. Finally, titin's C-zone does not extend all the way to the bare zone but instead terminates at the level of the second myosin crown. This study enhances insights in the molecular layout of the C-zone of titin, its relation to cMyBP-C, and its possible roles in cardiomyopathies.

Efficient Modulation of β-Amyloid Peptide Fibrillation with Polymer Nanoparticles Revealed by Super-Resolution Optical Microscopy.

β-Amyloid peptide (Aβ) aggregation is the essential hallmark of neurodegenerative disorders such as Alzheimer's disease. Efficient inhibitors are highly desired for the prevention of Aβ assembly that has been considered as the primary therapeutic strategy for neurodegenerative diseases. Apart from this, visualization of the aggregates and morphology at high spatial resolution is widely considered of crucial significance on biological treatment. In this work, we have developed small-sized (with diameter of ∼4.7 nm) and positively charged fluorescent conjugated polymer nanoparticles (CPNPs) with strong inhibition effect on Aβ1-40 peptides fibrillation. Interestingly, the CPNPs also possess excellent photophysical properties, including high photon counts, robust blinking, and repetitive fluorescence switching, that are especially suitable for localization-based super-resolution imaging. Spatial resolution of ∼20 nm for these blinking CPNPs is readily achieved. According to the optical microscopic results, it was found that binding of CPNPs to the terminal of seed fibrils can effectively inhibit the fibrillation process. Owing to these attractive biological and unique photophysical properties, the small-sized CPNPs show high potential in a variety of super-resolution based biological applications.

Preferred Formation of Heteromeric Channels between Coexpressed SK1 and IKCa Channel Subunits Provides a Unique Pharmacological Profile of Ca2+-Activated Potassium Channels.

Three small conductance calcium-activated potassium channel (SK) subunits have been cloned and found to preferentially form heteromeric channels when expressed in a heterologous expression system. The original cloning of the gene encoding the intermediate conductance calcium-activated potassium channel (IKCa) was termed SK4 because of the high homology between channel subtypes. Recent immunovisualization suggests that IKCa is expressed in the same subcellular compartments of some neurons as SK channel subunits. Stochastic optical reconstruction microscopy super-resolution microscopy revealed that coexpressed IKCa and SK1 channel subunits were closely associated, a finding substantiated by measurement of fluorescence resonance energy transfer between coexpressed fluorophore-tagged subunits. Expression of homomeric SK1 channels produced current that displayed typical sensitivity to SK channel inhibitors, while expressed IKCa channel current was inhibited by known IKCa channel blockers. Expression of both SK1 and IKCa subunits gave a current that displayed no sensitivity to SK channel inhibitors and a decreased sensitivity to IKCa current inhibitors. Single channel recording indicated that coexpression of SK1 and IKCa subunits produced channels with properties intermediate between those observed for homomeric channels. These data indicate that SK1 and IKCa channel subunits preferentially combine to form heteromeric channels that display pharmacological and biophysical properties distinct from those seen with homomeric channels.

Super-resolution optical microscope: principle, instrumentation, and application

Over the past two decades, several fluorescence- and non-fluorescence-based optical microscopes have been developed to break the diffraction limited barrier. In this review, the basic principles implemented in microscopy for super-resolution are described. Furthermore, achievements and instrumentation for super-resolution are presented. In addition to imaging, other applications that use super-resolution optical microscopes are discussed.

Super-resolution retinal imaging using optically reassigned scanning laser ophthalmoscopy.

Super-resolution optical microscopy techniques have enabled the discovery and visualization of numerous phenomena in physics, chemistry and biology1-3. However, the highest resolution super-resolution techniques depend on nonlinear fluorescence phenomena and are thus inaccessible to the myriad applications that require reflective imaging4,5. One promising super-resolution technique is optical reassignment6, which so far has only shown potential for fluorescence imaging at low speeds. Here, we present novel advances in optical reassignment to adapt it for any scanning microscopy, including reflective imaging, and enable an order of magnitude faster image acquisition than previous optical reassignment techniques. We utilized these advances to implement optically reassigned scanning laser ophthalmoscopy, an in vivo super-resolution human retinal imaging device not reliant on confocal gating. Using this instrument, we achieved high-resolution imaging of living human retinal cone photoreceptor cells (determined by minimum foveal eccentricity) without adaptive optics or chemical dilation of the eye7.

