We sincerely appreciate the support from Philipp Kukura, Gavin Young, and the Refeyn software team and acknowledge their assistance by sharing parts of the image analysis code. We thank the Cryo-EM MPIB Core Facility for providing access to the commercial Refeyn mass photometer. F.S. gratefully acknowledges the support and funding granted by J&#252;rgen Plitzko and Wolfgang Baumeister. T.H. and P.S. received funding through the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - Project-ID 201269156 - SFB 1032 (A09). N.H. was supported by a DFG return grant HU 2462/3-1. P.S. acknowledges support through the research network MaxSynBio via the joint funding initiative of the German Federal Ministry of Education and Research (BMBF) and the Max Planck Society.

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The authors have no conflicts of interest.

The presented protocol extends mass photometry21, a technique that analyzes the mass of single biomolecules adsorbing on glass, to an even more versatile tool capable of simultaneously measuring the mass and diffusion of unlabeled membrane-interacting biomolecules. This analysis extension is achieved through the implementation of a modified background removal strategy adapted to the lateral motion of molecules24,25. In general, the background removal is of utmost importance for iSCAT-based approaches, since the strong scattering of the glass surface roughness represents the main analysis impediment, and the accurate determination of each pixel&#39;s local background is essential for quantification of particle mass and location. Besides the image-analysis adapted to particle motion, subsequent particle detection, trajectory linking, and data analysis complete the novel expansion of MP into mass-sensitive particle tracking (MSPT).
In general, thoroughly cleaned glass cover slides and a clean working environment are critical requirements for the successful performance of MSPT experiments. Due to the absence of macromolecule labeling, the acquired signal is inherently nonselective. Clean samples, as well as proper sample handling, are hence crucial to ensure that observations cannot be misinterpreted. In particular, when molecules of low molecular weight are examined, control measurements of protein-free membranes are endorsed to assess background contributions (Supplementary Figure 1). Besides the inclusion of control measurements, it is thus recommended to follow the preparation steps displayed in Figure 2 for each flow chamber. When combined, these safety measures will ensure that the detected signal originates from the biomolecule of interest and not, for example, a contaminated flow chamber, buffer, or membrane.
Besides the precautions regarding the experimental design, care needs to also be taken during MSPT image processing. During video processing, the value for three parameters should be chosen carefully to ensure correct results: i) the length of the median window for background removal, ii) the threshold for particle detection, and iii) the maximum search radius during the linking assignment. A larger median window (i) generally facilitates the separation of diffusing particles from the superimposed quasi-constant background. However, for too large window sizes sample drift will eventually become noticeable and diminish the accuracy of the background estimation. Optimal settings heavily depend on sample properties and measurement conditions. Nevertheless, a value of 1,001 can be used as a robust starting point. The threshold parameter (ii) must be tuned depending on the lowest molecular mass expected in the sample. A value below 0.0005 is not recommended for measurements taken with the mass photometer used in this study. To speed up analysis times, higher values can be chosen if a sample with high molecular weight is expected. The search radius in trajectory linking (iii) specifies the maximum radial distance in pixels in which the particle&#39;s shifted location will be searched for in consecutive frames. It should be adapted to the fastest particle in the sample, and, if favored, an adaptive search range (see documentation of trackpy) could be used instead to reduce computation time. Especially during the initial phase of a project, re-analyzing the movies with varying parameters is recommended to validate the obtained results.
In light of the single-molecule nature of MSPT, it should be avoided to measure at high membrane particle densities as those can interfere with accurate contrast and mass determination. It has been shown that densities below one particle per squared micrometer are favorable for MSPT measurements24. An additional consideration is the expected diffusion coefficients in the sample. Although applicable to a broad range of diffusion coefficients, MSPT has a lower limit of accessible diffusion coefficients. Local confinement to a region of few pixels during a significant portion of the median window period merges the particle with the static background. For the imaging conditions used in this protocol, measurement of diffusion coefficients below 0.01 &#181;m2/s is not recommended. At this diffusion speed, for example, the mean squared displacement of a particle during the median window half-size is about 4 pixels and hence of similar size as the extent of the PSF. As a consequence, the static background estimate is likely to contain signal contributions from the particle itself, which results in an apparently reduced contrast of the particle until it eventually approaches the noise level. However, macromolecule diffusion coefficients ranging between 0.05 and 10 &#181;m2/s can clearly be resolved.
To further extend the range of MSPT applications, one can envision an advancement of the median-based background algorithm through the elimination of pixels that are temporarily occupied with a particle, or by sample drift correction enabling larger median window sizes. Both approaches would alleviate the problems regarding measurements at high particle densities and slow diffusion. Improvements in terms of lower mass sensitivity are on the horizon with a new generation of mass photometers, which may provide access to biomolecules smaller than 50 kDa. Therefore, future MSPT experiments will be able to study single-molecule dynamics and membrane-related interactions for an even wider range of membrane mimicries such as cushioned bilayers and macromolecular systems.

