Spatially and temporally synchronized atomic force and total internal reflection fluorescence microscopy for imaging and manipulating cells and biomolecules

The atomic force microscope is a high-resolution scanning-probe instrument which has become an important tool for cellular and molecular biophysics in recent years but lacks the time resolution and functional specificities offered by fluorescence microscopic techniques. To exploit the advantages of both methods, here we developed a spatially and temporally synchronized total internal reflection fluorescence and atomic force microscope system. The instrument, which we hereby call STIRF-AFM, is a stage-scanning device in which the mechanical and optical axes are coaligned to achieve spatial synchrony. At each point of the scan the sample topography (atomic force microscope) and fluorescence (photon count or intensity) information are simultaneously recorded. The tool was tested and validated on various cellular (monolayer cells in which actin filaments and intermediate filaments were fluorescently labeled) and biomolecular (actin filaments and titin molecules) systems. We demonstrate that with the technique, correlated sample topography and fluorescence images can be recorded, soft biomolecular systems can be mechanically manipulated in a targeted fashion, and the fluorescence of mechanically stretched titin can be followed with high temporal resolution.     

Applications of the molecular dynamics flexible fitting method

In recent years, cryo-electron microscopy (cryo-EM) has established itself as a key method in structural biology, permitting the structural characterization of large biomolecular complexes in various functional states. The data obtained through single-particle cryo-EM has recently seen a leap in resolution thanks to landmark advances in experimental and computational techniques, resulting in sub-nanometer resolution structures being obtained routinely. The remaining gap between these data and revealing the mechanisms of molecular function can be closed through hybrid modeling tools that incorporate known atomic structures into the cryo-EM data. One such tool, molecular dynamics flexible fitting (MDFF), uses molecular dynamics simulations to combine structures from X-ray crystallography with cryo-EM density maps to derive atomic models of large biomolecular complexes. The structures furnished by MDFF can be used subsequently in computational investigations aimed at revealing the dynamics of the complexes under study. In the present work, recent applications of MDFF are presented, including the interpretation of cryo-EM data of the ribosome at different stages of translation and the structure of a membrane-curvature-inducing photosynthetic complex.     

Specialized pro-resolving lipid mediators in the inflammatory response: An update

A new genus of specialized pro-resolving mediators (SPM) which include several families of distinct local mediators (lipoxins, resolvins, protectins, and maresins) are actively involved in the clearance and regulation of inflammatory exudates to permit restoration of tissue homeostasis. Classic lipid mediators that are temporally regulated are formed from arachidonic acid, and novel local mediators were uncovered that are biosynthesized from ω-3 poly-unsaturated fatty acids, such as eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid. The biosynthetic pathways for resolvins are constituted by fatty acid lipoxygenases and cyclooxygenase-2 via transcellular interactions established by innate immune effector cells which migrate from the vasculature to inflamed tissue sites. SPM provide local control over the execution of an inflammatory response towards resolution, and include recently recognized actions of SPM such as tissue protection and host defense. The structural families of the SPM do not resemble classic eicosanoids (PG or LT) and are novel structures that function uniquely via pro-resolving cellular and molecular targets. The extravasation of inflammatory cells expressing SPM biosynthetic routes are matched by the temporal provision of essential fatty acids from circulation needed as substrate for the formation of SPM. The present review provides an update and overview of the biosynthetic pathways and actions of SPM, and examines resolution as an integrated component of the inflammatory response and its return to homeostasis via biochemically active resolution mechanisms.     

Dynamics of leading lamellae of living fibroblasts visualized by high-speed scanning probe microscopy

In this study, we aimed at improving the temporal resolution of scanning probe microscopy (SPM) for observing living cells by introducing soft cantilevers, low feedback-gain operations, and cantilever deflection imaging. We achieved visualization of the mechanical architecture in leading lamellae of living fibroblasts at a temporal resolution of around 10 s, which is higher than that of conventional contact-mode SPM. Time-lapse SPM could be used to monitor not only cytoskeletal dynamics but also the dynamics of numerous microgranules. Statistical analysis of microgranular motion revealed that the microgranules have superdiffusive behaviors and significant directional order of motion. We also found that the direction of their motion is correlated with the direction of growing actin stress fibers. The combination of SPM with fluorescence microscopy showed that vinculin, a component of cell-substratum adhesion sites, localizes at the microgranules. Our experimental data provides a new insight into the intracellular mechanical architecture and its structural dynamics, suggesting that high-speed live-cell SPM has great potential for investigating the structural origin of cellular dynamics.     

