scholarly journals Dynamic nanoscale morphology of the ER surveyed by STED microscopy

2018 ◽  
Vol 218 (1) ◽  
pp. 83-96 ◽  
Author(s):  
Lena K. Schroeder ◽  
Andrew E.S. Barentine ◽  
Holly Merta ◽  
Sarah Schweighofer ◽  
Yongdeng Zhang ◽  
...  
Keyword(s):  
Living Cells ◽  
Stimulated Emission ◽  
Membrane Structures ◽  
Spatiotemporal Resolution ◽  
Sted Microscopy ◽  
Superresolution Microscopy ◽  
Structure And Dynamics ◽  
First Time ◽  
Nanoscale Dynamics ◽  
Conventional Light Microscopy

The endoplasmic reticulum (ER) is composed of interconnected membrane sheets and tubules. Superresolution microscopy recently revealed densely packed, rapidly moving ER tubules mistaken for sheets by conventional light microscopy, highlighting the importance of revisiting classical views of ER structure with high spatiotemporal resolution in living cells. In this study, we use live-cell stimulated emission depletion (STED) microscopy to survey the architecture of the ER at 50-nm resolution. We determine the nanoscale dimensions of ER tubules and sheets for the first time in living cells. We demonstrate that ER sheets contain highly dynamic, subdiffraction-sized holes, which we call nanoholes, that coexist with uniform sheet regions. Reticulon family members localize to curved edges of holes within sheets and are required for their formation. The luminal tether Climp63 and microtubule cytoskeleton modulate their nanoscale dynamics and organization. Thus, by providing the first quantitative analysis of ER membrane structure and dynamics at the nanoscale, our work reveals that the ER in living cells is not limited to uniform sheets and tubules; instead, we suggest the ER contains a continuum of membrane structures that includes dynamic nanoholes in sheets as well as clustered tubules.

scholarly journals The in vivo mechanics of the magnetotactic backbone as revealed by correlative FLIM-FRET and STED microscopy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Erika Günther ◽  
André Klauß ◽  
Mauricio Toro-Nahuelpan ◽  
Dirk Schüler ◽  
Carsten Hille ◽  
...  
Keyword(s):  
Magnetic Particles ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Stimulated Emission ◽  
Fluorescence Lifetime Imaging ◽  
Sted Microscopy

AbstractProtein interaction and protein imaging strongly benefit from the advancements in time-resolved and superresolution fluorescence microscopic techniques. However, the techniques were typically applied separately and ex vivo because of technical challenges and the absence of suitable fluorescent protein pairs. Here, we show correlative in vivo fluorescence lifetime imaging microscopy Förster resonance energy transfer (FLIM-FRET) and stimulated emission depletion (STED) microscopy to unravel protein mechanics and structure in living cells. We use magnetotactic bacteria as a model system where two proteins, MamJ and MamK, are used to assemble magnetic particles called magnetosomes. The filament polymerizes out of MamK and the magnetosomes are connected via the linker MamJ. Our system reveals that bacterial filamentous structures are more fragile than the connection of biomineralized particles to this filament. More importantly, we anticipate the technique to find wide applicability for the study and quantification of biological processes in living cells and at high resolution.

scholarly journals Blinking fluorescent probes for tubulin nanoscopy in living and fixed cells

2021 ◽  
Author(s):  
Ruta Gerasimaite ◽  
Jonas Bucevicius ◽  
Kamila A. Kiszka ◽  
Georgij Kostiuk ◽  
Tanja Koenen ◽  
...  
Keyword(s):  
Small Molecule ◽  
Single Molecule ◽  
Fluorescent Probes ◽  
Living Cells ◽  
Stimulated Emission ◽  
Sted Microscopy ◽  
Linker Length ◽  
Localisation Microscopy ◽  
Red Dye ◽  
Small Molecule Probe

Here we report a small molecule probe for single molecule localisation microscopy (SMLM) of tubulin in living and fixed cells. We explored a series of constructs composed of taxanes and spontaneously blinking far-red dye hydroxymethyl silicon-rhodamine (HMSiR). We found that the linker length profoundly affects the probe permeability and off-targeting. The best performing probe, HMSiR-tubulin, is composed of cabazitaxel and 6'-regioisomer of HMSiR bridged by a C6 linker. Microtubule diameters of <50 nm can be routinely measured in SMLM experiments on living and fixed cells. HMSiR-tubulin also performs well in 3D stimulated emission depletion (STED) microscopy, allowing a complementary use of both nanoscopy methods for investigating microtubule functions in living cells.