Once merely perceived as a barrier against the wide range of ambient physical conditions, biological membranes are nowadays considered functional entities and catalytic platforms1,2. Based on their ability to localize, amplify, and direct signals in response to membrane-associated macromolecule reactions, lipid interfaces constitute a crucial element for a wide variety of cellular processes such as membrane trafficking and signaling cascades3,4,5. Serving as a nucleation site for the assembly of stable complexes, membrane attachment often relies on a dynamic equilibrium between membrane-associated and cytosolic forms of macromolecules and is hence of transient nature6,7.
Despite their great importance in biology, it has so far been challenging to develop methods that can provide access to the compositional, spatial, and temporal heterogeneities of membrane-associated macromolecule reactions in real-time7,8. To resolve the underlying molecular processes, two experimental aspects are decisive: sufficient time resolution and single-particle sensitivity. Therefore, ensemble average techniques such as fluorescence recovery after photobleaching (FRAP) but also the much more sensitive fluorescence correlation spectroscopy (FCS) do have limitations, since they largely uncouple spatial information from temporal information9. An important step toward the characterization of individual molecule dynamics has thus been the advent of single-particle tracking (SPT) in combination with highly sensitive microscopy. In particular, two SPT approaches have proven to be effective in this regard. Firstly, the utilization of fluorophores as labels and the corresponding fluorescence detection systems paved the way for nanometer precision and millisecond time resolution10,11,12. Secondly, scattering-based detection using gold nanoparticles improved both localization precision and time resolution to the sub-nanometer and microsecond range, respectively13,14,15,16. Despite the many advantages of both approaches and their significant contributions regarding the mechanistic understanding of membrane-associated systems17,18, both techniques have so far been limited:&#160;they require labeling of the molecules of interest, which potentially perturbs their native behavior and are&#160;insensitive to the molecular composition of membrane-associated particles19,20.
Both these limitations have recently been overcome by the introduction of a novel interferometric scattering (iSCAT)-based approach termed mass photometry (MP)21,22,23. This technique allows the determination of in-solution mass distributions of biomolecules according to their iSCAT contrast when landing on a glass interface. However, for the detection and characterization of mobile molecules diffusing on lipid membranes, a more sophisticated image analysis approach had to be developed. This has meanwhile been successfully implemented and allows to detect, track, and determine the molecular mass of single unlabeled biomolecules diffusing on a lipid interface24,25. Referred to as dynamic mass photometry or mass-sensitive particle tracking (MSPT), this technique now enables the assessment of complex macromolecule interactions by directly recording changes in the molecular mass of the tracked entities and hence opens up new possibilities for the mechanistic analysis of membrane-associated molecular dynamics.
Here, a detailed protocol for sample preparation, imaging, and the data analysis pipeline required for MSPT is presented. In particular, sample requirements and potential problems that may occur during measurement and analysis are discussed. Furthermore, the unparalleled potential to analyze membrane-interacting macromolecule systems is showcased through various representative results.

