Diffusion analysis within single nanometric apertures reveals the ultrafine cell membrane organization

We describe the development of a new methodology to probe the plasma membrane organization of living cells at the nanometric scale. Single nanometric apertures in a metallic film limit the observed membrane area below the optical diffraction barrier. The new approach performs fluorescence correlation spectroscopy with increasing aperture sizes and extracts information on the diffusion process from the whole set of data. In particular, transient diffusion regimes are clearly observed when the probed area comes close to the size of the confining structures. First, this strategy allows identification of the mechanism controlling the diffusion of various fluorescent lipid analogs and green fluorescent protein-tagged proteins. Second, it gives an estimate of the characteristic size of the nanometric membrane heterogeneities, allowing a quantitative study of membrane domains such as lipid rafts. Compared to other optical techniques, this method combines the advantages of high spatio-temporal resolution and direct statistical analysis.     

Super-resolution Microscopy Approaches for Live Cell Imaging

By delivering optical images with spatial resolutions below the diffraction limit, several super-resolution fluorescence microscopy techniques opened new opportunities to study biological structures with details approaching molecular structure sizes. They have now become methods of choice for imaging proteins and their nanoscale dynamic organizations in live cells. In this mini-review, we describe and compare the main far-field super-resolution approaches that allow studying endogenous or overexpressed proteins in live cells.     

Super-resolution Microscopy Approaches for Live Cell Imaging

By delivering optical images with spatial resolutions below the diffraction limit, several super-resolution fluorescence microscopy techniques opened new opportunities to study biological structures with details approaching molecular structure sizes. They have now become methods of choice for imaging proteins and their nanoscale dynamic organizations in live cells. In this mini-review, we describe and compare the main far-field super-resolution approaches that allow studying endogenous or overexpressed proteins in live cells.     

Light waveguiding in bioinspired peptide nanostructures

Basic optical properties of bioinspired peptide nanostructures are deeply modified by thermally mediated refolding of peptide secondary structure from α‐helical to β‐sheet. This conformational transition is followed by the appearance in the β‐sheet structures of a wideband optical absorption and fluorescence in the visible region. We demonstrate that a new biophotonic effect of optical waveguiding recently observed in peptide/protein nanoensembles is a structure‐sensitive bimodal phenomenon. In the primary α‐helical structure input, light propagates via optical transmission window demonstrating conventional passive waveguiding, based on classical optics. In the β‐sheet structure, fluorescent (active) light waveguiding is revealed. The latter can be attributed to completely different physical mechanism of exciton‐polariton propagation, characterized by high effective refractive index, and can be observed in nanoscale fibers below diffraction limit. It has been shown that peptide material requirements for passive and active waveguiding are dissimilar. Original biocompatibility and biodegradability indicate high potential future applications of these bioinspired waveguiding materials in precise photobiomedicine towards advanced highly selective bioimaging, photon diagnostics, and optogenetics.     

Mikroskopie jenseits der Auflösungsgrenze

Super Resolution Microscopy Resolution in conventional light microscopy is restricted by the borders of physics. The limiting factor is the wavelength of the used light. Nevertheless, many essential intracellular components such as the cytoskeleton or secretory vesicles are in size well below this diffraction limit. Super Resolution Microscopy techniques such as Photoactivated Localization Microscopy make it possible to break through this diffraction barrier. Controlled activation and sampling of a subpopulation of photoactivatable fluorophores enables localization of probes in a nanometric scale.     

STED microscopy of living cells – new frontiers in membrane and neurobiology

Recent developments in fluorescence far‐field microscopy such as STED microscopy have accomplished observation of the living cell with a spatial resolution far below the diffraction limit. Here, we briefly review the current approaches to super‐resolution optical microscopy and present the implementation of STED microscopy for novel insights into live cell mechanisms, with a focus on neurobiology and plasma membrane dynamics.     

