scholarly journals Quantification of protein mobility and associated reshuffling of cytoplasm during chemical fixation

2018 ◽  
Author(s):  
Jan Huebinger ◽  
Jessica Spindler ◽  
Kristin J. Holl ◽  
Björn Koos
Keyword(s):  
Technological Progress ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Cultured Cells ◽  
Chemical Fixation ◽  
Cellular Processes ◽  
Cytoplasmic Proteins ◽  
Resolution Imaging ◽  
Spatio Temporal ◽  
Key Parameter

AbstractTo understand cellular functionalities, it is essential to unravel spatio-temporal patterns of molecular distributions and interactions within living cells. The technological progress in fluorescence microscopy now allows in principle to measure these patterns with sufficient spatial resolution. However, high resolution imaging comes along with long acquisition times and high phototoxicity. Physiological live cell imaging is therefore often unfeasible and chemical fixation is employed. However, fixation methods have not been rigorously reviewed to preserve patterns at the resolution at which they can be nowadays imaged. A key parameter for this is the time span until fixation is completed. During this time, cells are under unphysiological conditions and patterns decay. We demonstrate here that formaldehyde fixation takes more than one hour for cytosolic proteins in cultured cells. Associated with this, we found a distinct displacement of proteins and lipids, including their loss from the cells. Other small aldehydes like glyoxal or acrolein showed inferior results. Fixations using glutaraldehyde were faster than four minutes and retained most cytoplasmic proteins. Surprisingly, autofluorescence produced by glutaraldehyde was almost completely antagonized by supplementary addition of formaldehyde without compromising fixation speed. These findings indicate, which cellular processes can actually be reliably imaged after a certain chemical fixation.

scholarly journals A general fluorescent light-up probe for staining and quantifying protein

2019 ◽  
Vol 6 (8) ◽  
pp. 190580 ◽  
Author(s):  
Jiawei Zou ◽  
Gangyi Chen ◽  
Feng Du ◽  
Yi Yuan ◽  
Xin Huang ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Visual Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fluorescent Light ◽  
Cell Penetration ◽  
Cellular Processes ◽  
Low Toxicity ◽  
Protein Rich

Proteins are the primary functional agents in all cellular processes, facilitating various functions such as enzymes and structure-forming or signal-transducing molecules. In this work, we report a fluorescent dye, PyMDI-Zn, which could specifically bind with proteins and provide a red-shifted fluorescent emission. The visual analysis of protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis could be realized in 5 min by using PyMDI-Zn as a light-up dye. Based on its cell penetration and low toxicity, PyMDI-Zn could also be applied to locate protein-rich regions and organelles in live cell imaging. Moreover, the direct protein quantitation can be realized based on PyMDI-Zn, providing a method of screening for food adulteration by nitrogen-rich compounds.

scholarly journals Live cell imaging of the hyperthermophilic archaeon Sulfolobus acidocaldarius identifies complementary roles for two ESCRTIII homologues in ensuring a robust and symmetric cell division

2020 ◽  
Author(s):  
Andre Arashiro Pulschen ◽  
Delyan R. Mutavchiev ◽  
Kim Nadine Sebastian ◽  
Jacques Roubinet ◽  
Marc Roubinet ◽  
...  
Keyword(s):  
Cell Division ◽  
Cell Biology ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Halophilic Archaea ◽  
Live Cell ◽  
Tight Coupling ◽  
Dna Compaction ◽  
Cellular Processes ◽  
Daughter Cells

Live-cell imaging has revolutionized our understanding of dynamic cellular processes in bacteria and eukaryotes. While similar techniques have recently been applied to the study of halophilic archaea, our ability to explore the cell biology of thermophilic archaea is limited, due to the technical challenges of imaging at high temperatures. Here, we report the construction of the Sulfoscope, a heated chamber that enables live-cell imaging on an inverted fluorescent microscope. Using this system combined with thermostable fluorescent probes, we were able to image Sulfolobus cells as they divide, revealing a tight coupling between changes in DNA compaction, segregation and cytokinesis. By imaging deletion mutants, we observe important differences in the function of the two ESCRTIII proteins recently implicated in cytokinesis. The loss of CdvB1 compromises cell division, causing occasional division failures and fusion of the two daughter cells, whereas the deletion of cdvB2 leads to a profound loss of division symmetry, generating daughter cells that vary widely in size and eventually generating ghost cells. These data indicate that DNA separation and cytokinesis are coordinated in Sulfolobus, as is the case in eukaryotes, and that two contractile ESCRTIII polymers perform distinct roles to ensure that Sulfolobus cells undergo a robust and symmetrical division. Taken together, the Sulfoscope has shown to provide a controlled high temperature environment, in which cell biology of Sulfolobus can be studied in unprecedent details.