Quantitative Analysis of Interactive Behavior of Mitochondria and Lysosomes using Structured Illumination Microscopy

Super-resolution optical microscopy has extended the scope of cell biology from the cellular level to the nanoscale, giving researchers the ability to observe the interactive behavior of single mitochondria and lysosomes[1]. However, we are still without an applicable quantitative analysis system to assist us in understanding interactive behavior of mitochondria and lysosomes under super-resolution optical microscopy. Here, we introduce an M-value to quantitatively investigate mitochondria and lysosome contact (MLC) and mitophagy under structured illumination microscopy. We found that the M-value for an MLC is typically less than 0.4, whereas in mitophagy it ranges from 0.5 to 1.0. This system permits further investigation of the detailed molecular mechanism governing the interactive behavior of mitochondria and lysosomes. [1] Q X Chen, C Z Jin, X T Shao, R L Guan, Z Q Tian, C R Wang, F Liu, P X Ling, J L Guan, L N Ji, F S Wang, H Chao, and J J Diao, Small, 1802166 (2018).

Structured illumination imaging without grating rotation based on mirror operation on 1D Fourier spectrum.

Structured illumination microscopy (SIM) is a rapidly developing a super-resolution optical microscopy technique. With SIM, the grating is needed in order to rotate several angles for illuminating the sample in different directions. Multiple rotations reduce the imaging speed and grating rotation angle errors damage the image recovery quality. We introduce mirror transformation on one-dimension (1D) Fourier spectrum to SIM for resolving the problems of low imaging speed and severe impact on image reconstruction quality by grating rotation angle errors. When mirror operation and SIM are combined, the grating is placed at an orientation for obtaining three shadow images. The three shadow images are acquired by CCD at three different phase shift for a direction of grating. Thus, the SIM imaging speed is faster and the effect on image reconstruction quality by grating rotation angle errors is greatly reduced.

Molecular and living cell dynamic assays with optical microscopy imaging techniques.

Generally, the message elucidated by the conventional analytical methods overlooks the heterogeneity of single objects, where the behavior of individual molecules is shielded. With the advent of optical microscopy imaging techniques, it is possible to identify, visualize and track individual molecules or nanoparticles under a biological environment with high temporal and spatial resolution. In this work, we summarize the commonly adopted optical microscopy techniques for bio-analytical assays in living cells, including total internal reflection fluorescence microscopy (TIRFM), super-resolution optical microscopy (SRM), and dark-field optical microscopy (DFM). The basic principles of these methods and some recent interesting applications in molecular detection and single-particle tracking are introduced. Moreover, the development in high-dimensional optical microscopy to achieve three-dimensional (3-D) as well as sub-diffraction localization and tracking of biomolecules is also highlighted.

Engineering arrays of diffraction-limited hollow tubes and doughnut spots with prescribed distributions

Based on the principle of antenna pattern multiplication as the product of the element pattern and the array factor, a simple and flexible method to generate diffraction-limited optical hollow-tube and doughnut-spot arrays with predetermined properties is proposed. This approach can be realized in a 4Pi focusing system by reversing the field radiated from the collinear antenna array whose elements are isotropic magnetic current sources. Both hollow tubes and doughnut spots produced by this method are pure azimuthally polarized fields whose intensity distribution is nearly unchanged along the entire depth of focus. Numerical results show that the appearance and position of the focal field is determined by the element pattern and the array factor, respectively. These peculiar optical focal fields may find applications in multi-particle trapping and manipulation, materials processing, data storage, super-resolution optical microscopy and optical lithography.

Nanoscale deformation in polymers revealed by single-molecule super-resolution localization–orientation microscopy

Mechanical deformation can be detected at the nanoscale by determining fluorophore orientation in single-molecule super-resolution optical microscopy.