分子生物光子学的进展

随着分子生物学和标记技术的进步,光学成像和分析有了很快的发展,无论是从理论上还是应用上都有所突破,展现了良好的前景。本文综述了近年来在分子生物探测中光子学的情况,简介了几种突破光学衍射极限的探测方法,着重阐明了与生化技术密切相关的荧光共振能量转移和荧光相关光谱显微术及其在分子生物学中的应用。     

Optical Saturation as a Versatile Tool to Enhance Resolution in Confocal Microscopy

One of the most actively developing areas in fluorescence microscopy is the achievement of spatial resolution below Abbe's diffraction limit, which restricts the resolution to several hundreds of nanometers. Most of the approaches in use at this time require a complex optical setup, a difficult mathematical treatment, or usage of dyes with special photophysical properties. In this work, we present a new, to our knowledge, approach in confocal microscopy that enhances the resolution moderately but is both technically and computationally simple. As it is based on the saturation of the transition from the ground state to the first excited state, it is universally applicable with respect to the dye used. The idea of the method presented is based on a principle similar to that underlying saturation excitation microscopy, but instead of applying harmonically modulated excitation light, the fluorophores are excited by picosecond laser pulses at different intensities, resulting in different levels of saturation. We show that the method can be easily combined with the concept of triplet relaxation, which by tuning the dark periods between pulses helps to suppress the formation of a photolabile triplet state and effectively reduces photobleaching. We demonstrate our approach imaging GFP-labeled protein patches within the plasma membrane of yeast cells.     

A Highlights from MBoC Selection: Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics

Most current superresolution (SR) microscope techniques surpass the diffraction limit at the expense of temporal resolution, compromising their applications to live-cell imaging. Here we describe a new SR fluorescence microscope based on confocal microscope optics, which we name the spinning disk superresolution microscope (SDSRM). Theoretically, the SDSRM is equivalent to a structured illumination microscope (SIM) and achieves a spatial resolution of 120 nm, double that of the diffraction limit of wide-field fluorescence microscopy. However, the SDSRM is 10 times faster than a conventional SIM because SR signals are recovered by optical demodulation through the stripe pattern of the disk. Therefore a single SR image requires only a single averaged image through the rotating disk. On the basis of this theory, we modified a commercial spinning disk confocal microscope. The improved resolution around 120 nm was confirmed with biological samples. The rapid dynamics of micro­tubules, mitochondria, lysosomes, and endosomes were observed with temporal resolutions of 30–100 frames/s. Because our method requires only small optical modifications, it will enable an easy upgrade from an existing spinning disk confocal to a SR microscope for live-cell imaging.     

Beyond the diffraction limit: far-field fluorescence imaging with ultrahigh resolution

Fluorescence microscopy is an important and extensively utilised tool for imaging biological systems. However, the image resolution that can be obtained has a limit as defined through the laws of diffraction. Demand for improved resolution has stimulated research into developing methods to image beyond the diffraction limit based on far-field fluorescence microscopy techniques. Rapid progress is being made in this area of science with methods emerging that enable fluorescence imaging in the far-field to possess a resolution well beyond the diffraction limit. This review outlines developments in far-field fluorescence methods which enable ultrahigh resolution imaging and application of these techniques to biology. Future possible trends and directions in far-field fluorescence imaging with ultrahigh resolution are also outlined.     

Scanning Super-Resolution Imaging in Enclosed Environment by Laser Tweezer Controlled Superlens

Super-resolution imaging using microspheres has attracted tremendous scientific attention recently because it has managed to overcome the diffraction limit and allowed direct optical imaging of structures below 100 nm without the aid of fluorescent microscopy. To allow imaging of specific areas on the surface of samples, the migration of the microspheres to specific locations on two-dimensional planes should be controlled to be as precise as possible. The common approach involves the attachment of microspheres on the tip of a probe. However, this technology requires additional space for the probe and could not work in an enclosed environment, e.g., in a microfluidic enclosure, thereby reducing the range of potential applications for microlens-based super-resolution imaging. Herein, we explore the use of laser trapping to manipulate microspheres to achieve super-resolution imaging in an enclosed microfluidic environment. We have demonstrated that polystyrene microsphere lenses could be manipulated to move along designated routes to image features that are smaller than the optical diffraction limit. For example, a silver nanowire with a diameter of 90 nm could be identified and imaged. In addition, a mosaic image could be constructed by fusing a sequence of images of a sample in an enclosed environment. Moreover, we have shown that it is possible to image Escherichia coli bacteria attached on the surface of an enclosed microfluidic device with this method. This technology is expected to provide additional super-resolution imaging opportunities in enclosed environments, including microfluidic, lab-on-a-chip, and organ-on-a-chip devices.     

