scholarly journals High photon count rates improve the quality of super-resolution fluorescence fluctuation spectroscopy

2019 ◽  
Author(s):  
Falk Schneider ◽  
Pablo Hernandez-Varas ◽  
B. Christoffer Lagerholm ◽  
Dilip Shrestha ◽  
Erdinc Sezgin ◽  
...  
Keyword(s):  
Data Quality ◽  
Super Resolution ◽  
Correlation Spectroscopy ◽  
Stimulated Emission ◽  
Three Dimensions ◽  
High Count ◽  
Photon Count ◽  
Diffusion Dynamics ◽  
Wide Range

AbstractProbing the diffusion of molecules has become a routine measurement across the life sciences, chemistry and physics. It provides valuable insights into reaction dynamics, oligomerisation, molecular (re-)organisation or cellular heterogeneities. Fluorescence correlation spectroscopy (FCS) is one of the widely applied techniques to determine diffusion dynamics in two and three dimensions. This technique relies on the temporal autocorrelation of intensity fluctuations but recording these fluctuations has thus far been limited by the detection electronics, which could not efficiently and accurately time-tag photons at high count rates. This has until now restricted the range of measurable dye concentrations, as well as the data quality of the FCS recordings, especially in combination with super-resolution stimulated emission depletion (STED) nanoscopy.Here, we investigate the applicability and reliability of (STED-)FCS at high photon count rates (average intensities of up to 40 MHz) using novel detection equipment, namely hybrid detectors and real-time gigahertz sampling of the photon streams implemented on a commercial microscope. By measuring the diffusion of fluorophores in solution and cytoplasm of live cells, as well as in model and cellular membranes, we show that accurate diffusion and concentration measurements are possible in these previously inaccessible high photon count regimes. Specifically, it offers much greater flexibility of experiments with biological samples with highly variable intensity, e.g. due to a wide range of expression levels of fluorescent proteins. In this context, we highlight the independence of diffusion properties of cytosolic GFP in a concentration range of approx. 0.01–1 μM. We further show that higher photon count rates also allow for much shorter acquisition times, and improved data quality. Finally, this approach also pronouncedly increases the robustness of challenging live cell STED-FCS measurements of nanoscale diffusion dynamics, which we testify by confirming a free diffusion pattern for a fluorescent lipid analogue on the apical membrane of adherent cells.

scholarly journals Background reduction in STED-FCS using coherent-hybrid STED

2020 ◽  
Author(s):  
Aurélien Barbotin ◽  
Iztok Urbančič ◽  
Silvia Galiani ◽  
Christian Eggeling ◽  
Martin Booth
Keyword(s):  
Diffusion Processes ◽  
Super Resolution ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Stimulated Emission ◽  
Three Dimensions ◽  
Signal Quality ◽  
Optical Aberrations ◽  
Observation Volume ◽  
Supramolecular Arrangements

AbstractFluorescence correlation spectroscopy (FCS) is a valuable tool to study the molecular dynamics of living cells. When used together with a super-resolution stimulated emission depletion (STED) microscope, STED-FCS can measure diffusion processes at the nanoscale in living cells. In twodimensional (2D) systems like the cellular plasma membrane, a ring-shaped depletion focus is most commonly used to increase the lateral resolution, leading to more than 25-fold decrease in the observation volumee, reaching the relevant scale of supramolecular arrangements. However, STED-FCS faces severe limitations when measuring diffusion in three dimensions (3D), largely due to the spurious background contributions from undepleted areas of the excitation focus that reduce the signal quality and ultimately limit the resolution. In this paper, we investigate how different STED confinement modes can mitigate this issue. By simulations as well as experiments with fluorescent probes in solution and in cells, we demonstrate that the coherent-hybrid (CH) depletion pattern reduces background most efficiently and thus provides superior signal quality under comparable reduction of the observation volume. Featuring also the highest robustness to common optical aberrations, CH-STED can be considered the method of choice for reliable STED-FCS based investigations of 3D diffusion on the sub-diffraction scale.

Super-resolution optical microscopy of lipid plasma membrane dynamics

2015 ◽  
Vol 57 ◽  
pp. 69-80 ◽  
Author(s):  
Christian Eggeling
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Molecular Diffusion ◽  
Super Resolution ◽  
Optical Microscope ◽  
Membrane Dynamics ◽  
Correlation Spectroscopy ◽  
Stimulated Emission ◽  
Diffusion Dynamics ◽  
Lipid Protein Interactions