超分辨光学显微的成像原理及应用进展

超分辨光学显微的成像原理及应用进展

Fabrication and physical measurement accuracy of nanoscale standard samples for vector beams confocal laser scanning microscopy

Various kinds of super-resolution optical microscope techniques have been developed to break the diffraction barrier in the past decades. Confocal laser scanning microscopy is the super-resolution microscopy. It is widely used due to high resolution and depth selectivity in obtaining images. However, there are neither accurate nor rigorous measurement methods with a nanoscale resolution. In order to measure the resolution of vector beam confocal laser scanning microscopy accurately and rigorously, a nanoscale resolution standard sample is proposed and experimentally realized. This sample is composed of a series of accurate measure patterns and a couple of arrays of triangle finding structures. It allows a wide measurement range between 40 nm to 1000 nm, and provides appropriate measurement steps and high measurement accuracy. The measurement patterns can be efficiently figured out by using the found structures, and their structure line width can be easily calculated. The first standard sample is produced on a piece of amorphous silicon by electron beam lithography and inductive coupled plasma etching technology, and measured by the scanning electron microscopy. According to the test, the sample meets the requirements of accuracy for nanoscale resolution measurement. Optical testing is applied to the sample by a vector beam confocal laser scanning microscope. And the sample shows that the resolution is 96 nm (oil immersion, refractive index 1.52) under the irradiation of 405 nm radially polarized beams, which is far beyond the diffraction barrier. Furthermore, a metal structure standard sample, which is based on a piece of indium tin oxide glass, is produced to improve the signal contrast ratio of the silicon standard sample. The measurement patterns are fabricated by electron beam lithography and electron beam evaporation and made of 10 nm titanium and 100 nm gold. It works for both reflective and transmissive confocal laser scanning microscopy, and would obtain high resolution images with a better contrast ratio. These standard samples are able to test the performance of microscope system efficiently, and provide a more rigorous way to make sub-100 nm resolution measurement and a calibration guidance for point scanning super-resolution microscope. In the meantime, we find that nanoscale opticalimaging is affected not only by sample morphology, but also by the photoelectron property of the sample. Further study is required to understand the underlying mechanism.

Optical super-resolution microscopy unravels the molecular composition of functional protein complexes.

Optical super-resolution microscopy has revolutionized our understanding of cell biology. Next to visualizing cellular structures with near-molecular spatial resolution, an additional benefit is the molecular characterization of biomolecular complexes directly in an intact cell. Single-molecule localization microscopy, as one technology out of the toolbox of super-resolution methods, generates images by detecting the position of single fluorophore labels and is particularly suited for molecular quantification. We review imaging and analysis methods employing single-molecule localization microscopy and extract molecule numbers.

Light Concentration by Metal-Dielectric Micro-Resonators for SERS Sensing.

Metal-dielectric micro/nano-composites have surface plasmon resonances in visible and near-infrared domains. Excitation of coupled metal-dielectric resonances is also important. These different resonances can allow enhancement of the electromagnetic field at a subwavelength scale. Hybrid plasmonic structures act as optical antennae by concentrating large electromagnetic energy in micro- and nano-scales. Plasmonic structures are proposed for various applications such as optical filters, investigation of quantum electrodynamics effects, solar energy concentration, magnetic recording, nanolasing, medical imaging and biodetection, surface-enhanced Raman scattering (SERS), and optical super-resolution microscopy. We present the review of recent achievements in experimental and theoretical studies of metal-dielectric micro and nano antennae that are important for fundamental and applied research. The main impact is application of metal-dielectric optical antennae for the efficient SERS sensing.

Structured illumination microscopy combined with machine learning enables the high throughput analysis and classification of virus structure.

Optical super-resolution microscopy techniques enable high molecular specificity with high spatial resolution and constitute a set of powerful tools in the investigation of the structure of supramolecular assemblies such as viruses. Here, we report on a new methodology which combines Structured Illumination Microscopy (SIM) with machine learning algorithms to image and classify the structure of large populations of biopharmaceutical viruses with high resolution. The method offers information on virus morphology that can ultimately be linked with functional performance. We demonstrate the approach on viruses produced for oncolytic viriotherapy (Newcastle Disease Virus) and vaccine development (Influenza). This unique tool enables the rapid assessment of the quality of viral production with high throughput obviating the need for traditional batch testing methods which are complex and time consuming. We show that our method also works on non-purified samples from pooled harvest fluids directly from the production line.