Fully Automated Whole-Head Segmentation with Improved Smoothness and Continuity,with Theory Reviewed

Individualized current-flow models are needed for precise targeting of brain structures using transcranial electrical or magnetic stimulation (TES/TMS). The same is true for current-source reconstruction in electroencephalography and magnetoencephalography (EEG/MEG). The first step in generating such models is to obtain an accurate segmentation of individual head anatomy, including not only brain but also cerebrospinal fluid (CSF), skull and soft tissues, with a field of view (FOV) that covers the whole head. Currently available automated segmentation tools only provide results for brain tissues, have a limited FOV, and do not guarantee continuity and smoothness of tissues, which is crucially important for accurate current-flow estimates. Here we present a tool that addresses these needs. It is based on a rigorous Bayesian inference framework that combines image intensity model, anatomical prior (atlas) and morphological constraints using Markov random fields (MRF). The method is evaluated on 20 simulated and 8 real head volumes acquired with magnetic resonance imaging (MRI) at 1 mm3 resolution. We find improved surface smoothness and continuity as compared to the segmentation algorithms currently implemented in Statistical Parametric Mapping (SPM). With this tool, accurate and morphologically correct modeling of the whole-head anatomy for individual subjects may now be feasible on a routine basis. Code and data are fully integrated into SPM software tool and are made publicly available. In addition, a review on the MRI segmentation using atlas and the MRF over the last 20 years is also provided, with the general mathematical framework clearly derived.     

Coordinate-based colocalization analysis of single-molecule localization microscopy data

Colocalization of differently labeled biomolecules is a valuable tool in fluorescence microscopy and can provide information on biomolecular interactions. With the advent of super-resolution microscopy, colocalization analysis is getting closer to molecular resolution, bridging the gap to other technologies such as fluorescence resonance energy transfer. Among these novel microscopic techniques, single-molecule localization-based super-resolution methods offer the advantage of providing single-molecule coordinates that, rather than intensity information, can be used for colocalization analysis. This requires adapting the existing mathematical algorithms for localization microscopy data. Here, we introduce an algorithm for coordinate-based colocalization analysis which is suited for single-molecule super-resolution data. In addition, we present an experimental configuration for simultaneous dual-color imaging together with a robust approach to correct for optical aberrations with an accuracy of a few nanometers. We demonstrate the potential of our approach for cellular structures and for two proteins binding actin filaments.     

Characterising biomolecular interactions and dynamics with mass photometry

We review recent advances in our ability to characterise biomolecular structure, interactions and associated dynamics by mass photometry (MP), the label-free detection and mass measurement of individual biomolecules in solution. Molecular counting and identification provides direct access to relative abundance, and thereby affinities, while associated dynamics yield on- and off-rates. The molecular resolution afforded by MP enables these measurements as a function of stoichiometry and assembly at equilibrium, as opposed to the majority of existing solution-based methods. Together with future improvements in terms of assays and technological performance, MP is likely to provide mechanistic details of complex biomolecular processes.     

Gold and silver nanoparticles for clinical diagnostics - From genomics to proteomics

Nanotechnology has prompted researchers to develop new and improved materials aimed at biomedical applications with particular emphasis in diagnostics and therapy. Special interest has been directed at providing enhanced biomolecular diagnostics, including SNP detection gene expression profiles and biomarker characterisation. These strategies have focused on the development of nanoscale devices and platforms that can be used for single molecule characterisation of nucleic acid, DNA or RNA, and protein at an increased rate when compared to traditional techniques. Also, several advances have been reported on DNA analysis in real time, at both high resolution and very high throughputs, suitable for biomedical diagnostics. Here, we shall provide a review of available nanotechnology-based platforms for biomolecular recognition, and their application to molecular diagnostics and genome analysis, with emphasis on the use of noble metal nanoparticles for simple and specific analysis systems. Particular focus will be put on those already being translated into clinical settings. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Probing the Machinery of Intracellular Trafficking with the Atomic Force Microscope

