Super-Resolution Three-Dimensional Imaging of Actin Filaments in Cultured Cells and the Brain via Expansion Microscopy

ACS Nano ◽  
2020 ◽  
Vol 14 (11) ◽  
pp. 14999-15010
Author(s):  
Chan E Park ◽  
Youngbin Cho ◽  
In Cho ◽  
Hyunsu Jung ◽  
Byeongyeon Kim ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Cultured Cells ◽  
Three Dimensional ◽  
Super Resolution ◽  
Three Dimensional Imaging ◽  
The Brain

Super Resolution Optic Three-dimensional Imaging Based on Compressed Sensing

2015 ◽  
Vol 44 (3) ◽  
pp. 328001
Author(s):  
王锋 WANG Feng ◽  
罗建军 LUO Jian-jun ◽  
唐兴佳 TANG Xing-jia ◽  
李立波 LI Li-bo ◽  
胡炳樑 HU Bin-liang
Keyword(s):  
Compressed Sensing ◽  
Three Dimensional ◽  
Super Resolution ◽  
Three Dimensional 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.

Function of spindle microtubules in directing cortical movement and actin filament organization in dividing cultured cells

1996 ◽  
Vol 109 (8) ◽  
pp. 2041-2051 ◽  
Author(s):  
D.J. Fishkind ◽  
J.D. Silverman ◽  
Y.L. Wang
Keyword(s):  
Cell Division ◽  
Actin Filaments ◽  
Cultured Cells ◽  
Three Dimensional ◽  
Linear Dichroism ◽  
Single Particle Tracking ◽  
Contractile Apparatus ◽  
Cortical Actin ◽  
Surface Receptors ◽  
Spindle Microtubules

The mitotic spindle has long been recognized to play an essential role in determining the position of the cleavage furrow during cell division, however little is known about the mechanisms involved in this process. One attractive hypothesis is that signals from the spindle may function to induce reorganization of cortical structures and transport of actin filaments to the equator during cytokinesis. While an important idea, few experiments have directly tested this model. In the present study, we have used a variety of experimental approaches to identify microtubule-dependent effects on key cortical events during normal cell cleavage, including cortical flow, reorientation of actin filaments, and formation of the contractile apparatus. Single-particle tracking experiments showed that the microtubule disrupting drug nocodazole induces an inhibition of the movements of cell surface receptors following anaphase onset, while the microtubule stabilizing drug taxol causes profound changes in the overall pattern of receptor movements. These effects were accompanied by a related set of changes in the organization of the actin cytoskeleton. In nocodazole-treated cells, the three-dimensional organization of cortical actin filaments appeared less ordered than in controls. Measurements with fluorescence-detected linear dichroism indicated a decrease in the alignment of filaments along the spindle axis. In contrast, actin filaments in taxol-treated cells showed an increased alignment along the equator on both the ventral and dorsal cortical surfaces, mirroring the redistribution pattern of surface receptors. Together, these experiments show that spindle microtubules are involved in directing bipolar flow of surface receptors and reorganization of actin filaments during cell division, thus acting as a stimulus for positioning cortical cytoskeletal components and organizing the contractile apparatus of dividing tissue culture cells.

scholarly journals Three-Dimensional Imaging of Nitric Oxide Production in the Rat Brain Subjected to Ischemia—Hypoxia

1995 ◽  
Vol 15 (6) ◽  
pp. 899-903 ◽  
Author(s):  
Periannan Kuppusamy ◽  
S. Tsuyoshi Ohnishi ◽  
Yoshihiro Numagami ◽  
Tomoko Ohnishi ◽  
Jay L. Zweier
Keyword(s):  
Nitric Oxide ◽  
Right Hemisphere ◽  
Three Dimensional ◽  
Paramagnetic Resonance ◽  
Three Dimensional Imaging ◽  
Electron Paramagnetic Resonance Imaging ◽  
The Right ◽  
Oxide Synthase ◽  
Electron Paramagnetic ◽  
The Brain

By the systemic administration of diethyldithiocarbamate and iron into the rat, nitric oxide radicals produced in the brain during ischemia–hypoxia were trapped. The right hemisphere of the brain was then removed and frozen with liquid nitrogen. With use of recently developed electron paramagnetic resonance imaging instrumentation and techniques, three-dimensional imaging of the production of the nitric oxide radicals in several brains was performed. The results suggest that nitric oxide radicals were produced and trapped in the areas that are known to have high nitric oxide synthase activity, such as cortex, hippocampus, hypothalamus, amygdala, and substantia nigra. In this ischemia–hypoxia model, which did not interrupt the posterior circulation, the production and trapping of nitric oxide in the cerebellum were ∼30% of those in the cerebrum.

scholarly journals Orientation and three-dimensional organization of actin filaments in dividing cultured cells.