Bayesian localization microscopy based on intensity distribution of fluorophores

Super-resolution microscopy techniques have overcome the limit of optical diffraction. Recently, the Bayesian analysis of Bleaching and Blinking data (3B) method has emerged as an important tool to obtain super-resolution fluorescence images. 3B uses the change in information caused by adding or removing fluorophores in the cell to fit the data. When adding a new fluorophore, 3B selects a random initial position, optimizes this position and then determines its reliability. However, the fluorophores are not evenly distributed in the entire image region, and the fluorescence intensity at a given position positively correlates with the probability of observing a fluorophore at this position. In this paper, we present a Bayesian analysis of Bleaching and Blinking microscopy method based on fluorescence intensity distribution (FID3B). We utilize the intensity distribution to select more reliable positions as the initial positions of fluorophores. This approach can improve the reconstruction results and significantly reduce the computational time. We validate the performance of our method using both simulated data and experimental data from cellular structures. The results confirm the effectiveness of our method.     

Fluorophores for single-molecule localization microscopy

Super-resolution fluorescence microscopy allows for obtaining images with a resolution of 10–20 nm, far exceeding the diffraction limit of conventional optical microscopy (200–350 nm), and provides an opportunity to study in detail the subcellular structures and individual proteins in both living and fixed cells. Among these methods, single-molecule localization microscopy (SMLM) has become widespread. SMLM techniques are based on special fluorophores capable of photoswitching. The paper presents a classification of such fluorophores and describes their photoswitching mechanisms and successful practical applications. We discuss recent progress and prospects for the development of new effective labels suitable for SMLM.     

Cell structure imaging with bright and homogeneous nanometric light source

Label‐free optical nano‐imaging of dendritic structures and intracellular granules in biological cells is demonstrated using a bright and homogeneous nanometric light source. The optical nanometric light source is excited using a focused electron beam. A zinc oxide (ZnO) luminescent thin film was fabricated by atomic layer deposition (ALD) to produce the nanoscale light source. The ZnO film formed by ALD emitted the bright, homogeneous light, unlike that deposited by another method. The dendritic structures of label‐free macrophage receptor with collagenous structure‐expressing CHO cells were clearly visualized below the diffraction limit. The inner fiber structure was observed with 120 nm spatial resolution. Because the bright homogeneous emission from the ZnO film suppresses the background noise, the signal‐to‐noise ratio (SNR) for the imaging results was greater than 10. The ALD method helps achieve an electron beam excitation assisted microscope with high spatial resolution and high SNR.

     

Visualization of RecA filaments and DNA by fluorescence microscopy

We have developed two experimental methods for observing Escherichia coli RecA-DNA filament under a fluorescence microscope. First, RecA-DNA filaments were visualized by immunofluorescence staining with anti-RecA monoclonal antibody. Although the detailed filament structures below submicron scale were unable to be measured accurately due to optical resolution limit, this method has an advantage to analyse a large number of RecA-DNA filaments in a single experiment. Thus, it provides a reliable statistical distribution of the filament morphology. Moreover, not only RecA filament, but also naked DNA region was visualized separately in combination with immunofluorescence staining using anti-DNA monoclonal antibody. Second, by using cysteine derivative RecA protein, RecA-DNA filament was directly labelled by fluorescent reagent, and was able to observe directly under a fluorescence microscope with its enzymatic activity maintained. We showed that the RecA-DNA filament disassembled in the direction from 5' to 3' of ssDNA as dATP hydrolysis proceeded.     

