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 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 3D super-resolution deep-tissue imaging in living mice

2019 ◽  
Author(s):  
Mary Grace M. Velasco ◽  
Mengyang Zhang ◽  
Jacopo Antonello ◽  
Peng Yuan ◽  
Edward S. Allgeyer ◽  
...  
Keyword(s):  
Adaptive Optics ◽  
Organic Dyes ◽  
3D Visualization ◽  
Water Immersion ◽  
Three Dimensional ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Tissue Imaging ◽  
Objective Lens ◽  
Working Distance

Stimulated emission depletion (STED) microscopy enables the three-dimensional (3D) visualization of dynamic nanoscale structures in living cells, offering unique insights into their organization. However, 3D-STED imaging deep inside biological tissue is obstructed by optical aberrations and light scattering. We present a STED system that overcomes these challenges. Through the combination of 2-photon excitation, adaptive optics, far-red emitting organic dyes, and a long-working distance water-immersion objective lens, our system achieves aberration-corrected 3D super-resolution imaging, which we demonstrate 164 µm deep in fixed mouse brain tissue and 76 µm deep in the brain of a living mouse.

scholarly journals Numerically enhanced adaptive optics-based 3D STED microscopy for deep-tissue super-resolved imaging

2019 ◽  
Author(s):  
Piotr Zdankowski ◽  
Maciej Trusiak ◽  
David McGloin ◽  
Jason R. Swedlow
Keyword(s):  
Adaptive Optics ◽  
Signal To Noise Ratio ◽  
Super Resolution ◽  
Thick Layer ◽  
Block Matching ◽  
Stimulated Emission ◽  
Optical Aberrations ◽  
Deep Tissue ◽  
Resolution Imaging ◽  
Induced Pluripotent

AbstractIn stimulated emission depletion (STED) nanoscopy, the major origin of decreased signal-to-noise ratio within images can be attributed to sample photobleaching and strong optical aberrations. This is due to STED utilising both a high power depletion laser (increasing risk of photodamage), while the depletion beam is very sensitive to sample-induced aberrations. Here we demonstrate a custom-built 3D STED microscope with automated aberration correction that is capable of 3D super-resolution imaging through thick, highly aberrating, tissue. We introduce and investigate image denoising by block-matching and collaborative filtering (BM3D) to numerically enhance fine object details otherwise mixed with noise. Numerical denoising provides an increase in the final effective resolution of the STED imaging of 31% using the well-established Fourier ring correlation metric. Experimental validation of the proposed method is achieved through super-resolved 3D imaging of axons in differentiated induced pluripotent stem cells growing under a 80µm thick layer of tissue with lateral and axial resolution of 256nm and 300nm, respectively.

scholarly journals Super-Resolution Imaging by Dual Iterative Structured Illumination Microscopy

2021 ◽  
Author(s):  
Anna Loeschberger ◽  
Yauheni Novikau ◽  
Ralf Netz ◽  
Marie-Christin Spindler ◽  
Ricardo Benavente ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Brain Slices ◽  
Super Resolution ◽  
Reconstruction Algorithm ◽  
Living Cells ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Spatiotemporal Resolution ◽  
Coated Pits ◽  
Resolution Imaging

Three-dimensional (3D) multicolor super-resolution imaging in the 50-100 nm range in fixed and living cells remains challenging. We extend the resolution of structured illumination microscopy (SIM) by an improved nonlinear iterative reconstruction algorithm that enables 3D multicolor imaging with improved spatiotemporal resolution at low illumination intensities. We demonstrate the performance of dual iterative SIM (diSIM) imaging cellular structures in fixed cells including synaptonemal complexes, clathrin coated pits and the actin cytoskeleton with lateral resolutions of 60-100 nm with standard fluorophores. Furthermore, we visualize dendritic spines in 70 micrometer thick brain slices with an axial resolution < 200 nm. Finally, we image dynamics of the endoplasmatic reticulum and microtubules in living cells with up to 255 frames/s.