scholarly journals Dissecting tripartite synapses with STED microscopy

2014 ◽  
Vol 369 (1654) ◽  
pp. 20130597 ◽  
Author(s):  
Aude Panatier ◽  
Misa Arizono ◽  
U. Valentin Nägerl
Keyword(s):  
Imaging Modality ◽  
Stimulated Emission ◽  
Tripartite Synapse ◽  
Sted Microscopy ◽  
Superresolution Microscopy ◽  
Two Photon Microscopy ◽  
Synaptic Physiology ◽  
Dynamic Events ◽  
Neuronal Synapses ◽  
Synaptic Structures

The concept of the tripartite synapse reflects the important role that astrocytic processes are thought to play in the function and regulation of neuronal synapses in the mammalian nervous system. However, many basic aspects regarding the dynamic interplay between pre- and postsynaptic neuronal structures and their astrocytic partners remain to be explored. A major experimental hurdle has been the small physical size of the relevant glial and synaptic structures, leaving them largely out of reach for conventional light microscopic approaches such as confocal and two-photon microscopy. Hence, most of what we know about the organization of the tripartite synapse is based on electron microscopy, which does not lend itself to investigating dynamic events and which cannot be carried out in parallel with functional assays. The development and application of superresolution microscopy for neuron–glia research is opening up exciting experimental opportunities in this regard. In this paper, we provide a basic explanation of the theory and operation of stimulated emission depletion (STED) microscopy, outlining the potential of this recent superresolution imaging modality for advancing our understanding of the morpho-functional interactions between astrocytes and neurons that regulate synaptic physiology.

scholarly journals A photostable fluorescent marker for the superresolution live imaging of the dynamic structure of the mitochondrial cristae

2019 ◽  
Vol 116 (32) ◽  
pp. 15817-15822 ◽  
Author(s):  
Chenguang Wang ◽  
Masayasu Taki ◽  
Yoshikatsu Sato ◽  
Yasushi Tamura ◽  
Hideyuki Yaginuma ◽  
...  
Keyword(s):  
Dynamic Structure ◽  
Living Cells ◽  
Membrane Dynamics ◽  
Fluorescent Labeling ◽  
Spatiotemporal Resolution ◽  
Fluorescent Marker ◽  
Sted Microscopy ◽  
Fluorescent Markers ◽  
Enhanced Photostability ◽  
Single Mitochondrion

Stimulation emission depletion (STED) microscopy enables ultrastructural imaging of organelle dynamics with a high spatiotemporal resolution in living cells. For the visualization of the mitochondrial membrane dynamics in STED microscopy, rationally designed mitochondrial fluorescent markers with enhanced photostability are required. Herein, we report the development of a superphotostable fluorescent labeling reagent with long fluorescence lifetime, whose design is based on a structurally reinforced naphthophosphole fluorophore that is conjugated with an electron-donating diphenylamino group. The combination of long-lived fluorescence and superphotostable features of the fluorophore allowed us to selectively capture the ultrastructures of the mitochondrial cristae with a resolution of ∼60 nm when depleted at 660 nm. This chemical tool provides morphological information of the cristae, which has so far only been observed in fixed cells using electron microscopy. Moreover, this method gives information about the dynamic ultrastructures such as the intermembrane fusion in different mitochondria as well as the intercristae mergence in a single mitochondrion during the apoptosis-like mitochondrial swelling process.

Carbon dots derived from Fusobacterium nucleatum for intracellular determination of Fe3+ and bioimaging both in vitro and in vivo

2021 ◽  
Author(s):  
Lijuan Liu ◽  
Shengting Zhang ◽  
Xiaodan Zheng ◽  
Hongmei Li ◽  
Qi Chen ◽  
...  
Keyword(s):  
Carbon Dots ◽  
Fusobacterium Nucleatum ◽  
Living Cells ◽  
First Time ◽  
Fluorescent Carbon Dots ◽  
Fluorescent Carbon

Fusobacterium nucleatum has been employed for the first time to synthesize fluorescent carbon dots which could be applied for the determination of Fe3+ ions in living cells and bioimaging in vitro and in vivo with excellent biocompatibility.

