scholarly journals A comparative study on the use of microscopy in pharmacology and cell biology research

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245795
Author(s):  
Agatha M. Reigoto ◽  
Sarah A. Andrade ◽  
Marianna C. R. R. Seixas ◽  
Manoel L. Costa ◽  
Claudia Mermelstein
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Biomedical Sciences ◽  
Bright Field ◽  
Optical And Electron Microscopy ◽  
Microscopy Techniques ◽  
The Impact

Microscopy is the main technique to visualize and study the structure and function of cells. The impact of optical and electron microscopy techniques is enormous in all fields of biomedical research. It is possible that different research areas rely on microscopy in diverse ways. Here, we analyzed comparatively the use of microscopy in pharmacology and cell biology, among other biomedical sciences fields. We collected data from articles published in several major journals in these fields. We analyzed the frequency of use of different optical and electron microscopy techniques: bright field, phase contrast, differential interference contrast, polarization, conventional fluorescence, confocal, live cell imaging, super resolution, transmission and scanning electron microscopy, and cryoelectron microscopy. Our analysis showed that the use of microscopy has a distinctive pattern in each research area, and that nearly half of the articles from pharmacology journals did not use any microscopy method, compared to the use of microscopy in almost all the articles from cell biology journals. The most frequent microscopy methods in all the journals in all areas were bright field and fluorescence (conventional and confocal). Again, the pattern of use was different: while the most used microscopy methods in pharmacology were bright field and conventional fluorescence, in cell biology the most used methods were conventional and confocal fluorescence, and live cell imaging. We observed that the combination of different microscopy techniques was more frequent in cell biology, with up to 6 methods in the same article. To correlate the use of microscopy with the research theme of each article, we analyzed the proportion of microscopy figures with the use of cell culture. We analyzed comparatively the vocabulary of each biomedical sciences field, by the identification of the most frequent words in the articles. The collection of data described here shows a vast difference in the use of microscopy among different fields of biomedical sciences. The data presented here could be valuable in other scientific and educational contexts.

scholarly journals Customizable live-cell imaging chambers for multimodal and multiplex fluorescence microscopy

2020 ◽  
Vol 98 (5) ◽  
pp. 612-623
Author(s):  
Adam Tepperman ◽  
David Jiao Zheng ◽  
Maria Abou Taka ◽  
Angela Vrieze ◽  
Austin Le Lam ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Live Cell ◽  
Imaging Modalities ◽  
Experimental Conditions ◽  
Microscopy Imaging ◽  
Glass Coverslip ◽  
Multiple Imaging ◽  
Microscopy Techniques

Using multiple imaging modalities while performing independent experiments in parallel can greatly enhance the throughput of microscopy-based research, but requires the provision of appropriate experimental conditions in a format that meets the optical requirements of the microscope. Although customized imaging chambers can meet these challenges, the difficulty of manufacturing custom chambers and the relatively high cost and design inflexibility of commercial chambers has limited the adoption of this approach. Herein, we demonstrate the use of 3D printing to produce inexpensive, customized, live-cell imaging chambers that are compatible with a range of imaging modalities, including super-resolution microscopy. In this approach, biocompatible plastics are used to print imaging chambers designed to meet the specific needs of an experiment, followed by adhesion of the printed chamber to a glass coverslip, producing a chamber that is impermeant to liquids and that supports the growth and imaging of cells over multiple days. This approach can also be used to produce moulds for casting microfluidic devices made of polydimethylsiloxane. The utility of these chambers is demonstrated using designs for multiplex microscopy, imaging under shear, chemotaxis, and general cellular imaging. Together, this approach represents an inexpensive yet highly customizable approach for producing imaging chambers that are compatible with modern microscopy techniques.

scholarly journals Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein

mSphere ◽  
2016 ◽  
Vol 1 (4) ◽  
Author(s):  
H. M. van der Schaar ◽  
C. E. Melia ◽  
J. A. C. van Bruggen ◽  
J. R. P. M. Strating ◽  
M. E. D. van Geenen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Lipid Composition ◽  
Viral Protein ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Living Cells ◽  
Live Cell ◽  
Genome Replication ◽  
Fluorescent Reporter ◽  
Unique Protein

ABSTRACT Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for genome replication. Electron microscopy has revealed the morphology of enterovirus ROs, and immunofluorescence studies have been conducted to investigate their origin and formation. Yet, immunofluorescence analysis of fixed cells results in a rather static view of RO formation, and the results may be compromised by immunolabeling artifacts. While live-cell imaging of ROs would be preferred, enteroviruses encoding a membrane-anchored viral protein fused to a large fluorescent reporter have thus far not been described. Here, we tackled this constraint by introducing a small tag from a split-GFP system into an RO-resident enterovirus protein. This new tool bridges a methodological gap by circumventing the need for immunolabeling fixed cells and allows the study of the dynamics and formation of enterovirus ROs in living cells. Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues. The GFP(S11) (GFP with S11 fragment) fragment was inserted into the 3A protein of the enterovirus coxsackievirus B3 (CVB3), while the large fragment was supplied by transient or stable expression in cells. The introduction of GFP(S11) did not affect the known functions of 3A when expressed in isolation. Using correlative light electron microscopy (CLEM), we showed that GFP fluorescence was detected at ROs, whose morphologies are essentially identical to those previously observed for wild-type CVB3, indicating that GFP(S11)-tagged 3A proteins assemble with GFP(S1-10) to form GFP for illumination of bona fide ROs. It is well established that enterovirus infection leads to Golgi disintegration. Through live-cell imaging of infected cells expressing an mCherry-tagged Golgi marker, we monitored RO development and revealed the dynamics of Golgi disassembly in real time. Having demonstrated the suitability of this virus for imaging ROs, we constructed a CVB3 encoding GFP(S1-10) and GFP(S11)-tagged 3A to bypass the need to express GFP(S1-10) prior to infection. These tools will have multiple applications in future studies on the origin, location, and function of enterovirus ROs. IMPORTANCE Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for genome replication. Electron microscopy has revealed the morphology of enterovirus ROs, and immunofluorescence studies have been conducted to investigate their origin and formation. Yet, immunofluorescence analysis of fixed cells results in a rather static view of RO formation, and the results may be compromised by immunolabeling artifacts. While live-cell imaging of ROs would be preferred, enteroviruses encoding a membrane-anchored viral protein fused to a large fluorescent reporter have thus far not been described. Here, we tackled this constraint by introducing a small tag from a split-GFP system into an RO-resident enterovirus protein. This new tool bridges a methodological gap by circumventing the need for immunolabeling fixed cells and allows the study of the dynamics and formation of enterovirus ROs in living cells.