Nanoscale Imaging of Caveolin-1 Membrane Domains In Vivo

Light microscopy enables noninvasive imaging of fluorescent species in biological specimens, but resolution is generally limited by diffraction to ~200–250 nm. Many biological processes occur on smaller length scales, highlighting the importance of techniques that can image below the diffraction limit and provide valuable single-molecule information. In recent years, imaging techniques have been developed which can achieve resolution below the diffraction limit. Utilizing one such technique, fluorescence photoactivation localization microscopy (FPALM), we demonstrated its ability to construct super-resolution images from single molecules in a living zebrafish embryo, expanding the realm of previous super-resolution imaging to a living vertebrate organism. We imaged caveolin-1 in vivo, in living zebrafish embryos. Our results demonstrate the successful image acquisition of super-resolution images in a living vertebrate organism, opening several opportunities to answer more dynamic biological questions in vivo at the previously inaccessible nanoscale.     

Imaging of single molecules in live cells

Progress in optical microscopy, combined to the emergence of new fluorescent probes and advanced instrumentation, now permits the imaging of single molecules in fixed and live cells. This extreme detection sensitivity has opened new modalities in cellular imaging. On the one hand, optical images with an unprecedented resolution in the 10-50 nm range, well below the diffraction limit of light, can be recorded. These super-resolution images give new insights into the properties of cellular structures. On the other hand, proteins, either in the membrane or intracellular, can be tracked in live cells and in physiological conditions. Their individual trajectories provide invaluable information on the molecular interactions that control their dynamics and their spatial organization. Single molecule imaging is rapidly becoming a unique tool to understand the biochemical and biophysical processes that determine the properties of molecular assemblies in a cellular context.     

High-resolution near-field optical imaging of single nuclear pore complexes under physiological conditions

Scanning near-field optical microscopy (SNOM) circumvents the diffraction limit of conventional light microscopy and is able to achieve optical resolutions substantially below 100 nm. However, in the field of cell biology SNOM has been rarely applied, probably because previous techniques for sample-distance control are less sensitive in liquid than in air. Recently we developed a distance control based on a tuning fork in tapping mode, which is also well-suited for imaging in solution. Here we show that this approach can be used to visualize single membrane protein complexes kept in physiological media throughout. Nuclear envelopes were isolated from Xenopus laevis oocytes at conditions shown recently to conserve the transport functions of the nuclear pore complex (NPC). Isolated nuclear envelopes were fluorescently labeled by antibodies against specific proteins of the NPC (NUP153 and p62) and imaged at a resolution of approximately 60 nm. The lateral distribution of epitopes within the supramolecular NPC could be inferred from an analysis of the intensity distribution of the fluorescence spots. The different number densities of p62- and NUP153-labeled NPCs are determined and discussed. Thus we show that SNOM opens up new possibilities for directly visualizing the transport of single particles through single NPCs and other transporters.     

Fluorescence ‘off–on’ probe for lead (II) detection based on Atractylodes III CQDs and bioimaging

In this work, a type of carbon quantum dots (CQDs) with bright blue emission was readily fabricated through one-step hydrothermal treatment from Atractylodes III. We explored the surface morphology and optical properties of the CQDs using transmission electron microscopy, X-ray diffraction patterns, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy and ultraviolet–visible light spectrophotometry. The obtained CQDs possessed good photoluminescence properties, water solubility, and biocompatibility. The fluorescence quantum yield of these was 3.72%. It was found that the fluorescence intensity of CQDs was quenched by picric acid. After adding lead (II), the fluorescence could be effectively recovered. Therefore, an ‘off–on’ fluorescence probe was designed to detect lead (II) in the range 0–580 μM and the limit of detection was 0.068 μM. In addition, the experiments showed that the CQDs could be successfully used in bioimaging and as a hidden fluorescent ink.