scholarly journals Localization of CD4 and CCR5 in Living Cells

2003 ◽  
Vol 77 (8) ◽  
pp. 4985-4991 ◽  
Author(s):  
Carolyn M. Steffens ◽  
Thomas J. Hope
Keyword(s):  
Human Immunodeficiency Virus ◽  
High Resolution ◽  
Cell Membrane ◽  
Cell Surface ◽  
Fluorescent Microscopy ◽  
Living Cells ◽  
Stable Complex ◽  
Membrane Structures ◽  
Virus Fusion ◽  
Immunodeficiency Virus

ABSTRACT The events preceding human immunodeficiency virus fusion and entry are influenced by the concentration and distribution of receptor and coreceptor molecules on the cell surface. However, the extent to which these proteins colocalize with one another in the cell membrane remains unclear. Using high-resolution deconvolution fluorescent microscopy of living cells, we found that both CD4 and CCR5 accumulate in protruding membrane structures containing actin and ezrin. Although CD4 and CCR5 extensively colocalize in these structures, they do not exist in a stable complex.

Optobiochemistry: Genetically Encoded Control of Protein Activity by Light

2021 ◽  
Vol 90 (1) ◽  
Author(s):  
Jihye Seong ◽  
Michael Z. Lin
Keyword(s):  
Protein Function ◽  
Living Cells ◽  
Optical Methods ◽  
Annual Review ◽  
Publication Date ◽  
Protein Activity ◽  
Spatiotemporal Resolution ◽  
Protein Functions ◽  
Protein Classes ◽  
Control Protein

Optobiochemical control of protein activities allows the investigation of protein functions in living cells with high spatiotemporal resolution. Over the last two decades, numerous natural photosensory domains have been characterized and synthetic domains engineered and assembled into photoregulatory systems to control protein function with light.Here, we review the field of optobiochemistry, categorizing photosensory domains by chromophore, describing photoregulatory systems by mechanism of action, and discussing protein classes frequently investigated using optical methods. We also present examples of how spatial or temporal control of proteins in living cells has provided new insights not possible with traditional biochemical or cell biological techniques. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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.

Green/red pulsed vortex-beam oscillations in all-fiber lasers with visible-resonance gold nanorods

Nanoscale ◽  
2019 ◽  
Vol 11 (34) ◽  
pp. 15991-16000 ◽  
Author(s):  
Jinhai Zou ◽  
Zhe Kang ◽  
Rui Wang ◽  
Hongjian Wang ◽  
Jiaxing Liu ◽  
...  
Keyword(s):  
Solid State ◽  
Gold Nanorods ◽  
Fiber Lasers ◽  
Vortex Beam ◽  
Sted Microscopy ◽  
Visible Wavelength ◽  
Mode Division Multiplexing ◽  
First Time

We demonstrate visible-wavelength all-fiber pulsed vortex lasers for the first time that may serve as attractive alternatives to solid-state vortex lasers for a variety of applications, such as visible mode-division multiplexing and STED microscopy.

scholarly journals A Method to Generate Vector Beams with Adjustable Amplitude in the Focal Plane

2020 ◽  
Vol 10 (7) ◽  
pp. 2313 ◽  
Author(s):  
Alexandru Crăciun ◽  
Traian Dascălu
Keyword(s):  
Optical System ◽  
Focal Plane ◽  
Focal Point ◽  
Polarization State ◽  
Optical Component ◽  
Stimulated Emission ◽  
Matrix Formalism ◽  
Sted Microscopy ◽  
Diffraction Integral ◽  
Vector Beams

We design and investigate an original optical component made of a c-cut uniaxial crystal and an optical system to generate cylindrical vector beams with an adjustable polarization state. The original optical component has a specific, nearly conical shape which allows it to operate like a broadband wave retarder with the fast axis oriented radially with respect to the optical axis. We show via numerical simulations, using the Debye–Wolf diffraction integral, that the focal spot changes depending on the polarization state, thus enabling the control of the focal shape. Non-symmetrical shapes can be created although the optical system and incoming beam are circularly symmetric. We explained, using Jones matrix formalism, that this phenomenon is connected with the Gouy phase difference acquired by certain modes composing the beam due to propagation to the focal plane. We present our conclusions in the context of two potential applications, namely, stimulated emission depletion (STED) microscopy and laser micromachining. The optical system can potentially be used for STED microscopy for better control of the point-spread function of the microscope and to decrease the unwanted light emitted from the surroundings of the focal point. We give an analytical expression for the shape of the original component using the aspherical lens formula for the two versions of the component: one for each potential application.

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