Atomic force microscopy has emerged as a powerful tool for characterizing single biological macromolecules, macromolecular assemblies, and whole cells in aqueous buffer, in real time, and at molecular-scale spatial and force resolution. Many of the central elements of intracellular transport are tens to hundreds of nanometers in size and highly dynamic. Thus, atomic force microscopy provides a valuable means of addressing questions of structure and mechanism in intracellular transport. We begin this review of recent efforts to apply atomic force microscopy to problems in intracellular transport by discussing the technical principles behind atomic force microscopy. We then turn to three specific areas in which atomic force microscopy has been applied to problems with direct implications for intracellular trafficking: cytoskeletal structure and dynamics, vesicular transport, and receptor–ligand interactions. In each case, we discuss studies which use both intact cellular elements and reconstituted models. While many technical challenges remain, these studies point to several areas where atomic force microscopy can be used to provide valuable insight into intracellular transport at exquisite spatial and energetic resolution.     

Biomolecular structure prediction at a low resolution using a neural network and the double-iterated kalman filter technique

We report the application of an integrated computational approach for biomolecular structure determination at a low resolution. In particular, a neural network is trained to predict the spatial proximity of C-alpha atoms that are less than a given threshold apart, whereas a Kalman filter algorithm is employed to outline the biomolecular fold, with a constraints set that includes these pairwise atomic distances, and the distances and angles that define the structure as it is known from the protein's sequence. The results for Crambin demonstrate that this integrated approach is useful for molecular structure prediction at a low resolution and may also complement existing experimental distance data for a protein structure determination. © 1996 John Wiley & Sons, Inc.     

基于表面等离子共振的新型生物传感技术及其在生命科学中的应用

生物分子的活性功能是通过分子之间的相互作用来体现的,了解这种相互作用的过程对于生命科学领域的研究及揭示生命发生发展的基本机制具有重要的意义。基于表面等离子共振(surface plasmon resonance,SPR)的新型生物传感技术——BIAcore(biomolecular interaction analysis)是研究生物分子相互作用的理想工具。它可以实时跟踪检测生物分子间结合、解离的整个过程,已被广泛应用于蛋白质组学、信号转导、新药开发、遗传学分析和食品检测等领域,并且显示出广阔的应用前景。     

Fast optical monitoring of microscopic excitation patterns in cardiac muscle.

Many vital processes depend on the generation, changes, and conduction of cellular transmembrane potentials. Optical monitoring systems are well suited to detect such cellular electrical activities in networks of excitable cells and also tissues simultaneously at multiple sites. Here, an exceptionally fast array system (16 x 16 photodiodes, up to 4,000,000 samples per second, 12-bit resolution) for imaging voltage-sensitive dye fluorescence, permitted real time measurements of excitation patterns at a microscopic size scale (256 pixels within an area of 1.8-8 mm2), in rat cardiac muscle in vitro. Results emphasize a recent hypothesis for cardiac impulse conduction, based on cardiac structural complexities, that is contradictory to all continuous cable theory models.     

Perspective: a stirring role for metabolism in cells

Based on recent findings indicating that metabolism might be governed by a limit on the rate at which cells can dissipate Gibbs energy, in this Perspective, we propose a new mechanism of how metabolic activity could globally regulate biomolecular processes in a cell. Specifically, we postulate that Gibbs energy released in metabolic reactions is used to perform work, allowing enzymes to self‐propel or to break free from supramolecular structures. This catalysis‐induced enzyme movement will result in increased intracellular motion, which in turn can compromise biomolecular functions. Once the increased intracellular motion has a detrimental effect on regulatory mechanisms, this will establish a feedback mechanism on metabolic activity, and result in the observed thermodynamic limit. While this proposed explanation for the identified upper rate limit on cellular Gibbs energy dissipation rate awaits experimental validation, it offers an intriguing perspective of how metabolic activity can globally affect biomolecular functions and will hopefully spark new research.     

