Application and development of fluorescence probes in MINFLUX nanoscopy(invited paper)

The MINimal emission FLUXes(MINFLUX)technique in optical microscopy,widely recognized as the next innovative fluorescence microscopy method,claims a spatial resolution of 1-3 nm in both dead and living cells.To make use of the full resolution of the MINFLUX microscope,it is important to select appropriate fluorescence probes and labeling strategies,especially in living-cell imaging.This paper mainly focuses on recent applications and developments of fluorescence probes and the relevant labeling strategy for MINFLUX microscopy.Moreover,we discuss the deficiencies that need to be addressed in the future and a plan for the possible progression of MINFLUX to help investigators who have been involved in or are just starting in the field of super-resolution imaging microscopy with theoretical support.

Review of 4Pi Fluorescence Nanoscopy

Since the 1990s,continuous technical and scientific advances have defied the diffraction limit in microscopy and enabled three-dimensional(3D)super-resolution imaging.An important milestone in this pursuit is the coherent utilization of two opposing objectives(4Pi geometry)and its combination with superresolution microscopy.Herein,we review the recent progress in 4Pi nanoscopy,which provides a 3D,non-invasive,diffraction-unlimited,and isotropic resolution in transparent samples.This review includes both the targeted and stochastic switching modalities of 4Pi nanoscopy.The schematics,principles,applications,and future potential of 4Pi nanoscopy are discussed in detail.

Nanoscopy reveals surface-metallic black phosphorus

Black phosphorus(BP)is an emerging two-dimensional material with intriguing physical properties.It is highly anisotropic and highly tunable by means of both the number of monolayers and surface doping.Here,we experimentally investigate and theoretically interpret the near-field properties of a-few-atomic-monolayer nanoflakes of BP.We discover near-field patterns of bright outside fringes and a high surface polarizability of nanofilm BP consistent with its surface-metallic,plasmonic behavior at mid-infrared frequencies o1176 cm−1.We conclude that these fringes are caused by the formation of a highly polarizable layer at the BP surface.This layer has a thickness of~1 nm and exhibits plasmonic behavior.We estimate that it contains free carriers in a concentration of n≈1.1×10^(20) cm^(−3).Surface plasmonic behavior is observed for 10–40 nm BP thicknesses but absent for a 4-nm BP thickness.This discovery opens up a new field of research and potential applications in nanoelectronics,plasmonics and optoelectronics.

From microscopy to nanoscopy via visible light

The resolution of conventional optical equipment is always restricted by the diffraction limit,and improving on this was previously considered improbable.Optical super-resolution imaging,which has recently experienced rapid growth and attracted increasing global interest,will result in applications in many domains,benefiting fields such as biology,medicine and material research.This review discusses the contributions of different researchers who identified the diffractive barrier and attempted to realize optical super-resolution.This is followed by a personal viewpoint of the development of optical nanoscopy in recent decades and the road towards the next generation of optical nanoscopy.

Biocompatible carbon dots with low-saturation-intensity and highphotobleaching-resistance for STED nanoscopy imaging of the nucleolus and tunneling nanotubes in living cells

Many kinds of nano particles and organic dyes as fluorescent probes have been used in the stimulated emission depletion(STED)nanoscopy.Due to high toxicity,photobleaching and non-water solubility,these fluorescent probes are hard to apply in living cell imaging.Here,we reporta new fluorescence carbon dots(FNCDs)with high photoluminescence quantum yield(56%),low toxicity,anti-photobleaching and goodwater-solubility that suitable for live-cell imaging can be obtained by doping fluorine element.Moreover,the FNCDs can stain the nucleolusand tunneling nanotubes(TNTs)in the living cell.More importantly,for STED nanoscopy imaging,the FNCDs effectively depleted backgroundsignals and improved imaging resolution.Furthermore,the lateral resolution of single FNCDs size under the STED nanoscopy is up to 22.1 nm for FNCDs deposited on a glass slide was obtained.And because of their good water dispersibility,the higher resolution of single FNCDs sizein the nucleolus of a living cell can be up to 19.7 nm.After the image optimizati on steps,the fine fluoresce nee images of TNTs diameter with ca.75 nm resolution is obtained living cell,yielding a threefold enhancement compared with that in confocal imaging.Additionally,the FNCDs show excellent photobleaching resistance after 1,000 scan cycles in the STED model.All results show that FNCDs have significant potentialfor application in STED nanoscopy.