<table><tbody><tr><td>annexin V</td><td>Sigma Aldrich</td><td>#SRP8026</td><td>examplary membrane-interacting protein</td></tr><tr><td>Bio-Rad Protein Assay</td><td>Bio-Rad Laboratories Inc.</td><td>#5000006</td><td>bradford assay kit to determine protein stock concentrations</td></tr><tr><td>biotin labeled bovine albumin</td><td>Sigma Aldrich</td><td>#A8549</td><td>examplary protein that can be used as standard protein for MSPT</td></tr><tr><td>cholera toxin subunit B</td><td>Sigma Aldrich</td><td>#SAE0069</td><td>examplary membrane-interacting protein</td></tr><tr><td>cover glasses, #1.5, 24 x 24 mm</td><td>Paul Marienfeld GmbH &amp;amp; Co. KG</td><td>#0102062</td><td></td></tr><tr><td>cover glasses, #1.5, 24 x 60 mm</td><td>Paul Marienfeld GmbH &amp;amp; Co. KG</td><td>#0102242</td><td></td></tr><tr><td>dioleoyl-sn-glycero-3-phosphocholine (DOPC)</td><td>Avanti Polar Lipids</td><td>#850375</td><td>lipid - in the form of extruded small unilamellar vesicles required for supported lipid bilayer formation</td></tr><tr><td>dioleoyl-sn-glycero-3-phosphoethanolamine-N-cap biotinyl (18:1 Biotinyl Cap PE</td><td>Avanti Polar Lipids</td><td>#870273</td><td>lipid - in the form of extruded small unilamellar vesicles required for supported lipid bilayer formation</td></tr><tr><td>dioleoyl-sn-glycero-3-phosphoglycerol (DOPG)</td><td>Avanti Polar Lipids</td><td>#840475</td><td>lipid - in the form of extruded small unilamellar vesicles required for supported lipid bilayer formation</td></tr><tr><td>dioleoyl-sn-glycero-3-phospho-L-serine (DOPS)</td><td>Avanti Polar Lipids</td><td>#840035</td><td>lipid - in the form of extruded small unilamellar vesicles required for supported lipid bilayer formation</td></tr><tr><td>double-sided tape</td><td>tesa</td><td>#57912-00000-02</td><td>needed for the assembly of glass sample chambers</td></tr><tr><td>Extruder</td><td>Avanti Polar Lipids</td><td>#610023</td><td>Lipid extruder to enable monodisperse vesicle distributions</td></tr><tr><td>EZ-Link Maleimide-PEG2-Biotin</td><td>Thermo Fisher Scientific</td><td>#A39261</td><td>maileimide-fused biotin that can be used to biotinylate standard proteins for MSPT</td></tr><tr><td>Fibronectin (Biotinylated)</td><td>Cytoskeleton Inc.</td><td>#FNR03-A</td><td>examplary protein that can be used as standard protein for MSPT</td></tr><tr><td>Gel Filtration HMW Calibration Kit</td><td>Cytiva</td><td>#28403842</td><td>standard proteins, e.g. aldolase that can be biotinylated and used as molecular weight standards for MSPT</td></tr><tr><td>GM1 Ganglioside (Brain, Ovine-Sodium Salt)</td><td>Avanti Polar Lipids</td><td>#860065</td><td>lipid - in the form of extruded small unilamellar vesicles required for supported lipid bilayer formation</td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Biotin</td><td>Thermo Fisher Scientific, Waltham, USA)</td><td>#31820</td><td>examplary protein to highlight the existence of different protein states</td></tr><tr><td>Isopropanol, 99.5%, for spectroscopy</td><td>Thermo Fisher Scientific</td><td>#10003643</td><td></td></tr><tr><td>Low Autofluorescence Immersion Oil</td><td>Olympus K.K.</td><td>#IMMOIL-F30CC</td><td></td></tr><tr><td>pET21a-Streptavidin-Alive</td><td>Addgene</td><td>#20860</td><td>required to express and purify divalent streptavidin in combination with each other</td></tr><tr><td>pET21a-Streptavidin-Dead</td><td>Addgene</td><td>#20859</td><td>required to express and purify divalent streptavidin in combination with each other</td></tr><tr><td>Pierce Alkaline Phosphatase, biotinylated</td><td>Thermo Fisher Scientific</td><td>#29339</td><td>examplary protein that can be used as standard protein for MSPT</td></tr><tr><td>Pierce Protein A, Biotinylated</td><td>Thermo Fisher Scientific</td><td>#29989</td><td>examplary protein that can be used as standard protein for MSPT</td></tr><tr><td>Refeyn Acquire</td><td>Refeyn Ltd.</td><td></td><td>control software for Refeyn OneMP</td></tr><tr><td>Refeyn One</td><td>Refeyn Ltd.</td><td>-</td><td>mass photometer</td></tr><tr><td>sterile syringe filters 0.45 &amp;micro;m cellulose acetate membrane</td><td>VWR International</td><td>#514-0063</td><td>needed to filter particles from the buffer of interest</td></tr><tr><td>tetravalent streptavidin</td><td>Thermo Fisher Scientific</td><td>#SNN1001</td><td>tetravalent streptavidin to enable the presence of several biotin binding sites</td></tr><tr><td>Whatman Nuclepore Hydrophilic Membrane, 0.05 &amp;micro;m Pore Size, 25 mm Circle</td><td>Cytiva</td><td>#110603</td><td>a pore size of 50 nm is recommended for supported lipid bilayer formation in the context of MSPT</td></tr><tr><td>Zepto model 2 plasma cleaner</td><td>Diener electronic GmbH</td><td>-</td><td></td></tr></tbody></table>