Plasma membrane dynamics are an important ruler of cellular activity, particularly through the interaction and diffusion dynamics of membrane-embedded proteins and lipids. FCS (fluorescence correlation spectroscopy) on an optical (confocal) microscope is a popular tool for investigating such dynamics. Unfortunately, its full applicability is constrained by the limited spatial resolution of a conventional optical microscope. The present chapter depicts the combination of optical super-resolution STED (stimulated emission depletion) microscopy with FCS, and why it is an important tool for investigating molecular membrane dynamics in living cells. Compared with conventional FCS, the STED-FCS approach demonstrates an improved possibility to distinguish free from anomalous molecular diffusion, and thus to give new insights into lipid–protein interactions and the traditional lipid ‘raft’ theory.

scholarly journals The cortical actin network regulates avidity-dependent binding of hyaluronan by the lymphatic vessel endothelial receptor LYVE-1

2020 ◽  
Vol 295 (15) ◽  
pp. 5036-5050 ◽  
Author(s):  
Tess A. Stanly ◽  
Marco Fritzsche ◽  
Suneale Banerji ◽  
Dilip Shrestha ◽  
Falk Schneider ◽  
...  
Keyword(s):  
Lymphatic Vessel ◽  
Leukocyte Adhesion ◽  
Lymphatic Vessels ◽  
Super Resolution ◽  
Direct Interaction ◽  
Correlation Spectroscopy ◽  
Stimulated Emission ◽  
Actin Network ◽  
Cortical Actin ◽  
Limited Mobility

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) mediates the docking and entry of dendritic cells to lymphatic vessels through selective adhesion to its ligand hyaluronan in the leukocyte surface glycocalyx. To bind hyaluronan efficiently, LYVE-1 must undergo surface clustering, a process that is induced efficiently by the large cross-linked assemblages of glycosaminoglycan present within leukocyte pericellular matrices but is induced poorly by the shorter polymer alone. These properties suggested that LYVE-1 may have limited mobility in the endothelial plasma membrane, but no biophysical investigation of these parameters has been carried out to date. Here, using super-resolution fluorescence microscopy and spectroscopy combined with biochemical analyses of the receptor in primary lymphatic endothelial cells, we provide the first evidence that LYVE-1 dynamics are indeed restricted by the submembranous actin network. We show that actin disruption not only increases LYVE-1 lateral diffusion but also enhances hyaluronan-binding activity. However, unlike the related leukocyte HA receptor CD44, which uses ERM and ankyrin motifs within its cytoplasmic tail to bind actin, LYVE-1 displays little if any direct interaction with actin, as determined by co-immunoprecipitation. Instead, as shown by super-resolution stimulated emission depletion microscopy in combination with fluorescence correlation spectroscopy, LYVE-1 diffusion is restricted by transient entrapment within submembranous actin corrals. These results point to an actin-mediated constraint on LYVE-1 clustering in lymphatic endothelium that tunes the receptor for selective engagement with hyaluronan assemblages in the glycocalyx that are large enough to cross-bridge the corral-bound LYVE-1 molecules and thereby facilitate leukocyte adhesion and transmigration.

scholarly journals Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5

2021 ◽  
Author(s):  
Silvia Galiani ◽  
Katharina Reglinski ◽  
Pablo Carravilla ◽  
Aurelien Barbotin ◽  
Iztok Urbančič ◽  
...  
Keyword(s):  
Molecular Interactions ◽  
Super Resolution ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Interaction Dynamics ◽  
Diffusion Dynamics ◽  
Interaction Partners ◽  
Target Binding ◽  
Import Receptor

Measuring diffusion dynamics in living cells is essential for the understanding of molecular interactions. While various techniques have been used to explore such characteristics in the plasma membrane, this is less developed for measurements inside the cytosol. An example of cytosolic action is the import of proteins into peroxisomes, via the peroxisomal import receptor PEX5. Here, we combined advanced microscopy and spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and super-resolution STED microscopy to present a detailed characterization of the diffusion and interaction dynamics of PEX5. Among other features, we disclose a slow diffusion of PEX5, independent of aggregation or target binding, but associated with cytosolic interaction partners via its N-terminal domain. This sheds new light on the functionality of the receptor in the cytosol. Besides specific insights, our study highlights the potential of using complementary microscopy tools to decipher molecular interactions in the cytosol via studying their diffusion dynamics.

scholarly journals Adaptive optics allows 3D STED-FCS measurements in the cytoplasm of living cells

2019 ◽  
Author(s):  
Aurélien Barbotin ◽  
Silvia Galiani ◽  
Iztok Urbančič ◽  
Christian Eggeling ◽  
Martin Booth
Keyword(s):  
Adaptive Optics ◽  
Molecular Diffusion ◽  
Three Dimensional ◽  
Super Resolution ◽  
Correction Method ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Stimulated Emission ◽  
Optical Aberrations ◽  
Extreme Sensitivity

Fluorescence correlation spectroscopy in combination with super-resolution stimulated emission depletion microscopy (STED-FCS) is a powerful tool to investigate molecular diffusion with sub-diffraction resolution. It has been of particular use for investigations of two dimensional systems like cell membranes, but has so far seen very limited applications to studies of three-dimensional diffusion. One reason for this is the extreme sensitivity of the axial (3D) STED depletion pattern to optical aberrations. We present here an adaptive optics-based correction method that compensates for these aberrations and allows STED-FCS measurements in the cytoplasm of living cells.

