- 标题
- 摘要
- 关键词
- 实验方案
- 产品
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Microscopy of the Heart || Optical Sectioning Microscopy at ‘Temporal Super-Resolution’
摘要： Within the recent years several super-resolution microscopic methods were developed, where the super-resolution refers to bringing the optical resolution beyond the diffraction limit introduced by Ernst Abbe, which was believed to be a real limit for quite some time. The popularity of the method also in cardiac related research can be followed in the chapter ‘Quantitative super-resolution microscopy of cardiac myocytes’ in this book. In parallel to this spatial super-resolution progress, within the past two decades there was a dynamic development of high speed–high resolution imaging initially towards video-rate (30 frames per second, also referred to as ‘real time’-imaging) but soon to ever increasing frame rates reaching the kHz order of magnitude these days. Many processes, especially those in excitable cells such as neurons and cardiomyocytes [1] or cells in ?ow like erythrocytes or leukocytes [2], require even higher temporal resolution to elucidate the kinetics of processes like the Excitation-Contraction Coupling (ECC). Such ultra high speed recordings still require a diffraction limited spatial resolution to correlate function and subcellular structures [3]. Within this chapter we review optical sectioning microscopy and their application in cellular cardiology. In this approach we focus on methods that allow to access any part of the cell, i.e. we exclude methods that are intrinsically limited to surface investigations like total internal re?ection ?uorescence (TIRF) microscopy [4] or scanning near ?eld optical microscopy (SNOM) [5]. In similarity we exclude techniques that require several images to calculate an image section such as deconvolution microscopy [6] or structured illumination microscopy [7] (e.g., Apotome.2, Zeiss, Jena, Germany).
关键词： super-resolution microscopy,Excitation-Contraction Coupling,high-speed imaging,cardiomyocytes,optical sectioning
更新于2025-09-23 15:19:57
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Small-Molecule Fluorescent Probes for Live-Cell Super-Resolution Microscopy
摘要： Super-resolution fluorescence microscopy is a powerful tool to visualize biomolecules and cellular structures at the nanometer scale. Employing these techniques in living cells has opened up the possibility to study dynamic processes with unprecedented spatial and temporal resolution. Different physical approaches to super-resolution microscopy have been introduced over the last years. A bottleneck to apply these approaches for live-cell imaging has become the availability of appropriate fluorescent probes that can be specifically attached to biomolecules. In this perspective, we discuss the role of small-molecule fluorescent probes for live-cell super-resolution microscopy and the challenges that need to be overcome for their generation. Recent trends in the development of labeling strategies are reviewed together with the required chemical and spectroscopic properties of the probes. Finally, selected examples of the use of small-molecule fluorescent probes in live-cell super-resolution microscopy are given.
关键词： Bioorthogonal chemistry,Super-resolution microscopy,Live-cell imaging,Fluorogenic probes,Protein labeling
更新于2025-09-23 15:19:57
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Amplified Expansion Stimulated Emission Depletion Microscopy
摘要： Expansion microscopy (ExM) enhances spatial resolution using a swellable polymer that expands the sample volume by a factor of ~4 in one dimension and factor of ~20 in volume. Combining ExM with stimulated emission depletion (STED) microscopy, referred to as ExSTED, increases the resolution to up to 10 nm. However, photobleaching is a critical issue in ExSTED because sample expansion lowers fluorophore density and high-resolution STED requires high depletion intensity. To overcome these issues, we developed highly bright expansion nanoscopy using biotin-avidin signal amplification to increase the labeling density. Our method provides up to a 7-fold increase in fluorescence signal intensity in expanded samples, thus enabling the use of STED imaging with the maximum depletion intensity of a commercial microscope in the order of GW/cm2. We demonstrate the method using biotinylated antibodies and genetic incorporation approaches of biotin to a specific molecule or organelle.
关键词： stimulated emission depletion microscopy,super-resolution microscopy,expansion microscopy
更新于2025-09-19 17:15:36
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NanoJ: a high-performance open-source super-resolution microscopy toolbox
摘要： Super-resolution microscopy has become essential for the study of nanoscale biological processes. This type of imaging often requires the use of specialised image analysis tools to process a large volume of recorded data and extract quantitative information. In recent years, our team has built an open-source image analysis framework for super-resolution microscopy designed to combine high performance and ease of use. We named it NanoJ - a reference to the popular ImageJ software it was developed for. In this paper, we highlight the current capabilities of NanoJ for several essential processing steps: spatio-temporal alignment of raw data (NanoJ-Core), super-resolution image reconstruction (NanoJ-SRRF), image quality assessment (NanoJSQUIRREL), structural modelling (NanoJ-VirusMapper) and control of the sample environment (NanoJ-Fluidics). We expect to expand NanoJ in the future through the development of new tools designed to improve quantitative data analysis and measure the reliability of fluorescent microscopy studies.