Possible implications of dysregulated nicotinic acetylcholine receptor diffusion and nanocluster formation in myasthenia gravis

Myasthenia gravis is a rare and invalidating disease affecting the neuromuscular junction of voluntary muscles.The classical form of this autoimmune disease is characterized by the presence of antibodies against the most abundant protein in the neuromuscular junction,the nicotinic acetylcholine receptor.Other variants of the disease involve autoimmune attack of non-receptor scaffolding proteins or enzymes essential for building or maintaining the integrity of this peripheral synapse.This review summarizes the participation of the above proteins in building the neuromuscular junction and the destruction of this cholinergic synapse by autoimmune aggression in myasthenia gravis.The review also covers the application of a powerful biophysical technique,superresolution optical microscopy,to image the nicotinic receptor in live cells and follow its motional dynamics.The hypothesis is entertained that anomalous nanocluster formation by antibody crosslinking may lead to accelerated endocytic internalization and elevated turnover of the receptor,as observed in myasthenia gravis.

Nanoscale interactions between the nicotinic acetylcholine receptor and cholesterol

Cholesterol is a major lipid in biological membranes.It not only plays a structural role but also modulates a wide range of functional properties of neurotransmitter and hormone receptors and ion channels.The membraneembedded segments of the paradigm neurotransmitter receptor for acetylcholine(nAChR)contain linear sequences of amino acids with the capacity to recognize cholesterol.These cholesterol consensus domains have been designated as“CARC”and its mirror sequence“CRAC”.CARC preferentially occurs in the exoplasmic-facing membrane leaflet,and CRAC,in the cytoplasmic-facing hemilayer.Both motifs are highly conserved among ion-channel and neurotransmitter receptor proteins in vertebrate nervous systems,where they recognize cholesterol,and in prokaryotic homologues in bacteria,where they recognize hopanoids.This phylogenetically conserved trait is an indication that the hopanoids in some bacteria and cholesterol in eukaryotes subserve analogous functions,probably contributing to the stability of membrane-embedded protein domains.Structural studies from our laboratory using superresolution optical microscopy(“nanoscopy”)have disclosed other interrelated functional and structural properties exerted by cholesterol on the nAChR.The neutral lipid content at the cell surface influences both the macromolecular organization of the receptor and its translational mobility(diffusion)in the plane of the membrane.

A Discrete Model of the Evanescent Light Emission from Ultra-Thin Layers

A discrete model of the Differential Evanescent Light Intensity (DELI) technique was developed to calculate and map 3D nanolayers thicknesses from the evanescent light intensity captured from optical waveguides. The model was used for ultra-thin Pd nanometric layers sputtered on glass substrates. The layers thickness profiles were displayed in 3D and 1D profiles plots. The total thickness profiles of the ultra-thin Pd films obtained in the range of 1-10 nm were validated using AFM measurements. Based on the model developed the evanescent photon extraction parameter of the material was estimated.

A Perspective on Data Processing in Super-resolution Fluorescence Microscopy Imaging

With super-resolution microscopy,we attempt to visualize(biological)structures and processes at the sub-cellular level(i.e.,nanoscale).To obtain this information,the samples are labeled with fluorophores that have a stochastic on/off switching of their emissions,which help to overcome the optical diffraction limit of around 250 nm,related to the use of optical micro-scopes.However,nowadays,research focuses on the imaging of live cells and thicker samples.These investigations require a high amount of simultaneously active fluorophores(i.e.,high-density imaging)and are challenging due to the collapse of the single-molecule localization techniques and the increased background in the image.Therefore,recent efforts have shifted towards the development of new ways to process the data.This publication gives an introduction to wide-field super-resolution fluorescence microscopy,explaining the concepts of the technique,and then gives an overview of the recently developed methods to provide super-resolution images for high-density data of live cells and ways to overcome the issues related to the imaging of these samples.