scholarly journals Caught in the Act: Live-Cell Imaging of Plant Meiosis

2021 ◽  
Vol 12 ◽  
Author(s):  
Maria Ada Prusicki ◽  
Martina Balboni ◽  
Kostika Sofroni ◽  
Yuki Hamamura ◽  
Arp Schnittger
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Ex Vivo ◽  
Live Cell ◽  
Cellular Processes ◽  
Short Period ◽  
Molecular Forces ◽  
Cellular Components ◽  
Plant Meiosis ◽  
Imaging Conditions

Live-cell imaging is a powerful method to obtain insights into cellular processes, particularly with respect to their dynamics. This is especially true for meiosis, where chromosomes and other cellular components such as the cytoskeleton follow an elaborate choreography over a relatively short period of time. Making these dynamics visible expands understanding of the regulation of meiosis and its underlying molecular forces. However, the analysis of meiosis by live-cell imaging is challenging; specifically in plants, a temporally resolved understanding of chromosome segregation and recombination events is lacking. Recent advances in live-cell imaging now allow the analysis of meiotic events in plants in real time. These new microscopy methods rely on the generation of reporter lines for meiotic regulators and on the establishment of ex vivo culture and imaging conditions, which stabilize the specimen and keep it alive for several hours or even days. In this review, we combine an overview of the technical aspects of live-cell imaging in plants with a summary of outstanding questions that can now be addressed to promote live-cell imaging in Arabidopsis and other plant species and stimulate ideas on the topics that can be addressed in the context of plant meiotic recombination.

scholarly journals Archaeal imaging: leading the hunt for new discoveries

2018 ◽  
Vol 29 (14) ◽  
pp. 1675-1681 ◽  
Author(s):  
Alexandre W. Bisson-Filho ◽  
Jenny Zheng ◽  
Ethan Garner
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Structural Information ◽  
Eukaryotic Cell ◽  
Live Cell ◽  
Biological Processes ◽  
Cellular Processes ◽  
Archaeal Species ◽  
Eukaryotic Organisms ◽  
Cellular Components

Since the identification of the archaeal domain in the mid-1970s, we have collected a great deal of metagenomic, biochemical, and structural information from archaeal species. However, there is still little known about how archaeal cells organize their internal cellular components in space and time. In contrast, live-cell imaging has allowed bacterial and eukaryotic cell biologists to learn a lot about biological processes by observing the motions of cells, the dynamics of their internal organelles, and even the motions of single molecules. The explosion of knowledge gained via live-cell imaging in prokaryotes and eukaryotes has motivated an ever-improving set of imaging technologies that could allow analogous explorations into archaeal biology. Furthermore, previous studies of essential biological processes in prokaryotic and eukaryotic organisms give methodological roadmaps for the investigation of similar processes in archaea. In this perspective, we highlight a few fundamental cellular processes in archaea, reviewing our current state of understanding about each, and compare how imaging approaches helped to advance the study of similar processes in bacteria and eukaryotes.

scholarly journals Quantitative live-cell imaging reveals spatio-temporal dynamics and cytoplasmic assembly of the 26S proteasome

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Chan-Gi Pack ◽  
Haruka Yukii ◽  
Akio Toh-e ◽  
Tai Kudo ◽  
Hikaru Tsuchiya ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Temporal Dynamics ◽  
Live Cell ◽  
26S Proteasome ◽  
Spatio Temporal ◽  
Cytoplasmic Assembly

scholarly journals Low-Power Two-Color Stimulated Emission Depletion Microscopy for Live Cell Imaging

Biosensors ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 330
Author(s):  
Jia Zhang ◽  
Xinwei Gao ◽  
Luwei Wang ◽  
Yong Guo ◽  
Yinru Zhu ◽  
...  
Keyword(s):  
Low Power ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Live Cell ◽  
Stimulated Emission ◽  
Live Cells ◽  
Sted Microscopy ◽  
Resolution Imaging ◽  
Stimulated Emission Depletion