Cryo-electron microscopy for structural analysis of dynamic biological macromolecules

Background

Since the introduction of what became today's standard for cryo-embedding of biological macromolecules at native conditions more than 30 years ago, techniques and equipment have been drastically improved and the structure of biomolecules can now be studied at near atomic resolution by cryo-electron microscopy (cryo-EM) while capturing multiple dynamic states. Here we review the recent progress in cryo-EM for structural studies of dynamic biological macromolecules.

Biomolecular condensates in autophagy regulation

Autophagy is an intracellular degradation system that contributes to cellular homeostasis. Autophagosome formation is a landmark event in autophagy, which sequesters and delivers cytoplasmic components to the lysosome for degradation. Based on selectivity, autophagy can be classified into bulk and selective autophagy, which are mechanistically distinct from each other, especially in the requirement of cargos for autophagosome formation. Recent studies revealed that liquid-like biomolecular condensates, which are formed through liquid–liquid phase separation, regulate the autophagosome formation of both bulk and selective autophagy. Here, we focus on recent findings on the involvement of biomolecular condensates in autophagy regulation and discuss their significance.     

Single-Molecule Imaging at High Fluorophore Concentrations by Local Activation of Dye

Single-molecule fluorescence microscopy is a powerful tool for observing biomolecular interactions with high spatial and temporal resolution. Detecting fluorescent signals from individual labeled proteins above high levels of background fluorescence remains challenging, however. For this reason, the concentrations of labeled proteins in in vitro assays are often kept low compared to their in vivo concentrations. Here, we present a new fluorescence imaging technique by which single fluorescent molecules can be observed in real time at high, physiologically relevant concentrations. The technique requires a protein and its macromolecular substrate to be labeled each with a different fluorophore. Making use of short-distance energy-transfer mechanisms, only the fluorescence from those proteins that bind to their substrate is activated. This approach is demonstrated by labeling a DNA substrate with an intercalating stain, exciting the stain, and using energy transfer from the stain to activate the fluorescence of only those labeled DNA-binding proteins bound to the DNA. Such an experimental design allowed us to observe the sequence-independent interaction of Cy5-labeled interferon-inducible protein 16 with DNA and the sliding via one-dimensional diffusion of Cy5-labeled adenovirus protease on DNA in the presence of a background of hundreds of nanomolar Cy5 fluorophore.     

Scanning probe microscopy for Bio & Nanotechnology onboard the ISS

Since February 2002 Kayser-Threde GmbH, Munich (Germany) leads a study under ESA contract in order to study the technical feasibility and the applications of "Scanning Probe Microscopy for Bio & Nanotechnology onboard the ISS (SONOS)". The objective of this effort is to demonstrate the feasibility of an SPM instrument on the ISS. An appropriate breadboard model will be manufactured and tested within the present study. Its development will be based upon the developed pocket size SPM instrument by Professor W. Hecki of the Center for Crystallography and NanoScience (CeNS) at the Ludwig-Maximilians University (LMU) in Munich. Scanning probe microscopy (SPM) investigates surface structures at very high resolution and can perform nanoengineering. These techniques can be applied to non organic as well as to organic or biological materials.     

High-throughput quantification of phosphatidylcholine and sphingomyelin by electrospray ionization tandem mass spectrometry coupled with isotope correction algorithm

The choline head group containing phosphatidylcholine (PC) and sphingomyelin (SPM) are major eukaryotic lipid components playing an important role in forming membrane microdomains and serve as precursor of signaling molecules. Both lipids can be monitored by positive ion mode electrospray tandem mass spectrometry using a parent ion scan of m/z 184. Although PC species appear at even m/z and SPM species at odd m/z, there may be a significant overlap of their isotopes. In order to separate PC and SPM species, an isotope correction algorithm was established, which utilizes calculated isotope percentages to correct the measured peak intensities for their isotopic overlap. We could demonstrate that this approach was applicable to correct the isotope overlap resulting from spiked PC and SPM species. Quantification was achieved by addition of different PC and SPM species prior to lipid extraction. The developed assay showed a precision, detection limit and robustness sufficient for routine analysis. Furthermore, an analysis time of only 1.3 min combined with automated data analysis using self-programmed Excel Macros allows high-throughput analysis. In summary, this assay may be a valuable tool for detailed lipid analysis of PC and SPM species in a variety of sample materials.