关键词： Virus,Vaccinia,Archaea,Quantitative imaging,Sulfolobus acidocaldarius,Super-resolution microscopy,Fluidics,Modelling,Resolution,Image quality assessment,Pox,Image analysis,ImageJ,Fiji
更新于2025-09-19 17:15:36
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Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors
摘要： DNA point accumulation for imaging in nanoscale topography (PAINT) is a rapidly developing fluorescence super-resolution technique, which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20–25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using three nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the three targets with 20 nm resolution, and within only 35 minutes acquisition time.
关键词： DNA-PAINT,microfluidics,super-resolution microscopy,fluorescent proteins,molecular localization,multi-color imaging,multiplexing,single domain antibodies (sdAb),linkage error,nanobodies
更新于2025-09-19 17:15:36
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Fluorogenic probes for super-resolution microscopy
摘要： Fluorogenic probes efficiently reduce non-specific background signals, which often results in highly improved signal-to-noise ratios. Although this implies improved resolution, fluorogenic probes in the context of super-resolution microscopy are somewhat overlooked. Several excellent reviews summarize recent developments in SRM techniques, labeling techniques or different aspects of small synthetic fluorophores, however there is no comprehensive review on fluorogenic probes suitable for super-resolution microscopy. Herein we wish to fill this gap by providing the readers with an up-to-date summary of fluorogenic probes applied to super-resolution imaging of cellular structures.
关键词： signal-to-noise ratio,background reduction,super-resolution microscopy,cellular imaging,fluorogenic probes
更新于2025-09-19 17:15:36
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On the impact of competing intra- and intermolecular triplet-state quenching on photobleaching and photoswitching kinetics of organic fluorophores
摘要： While buffer cocktails remain the most commonly used method for photostabilization and photoswitching of fluorescent markers, intramolecular triplet-state quenchers emerge as an alternative strategy to impart fluorophores with 'self-healing' or even functional properties such as photoswitching. In this contribution, we evaluated combinations of both approaches and show that inter- and intramolecular triplet-state quenching processes compete with each other. We find that although the rate of triplet-state quenching is additive, the photostability is limited by the faster pathway. Often intramolecular processes dominate the photophysical situation for combinations of covalently-linked and solution-based photostabilizers and photoswitching agents. Furthermore we show that intramolecular photostabilizers can protect fluorophores from reversible off-switching events caused by solution-additives, which was previously misinterpreted as photobleaching. Our studies also provide practical guidance for usage of photostabilizer–dye conjugates for STORM-type super-resolution microscopy permitting the exploitation of their improved photophysics for increased spatio-temporal resolution. Finally, we provide evidence that the biochemical environment, e.g., proximity of aromatic amino-acids such as tryptophan, reduces the photostabilization efficiency of commonly used buffer cocktails. Not only have our results important implications for a deeper mechanistic understanding of self-healing dyes, but they will provide a general framework to select label positions for optimal and reproducible photostability or photoswitching kinetics in different biochemical environments.
关键词： photoswitching,intramolecular quenching,self-healing dyes,super-resolution microscopy,intermolecular quenching,photostabilization,fluorophores,triplet-state quenching,STORM
更新于2025-09-19 17:15:36
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Photoswitchable single-walled carbon nanotubes for super-resolution microscopy in the near-infrared
摘要： The design of single-molecule photoswitchable emitters was the first milestone toward the advent of single-molecule localization microscopy, setting a new paradigm in the field of optical imaging. Several photoswitchable emitters have been developed, but they all fluoresce in the visible or far-red ranges, missing the desirable near-infrared window where biological tissues are most transparent. Moreover, photocontrol of individual emitters in the near-infrared would be highly desirable for elementary optical molecular switches or information storage elements since most communication data transfer protocols are established in this spectral range. Here, we introduce a type of hybrid nanomaterials consisting of single-wall carbon nanotubes covalently functionalized with photoswitching molecules that are used to control the intrinsic luminescence of the single nanotubes in the near-infrared (beyond 1 mm). Through the control of photoswitching, we demonstrate super-localization imaging of nanotubes unresolved by diffraction-limited microscopy.
关键词： single-molecule localization microscopy,near-infrared,super-resolution microscopy,carbon nanotubes,photoswitchable emitters
更新于2025-09-12 10:27:22
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Multi-color live-cell super-resolution volume imaging with multi-angle interference microscopy
摘要： Imaging and tracking of near-surface three-dimensional volumetric nanoscale dynamic processes of live cells remains a challenging problem. In this paper, we propose a multi-color live-cell near-surface-volume super-resolution microscopy method that combines total internal reflection fluorescence structured illumination microscopy with multi-angle evanescent light illumination. We demonstrate that our approach of multi-angle interference microscopy is perfectly adapted to studying subcellular dynamics of mitochondria and microtubule architectures during cell migration.
关键词： super-resolution microscopy,live-cell imaging,multi-angle interference microscopy,mitochondria,microtubules
更新于2025-09-10 09:29:36
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Imaging cellular ultrastructures using expansion microscopy (U-ExM)
摘要： Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.
关键词： centriolar chirality,U-ExM,electron microscopy,super-resolution microscopy,ultrastructure expansion microscopy
更新于2025-09-09 09:28:46