mass-sensitive particle tracking to characterize membrane-associated macromolecule dynamics

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take place, are inherently difficult to assess with standard biophysical methods. The introduction of mass-sensitive particle tracking (MSPT) constitutes an important step toward a thorough quantitative characterization of such processes. Technically, this was made possible through the advent of interferometric scattering microscopy (iSCAT)-based mass photometry (MP). When the background removal strategy is optimized to reveal the two-dimensional motion of membrane-associated particles, this technique allows the real-time analysis of both diffusion and molecular mass of unlabeled macromolecules on biological membranes. Here, a detailed protocol to perform and analyze mass-sensitive particle tracking of membrane-associated systems is described. Measurements performed on a commercial mass photometer achieve time resolution in the millisecond regime and, depending on the MP system, a mass detection limit down to 50 kDa. To showcase the potential of MSPT for the in-depth analysis of membrane-catalyzed macromolecule dynamics in general, results obtained for exemplary protein systems such as the native membrane interactor annexin V are presented.

Watch this Scientific Journal Video about Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics at JoVE.com

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

This protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided alongside directions to prevent potential pitfalls.

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take ...

mass-sensitive-particle-tracking-to-characterize-membrane-associated

Research

JoVE Journal

Biochemistry

4.0K Views.  Max Planck Institute of Biochemistry. Met massagevoelige deeltjestracking kunnen we individuele biomoleculen observeren die zich verspreiden op lipidemembranen en hun dynamiek in realtime kwantificeren, allemaal volledig labelvrij. Omdat er geen labels nodig zijn, is er geen gevaar voor verstoring van het dynamische gedrag van de biomoleculen. Een ander voordeel van deze methode is de massagevoeligheid, waarmee we membraangebonden oligomere toestanden kunnen bepalen.Vanwege de veelzijdigheid kunnen we deze methode toepasse...

Video: Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and millisecond temporal resolution. These capabilities make single-molecule localization microscopy ideally suited to study molecular level biological functions in physiologically relevant environments. Here, we demonstrate an integrated protocol for both acquisition and processing/analysis of single-molecule tracking data to extract the different diffusive states a protein of interest may exhibit. This information can be used to quantify molecular complex formation in living cells. We provide a detailed description of a camera-based 3D single-molecule localization experiment, as well as the subsequent data processing steps that yield the trajectories of individual molecules. These trajectories are then analyzed using a numerical analysis framework to extract the prevalent diffusive states of the fluorescently labeled molecules and the relative abundance of these states. The analysis framework is based on stochastic simulations of intracellular Brownian diffusion trajectories that are spatially confined by an arbitrary cell geometry. Based on the simulated trajectories, raw single-molecule images are generated and analyzed in the same way as experimental images. In this way, experimental precision and accuracy limitations, which are difficult to calibrate experimentally, are explicitly incorporated into the analysis workflow. The diffusion coefficient and relative population fractions of the prevalent diffusive states are determined by fitting the distributions of experimental values using linear combinations of simulated distributions. We demonstrate the utility of our protocol by resolving the diffusive states of a protein that exhibits different diffusive states upon forming homo- and hetero-oligomeric complexes in the cytosol of a bacterial pathogen.