Stimulated emission depletion (STED) microscopy is a typical laser-scanning super-resolution imaging technology, the emergence of which has opened a new research window for studying the dynamic processes of live biological samples on a nanometer scale. According to the characteristics of STED, a high depletion power is required to obtain a high resolution. However, a high laser power can induce severe phototoxicity and photobleaching, which limits the applications for live cell imaging, especially in two-color STED super-resolution imaging. Therefore, we developed a low-power two-color STED super-resolution microscope with a single supercontinuum white-light laser. Using this system, we achieved low-power two-color super-resolution imaging based on digital enhancement technology. Lateral resolutions of 109 and 78 nm were obtained for mitochondria and microtubules in live cells, respectively, with 0.8 mW depletion power. These results highlight the great potential of the novel digitally enhanced two-color STED microscopy for long-term dynamic imaging of live cells.

scholarly journals Spatio-temporal modeling and live-cell imaging of proteolysis in the 4D microenvironment of breast cancer

2019 ◽  
Vol 38 (3) ◽  
pp. 445-454 ◽  
Author(s):  
Kyungmin Ji ◽  
Mansoureh Sameni ◽  
Kingsley Osuala ◽  
Kamiar Moin ◽  
Raymond R. Mattingly ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Temporal Modeling ◽  
Spatio Temporal

scholarly journals The ‘super-resolution’ revolution

2009 ◽  
Vol 37 (5) ◽  
pp. 1042-1044 ◽  
Author(s):  
Ilan Davis
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Light Sheet ◽  
Live Cell ◽  
Stimulated Emission ◽  
Structured Illumination ◽  
Light Sheet Microscopy ◽  
Resolution Imaging ◽  
New Instruments

We are currently in the midst of an exciting revolution in microscopy. In many ways, this has been happening for several decades, but it is the rate of development of new methods that has increased recently. The last few years have seen an impressive proliferation of new instruments for imaging at higher resolution, imaging single molecules and faster and more sensitive multidimensional live cell imaging. These include light sheet microscopy, stimulated emission depletion, structured illumination and live cell imaging on the OMX (optical microscopy experimental) platform. However, new probes and image analysis methods have also been crucial for the development of these revolutionary methods.

Live Cell Super-Resolution Imaging with N-SIM

2012 ◽  
Vol 20 (4) ◽  
pp. 18-21
Author(s):  
Christopher B. O'Connell
Keyword(s):  
Light Microscope ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Live Cell ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Specific Proteins ◽  
Resolution Imaging ◽  
Patterned Illumination

The ability to visualize the distributions of specific proteins with a light microscope and fluorescent probes is largely responsible for our current understanding of cellular structure. A major limitation of this approach arises from the blurring effects of diffraction, which decreases resolution and limits the ability to obtain information at the nanoscale. There has been a tremendous drive to develop optical and computational methods that improve the resolution of the light microscope, and structured illumination microscopy (SIM) is one solution. This method uses patterned illumination to double both lateral and axial resolution. Nikon's N-SIM is a commercial system that integrates the most desirable features of light microscopy, specific labeling of molecules, and live cell imaging, with structured illumination. This provides the ability to achieve super resolution suitable for a range of biological applications.

scholarly journals A live-cell imaging system for visualizing the transport of Marburg virus nucleocapsid-like structures

2019 ◽  
Author(s):  
Yuki Takamatsu ◽  
Takeshi Noda ◽  
Stephan Becker
Keyword(s):  
Live Cell Imaging ◽  
Ebola Virus ◽  
Cell Imaging ◽  
Temporal Dynamics ◽  
Imaging System ◽  
Marburg Virus ◽  
Live Cell ◽  
Infected Cells ◽  
Spatio Temporal ◽  
Living Organisms

AbstractLive-cell imaging is a powerful tool for visualization of the spatio-temporal dynamics of living organisms. Although this technique is utilized to visualize nucleocapsid transport in Marburg virus (MARV)- or Ebola virus-infected cells, the experiments require biosafety level-4 (BSL-4) laboratories, which are restricted to trained and authorized individuals. To overcome this limitation, we developed a live-cell imaging system to visualize MARV nucleocapsid-like structures using fluorescence-conjugated viral proteins, which can be conducted outside BSL-4 laboratories. Our experiments revealed that nucleocapsid-like structures have similar transport characteristics to nucleocapsids observed in MARV-infected cells. This system provides a safe platform to evaluate antiviral drugs that inhibit MARV nucleocapsid transport.

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