1993 ◽  
Vol 123 (4) ◽  
pp. 837-848 ◽  
Author(s):  
D J Fishkind ◽  
Y L Wang
Keyword(s):  
Actin Filaments ◽  
Cultured Cells ◽  
Three Dimensional ◽  
Dorsal Surface ◽  
Ventral Surface ◽  
Equatorial Region ◽  
Optical Sectioning ◽  
Rhodamine Phalloidin ◽  
Anaphase Onset ◽  
Spindle Axis

The current hypothesis of cytokinesis suggests that contractile forces in the cleavage furrow are generated by a circumferential band of actin filaments. However, relatively little is known about the global organization of actin filaments in dividing cells. To approach this problem we have used fluorescence-detected linear dichroism (FDLD) microscopy to measure filament orientation, and digital optical sectioning microscopy to perform three-dimensional reconstructions of dividing NRK cells stained with rhodamine-phalloidin. During metaphase, actin filaments in the equatorial region show a slight orientation along the spindle axis, while those in adjacent regions appear to be randomly distributed. Upon anaphase onset and through cytokinesis, the filaments become oriented along the equator in the furrow region, and along the spindle axis in adjacent regions. The degree of orientation appears to be dependent on cell-cell and cell-substrate adhesions. By performing digital optical sectioning microscopy on a highly spread NRK subclone, we show that actin filaments organize as a largely isotropic cortical meshwork in metaphase cells and convert into an anisotropic network shortly after anaphase onset, becoming more organized as cytokinesis proceeds. The conversion is most dramatic on the adhering ventral surface which shows little or no cleavage activity, and results in the formation of large bundles along the equator. On the dorsal surface, where cleavage occurs actively, actin filaments remain isotropic, showing only subtle alignment late in cytokinesis. In addition, stereo imaging has led to the discovery of a novel set of filaments that are associated with the cortex and traverse through the cytoplasm. Together, these studies provide important insights into the process of actin remodeling during cell division and point to possible additional mechanisms for force generation.

3-D imaging of freeze-dried actin filaments in fibroblast using high-resolution cryo-SEM

1993 ◽  
Vol 51 ◽  
pp. 120-121
Author(s):  
Ya Chen ◽  
Alexander B. Verkhovsky ◽  
Gary G. Borisy
Keyword(s):  
Grain Size ◽  
High Resolution ◽  
Low Temperature ◽  
Actin Filaments ◽  
Cultured Cells ◽  
Metal Layer ◽  
Three Dimensional ◽  
Fine Grain ◽  
Freeze Dried ◽  
Temperature Scanning

A new approach to image the macromolecular structure of actin filaments from detergent-extracted cultured cells using high resolution low-temperature scanning electron microscopy (cryo-SEM) is described. An advantage of studying the cytoskeleton by SEM is its large depth of focus which makes it easy to reveal the three-dimensional relationship between the cytoskeleton filaments. Recently, procedures have been investigated for high resolution visualization of biological samples by SEM. These studies have demonstrated the importance of cryo preparation techniques for preserving the morphology of the specimen and advanced metal coating techniques for revealing its topography.Coating for HRSEM requires a metal layer that will not obscure fine details while still yielding adequate SE signal. Magnetron-sputter coating of the samples with an ultrathin layer of metal at low temperature provides finer grain size than coating at ambient temperature. Chromium has many properties that make it suitable as coating metal for cytoskeleton: it has fine grain size, low BSE yield due to its low Z number, and it provides adequate SE signal.

scholarly journals Three-dimensional imaging of the brain cavities in human embryos

1995 ◽  
Vol 5 (4) ◽  
pp. 228-232 ◽  
Author(s):  
H.-G. Blaas ◽  
S. H. Eik-Nes ◽  
T. Kiserud ◽  
S. Berg ◽  
B. Angelsen ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Human Embryos ◽  
Three Dimensional Imaging ◽  
The Brain
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