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 PSF shaping using adaptive optics for three-dimensional single-molecule super-resolution imaging and tracking

2012 ◽  
Vol 20 (5) ◽  
pp. 4957 ◽  
Author(s):  
Ignacio Izeddin ◽  
Mohamed El Beheiry ◽  
Jordi Andilla ◽  
Daniel Ciepielewski ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Adaptive Optics ◽  
Single Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Resolution Imaging

scholarly journals POLArIS, a versatile probe for molecular orientation, revealed actin filaments associated with microtubule asters in early embryos

2021 ◽  
Vol 118 (11) ◽  
pp. e2019071118
Author(s):  
Ayana Sugizaki ◽  
Keisuke Sato ◽  
Kazuyoshi Chiba ◽  
Kenta Saito ◽  
Masahiko Kawagishi ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Molecular Orientation ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Super Resolution ◽  
Living Cells ◽  
Test Case ◽  
Polarization Microscopy ◽  
Universal Method ◽  
Early Embryos

Biomolecular assemblies govern the physiology of cells. Their function often depends on the changes in molecular arrangements of constituents, both in the positions and orientations. While recent advancements of fluorescence microscopy including super-resolution microscopy have enabled us to determine the positions of fluorophores with unprecedented accuracy, monitoring the orientation of fluorescently labeled molecules within living cells in real time is challenging. Fluorescence polarization microscopy (FPM) reports the orientation of emission dipoles and is therefore a promising solution. For imaging with FPM, target proteins need labeling with fluorescent probes in a sterically constrained manner, but because of difficulties in the rational three-dimensional design of protein connection, a universal method for constrained tagging with fluorophore was not available. Here, we report POLArIS, a genetically encoded and versatile probe for molecular orientation imaging. Instead of using a direct tagging approach, we used a recombinant binder connected to a fluorescent protein in a sterically constrained manner that can target specific biomolecules of interest by combining with phage display screening. As an initial test case, we developed POLArISact, which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArISact with FPM. In living starfish early embryos expressing POLArISact, we found actin filaments radially extending from centrosomes in association with microtubule asters during mitosis. By taking advantage of the genetically encoded nature, POLArIS can be used in a variety of living specimens, including whole bodies of developing embryos and animals, and also be expressed in a cell type/tissue specific manner.

scholarly journals Nano-scale size holes in ER sheets provide an alternative to tubules for highly-curved membranes

2017 ◽  
Author(s):  
Lena K. Schroeder ◽  
Andrew E. S. Barentine ◽  
Sarah Schweighofer ◽  
David Baddeley ◽  
Joerg Bewersdorf ◽  
...  
Keyword(s):  
Analytical Modeling ◽  
High Spatial Resolution ◽  
Super Resolution ◽  
Living Cells ◽  
Stimulated Emission ◽  
Fast Time ◽  
Nano Scale ◽  
Scale Size ◽  
Curved Membranes ◽  
Super Resolution Microscopy

AbstractThe endoplasmic reticulum (ER) is composed of interconnected membrane sheets and tubules. Super-resolution microscopy recently revealed densely packed, rapidly moving ER tubules, highlighting the importance of revisiting classical views of ER structure with high spatial resolution in living cells. Using live-cell Stimulated Emission Depletion (STED) microscopy, we show highly dynamic, subdiffraction-sized holes in ER sheets. Holes coexist with uniform sheet regions and are distinct from tubular ER structures. The curvature-stabilizing reticulon protein Rtn4 localizes to these holes and the ER luminal tether Climp63 controls their diameter and mobility. Analytical modeling demonstrates that holes in ER sheets can serve as reservoirs for curvature-stabilizing proteins to support ER tubule extension and retraction, thus providing an explanation for how the ER locally alters its morphology on fast time-scales.One Sentence SummaryDynamic nano-scale sized holes are prominent features of ER sheets that serve as reservoirs for curvature-stabilizing proteins to support ER tubule extension and retraction.

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