scholarly journals Evaluation of sted super-resolution image quality by image correlation spectroscopy (QuICS)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Elena Cerutti ◽  
Morgana D’Amico ◽  
Isotta Cainero ◽  
Gaetano Ivan Dellino ◽  
Mario Faretta ◽  
...  
Keyword(s):  
Autocorrelation Function ◽  
Signal To Noise Ratio ◽  
Super Resolution ◽  
Image Contrast ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Signal To Noise ◽  
Image Correlation Spectroscopy ◽  
Noise Ratio

AbstractQuantifying the imaging performances in an unbiased way is of outmost importance in super-resolution microscopy. Here, we describe an algorithm based on image correlation spectroscopy (ICS) that can be used to assess the quality of super-resolution images. The algorithm is based on the calculation of an autocorrelation function and provides three different parameters: the width of the autocorrelation function, related to the spatial resolution; the brightness, related to the image contrast; the relative noise variance, related to the signal-to-noise ratio of the image. We use this algorithm to evaluate the quality of stimulated emission depletion (STED) images of DNA replication foci in U937 cells acquired under different imaging conditions. Increasing the STED depletion power improves the resolution but may reduce the image contrast. Increasing the number of line averages improves the signal-to-noise ratio but facilitates the onset of photobleaching and subsequent reduction of the image contrast. Finally, we evaluate the performances of two different separation of photons by lifetime tuning (SPLIT) approaches: the method of tunable STED depletion power and the commercially available Leica Tau-STED. We find that SPLIT provides an efficient way to improve the resolution and contrast in STED microscopy.

scholarly journals Super-Resolution STED Microscopy-Based Mobility Studies of the Viral Env Protein at HIV-1 Assembly Sites of Fully Infected T-Cells

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 608
Author(s):  
Jakub Chojnacki ◽  
Christian Eggeling
Keyword(s):  
Molecular Dynamics ◽  
Plasma Membrane ◽  
Molecular Mobility ◽  
Virus Assembly ◽  
Methodological Approach ◽  
Super Resolution ◽  
Correlation Spectroscopy ◽  
Stimulated Emission ◽  
Model Systems ◽  
Hiv 1

The ongoing threat of human immunodeficiency virus (HIV-1) requires continued, detailed investigations of its replication cycle, especially when combined with the most physiologically relevant, fully infectious model systems. Here, we demonstrate the application of the combination of stimulated emission depletion (STED) super-resolution microscopy with beam-scanning fluorescence correlation spectroscopy (sSTED-FCS) as a powerful tool for the interrogation of the molecular dynamics of HIV-1 virus assembly on the cell plasma membrane in the context of a fully infectious virus. In this process, HIV-1 envelope glycoprotein (Env) becomes incorporated into the assembling virus by interacting with the nascent Gag structural protein lattice. Molecular dynamics measurements at these distinct cell surface sites require a guiding strategy, for which we have used a two-colour implementation of sSTED-FCS to simultaneously target individual HIV-1 assembly sites via the aggregated Gag signal. We then compare the molecular mobility of Env proteins at the inside and outside of the virus assembly area. Env mobility was shown to be highly reduced at the assembly sites, highlighting the distinct trapping of Env as well as the usefulness of our methodological approach to study the molecular mobility of specifically targeted sites at the plasma membrane, even under high-biosafety conditions.

Usability Data Quality

2006 ◽  
pp. 661-667 ◽  
Author(s):  
David R. Danielson
Keyword(s):  
Data Quality ◽  
Cost Effective ◽  
High Quality ◽  
Effective Work ◽  
Design Changes ◽  
Wide Range ◽  
Usability Data ◽  
The Right ◽  
Effective Design

A substantial portion of usability work involves the coordinated collection of data by a team of specialists with varied backgrounds, employing multiple collection methods, and observing users with a wide range of skills, work contexts, goals, and responsibilities. The desired result is an improved system design, and the means to that end are the successful detection of, and reaction to, real deficiencies in system usability that severely impact the quality of experience for a range of users. In the context of user-centered design processes, valid and reliable data from a representative user sample is simply not enough. High-quality usability data is not just representative of reality. It is useful. It is persuasive in the eyes of the right stakeholders. It results in verifiable improvements to the system for which it is intended to represent a deficiency. The data must be efficiently and effectively translated into development action items with appropriate priority levels, and it must result in effective work products downstream, leading to cost-effective design changes. The remainder of this article (a) briefly reviews basic usability data collection concepts, (b) examines the dimensions that make up high-quality usability data, and (c) suggests future trends in usability data quality research.

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