3D single-molecule localization microscopy is utilized to probe the spatial positions and motion trajectories of fluorescently labeled proteins in living bacterial cells. The experimental and data analysis protocol described herein determines the prevalent diffusive behaviors of cytosolic proteins based on pooled single-molecule trajectories.

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Bioengineering

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

Biology

Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using the analysis of cell membrane receptor clusters as a model, we present a detailed protocol for this image analysis task using Fiji (ImageJ) and Matlab routines to: 1) define regions of interest and design masks adapted to these regions; 2) track the particles in fluorescence microscopy videos; 3) analyze the diffusion and intensity characteristics of selected tracks. The quantitative analysis of the diffusion coefficients, types of motion, and cluster size obtained by fluorescence microscopy and image processing provides a valuable tool to objectively determine particle dynamics and the consequences of modifying environmental conditions. In this article we present detailed protocols for the analysis of these features. The method described here not only allows single-molecule tracking detection, but also automates the estimation of lateral diffusion parameters at the cell membrane, classifies the type of trajectory and allows complete analysis thus overcoming the difficulties in quantifying spot size over its entire trajectory at the cell membrane.

Here, we present a protocol for single particle tracking image analysis that allows quantitative evaluation of diffusion coefficients, types of motion and cluster sizes of single particles detected by fluorescence microscopy.

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using ...

Immunology and Infection

Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)1&#160;was developed to enable routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely available code to measure diffusion coefficients of proteins. kICS calculates a time correlation function from a fluorescence microscopy image stack after Fourier transformation of each image to reciprocal (k-) space. Subsequently, circular averaging, natural logarithm transform and linear fits to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS.
First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope. Then, a region of interest (ROI) avoiding intracellular organelles, moving vesicles or protruding membrane regions is selected. The ROI stack is imported into a freely available code and several defined parameters (see Method section) are set for kICS analysis. The program then generates a &#34;slope of slopes&#34; plot from the k-space time correlation functions, and the diffusion coefficient is calculated from the slope of the plot. Below is a step-by-step kICS procedure to measure the diffusion coefficient of a membrane protein using the renal water channel aquaporin-3 tagged with EGFP as a canonical example.

This paper provides a step by step guide to the fluctuation analysis technique k-Space Image Correlation Spectroscopy (kICS) for measuring diffusion coefficients of fluorescently labeled plasma membrane proteins in live mammalian cells.

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new ...

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed.
The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates.
High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results.
The chip technology is currently based on SiO2 surfaces, but further functionalization using gold-coated chip surfaces is also possible.

The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable ...

Probing Structural and Dynamic Properties of Trafficking Subcellular Nanostructures by Spatiotemporal Fluctuation Spectroscopy

Imaging-derived mean square displacement (iMSD) is used to address the structural and dynamic properties of subcellular nanostructures, such as vesicles involved in the endo/exocytotic trafficking of solutes and biomolecules. iMSD relies on standard time-lapse imaging, is compatible with any optical setup, and does not need to dwell on single objects to extract trajectories. From each iMSD trace, a unique triplet of average structural and dynamic parameters (i.e., size, local diffusivity, anomalous coefficient) is calculated and combined to build the "iMSD signature" of the nanostructure under study. 
The potency of this approach is proved here with the exemplary case of macropinosomes. These vesicles evolve in time, changing their average size, number, and dynamic properties passing from early to late stages of intracellular trafficking. As a control, insulin secretory granules (ISGs) are used as a reference for subcellular structures that live in a stationary state in which the average structural and dynamic properties of the whole population of objects are invariant in time. The iMSD analysis highlights these peculiar features quantitatively and paves the way to similar applications at the sub-cellular level, both in the physiological and pathological states.

Imaging-derived mean square displacement (iMSD) analysis is applied to macropinosomes to highlight their intrinsic time-evolving nature in terms of structural and dynamic properties. Macropinosomes are then compared to insulin secretory granules (ISGs) as a reference for subcellular structures with time-invariant average structural/dynamic properties.

Imaging-derived mean square displacement (iMSD) is used to address the structural and dynamic properties of subcellular nanostructures, such as vesicles ...

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

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