scholarly journals A drug-compatible and temperature-controlled microfluidic device for live-cell imaging

Open Biology ◽  
2016 ◽  
Vol 6 (8) ◽  
pp. 160156 ◽  
Author(s):  
Tong Chen ◽  
Blanca Gomez-Escoda ◽  
Javier Munoz-Garcia ◽  
Julien Babic ◽  
Laurent Griscom ◽  
...  
Keyword(s):  
Cell Biology ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Critical Issue ◽  
Growth Conditions ◽  
Medium Composition ◽  
Live Cell ◽  
Model Systems ◽  
Dynamic Regulation ◽  
Cellular Environment

Monitoring cellular responses to changes in growth conditions and perturbation of targeted pathways is integral to the investigation of biological processes. However, manipulating cells and their environment during live-cell-imaging experiments still represents a major challenge. While the coupling of microfluidics with microscopy has emerged as a powerful solution to this problem, this approach remains severely underexploited. Indeed, most microdevices rely on the polymer polydimethylsiloxane (PDMS), which strongly absorbs a variety of molecules commonly used in cell biology. This effect of the microsystems on the cellular environment hampers our capacity to accurately modulate the composition of the medium and the concentration of specific compounds within the microchips, with implications for the reliability of these experiments. To overcome this critical issue, we developed new PDMS-free microdevices dedicated to live-cell imaging that show no interference with small molecules. They also integrate a module for maintaining precise sample temperature both above and below ambient as well as for rapid temperature shifts. Importantly, changes in medium composition and temperature can be efficiently achieved within the chips while recording cell behaviour by microscopy. Compatible with different model systems, our platforms provide a versatile solution for the dynamic regulation of the cellular environment during live-cell imaging.

scholarly journals From imaging to understanding: Frontiers in Live Cell Imaging, Bethesda, MD, April 19–21, 2006

2006 ◽  
Vol 174 (4) ◽  
pp. 481-484 ◽  
Author(s):  
Yu-li Wang ◽  
Klaus M. Hahn ◽  
Robert F. Murphy ◽  
Alan F. Horwitz
Keyword(s):  
Cell Biology ◽  
Live Cell Imaging ◽  
Recent Progress ◽  
Cell Imaging ◽  
Live Cell ◽  
The Other ◽  
Biological Applications ◽  
Research Fields ◽  
Recent Meeting ◽  
The One

A recent meeting entitled Frontiers in Live Cell Imaging was attended by more than 400 cell biologists, physicists, chemists, mathematicians, and engineers. Unlike typical special topics meetings, which bring together investigators in a defined field primarily to review recent progress, the purpose of this meeting was to promote cross-disciplinary interactions by introducing emerging methods on the one hand and important biological applications on the other. The goal was to turn live cell imaging from a “technique” used in cell biology into a new exploratory science that combines a number of research fields.

scholarly journals Traffic watch: Live-cell imaging

2003 ◽  
Vol 25 (3) ◽  
pp. 15-17
Author(s):  
David J. Stephens
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Living Cells ◽  
Robert Hooke ◽  
The Core ◽  
Biology Research ◽  
Insight Into ◽  
Dynamics Process

Microscopy has been at the core of cell biology research ever since the coining of the term ‘cell’ by Robert Hooke in the 17th Century1. For many years, it has been possible to gain insight into ‘steady-state’ cellular function from the analysis of fixed samples, but it is only relatively recently that imaging of living cells has become a widely used tool to support biochemical and electron microscopy studies. Membrane traffic research, which by its very nature is a highly dynamics process, has benefited hugely from the ability to image specific processes in living cells and tissues.

scholarly journals Sortase-Modified Cholera Toxoids Show Specific Golgi Localization

2019 ◽  
Author(s):  
Darren Machin ◽  
Daniel Williamson ◽  
Peter Fisher ◽  
victoria miller ◽  
Gemma Wildsmith ◽  
...  
Keyword(s):  
Lipid Rafts ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Neuronal Tracing ◽  
Future Role ◽  
Golgi Localization

Cholera toxoid is an established tool for use in cellular tracing in neuroscience and cell biology. We use a sortase-labelling approach to generate site-specifically <i>N</i>-terminally modified variants of both the A2-B<sub>5</sub> heterohexamer and B<sub>5</sub> pentamer forms of the toxoid. Both forms of the toxoid are endocytosed by GM1-positive mammalian cells, and while the heterohexameric toxoid was principally localized in the ER, the B<sub>5</sub> pentamer showed an unexpected localization in the <i>medial/trans</i> Golgi. This study suggests a future role for specifically-labelled cholera toxoids in live-cell imaging beyond their current applications in neuronal tracing and labelling of lipid-rafts in fixed cells.

scholarly journals Holotomography: refractive index as an intrinsic imaging contrast for 3-D label-free live cell imaging

2017 ◽  
Author(s):  
Doyeon Kim ◽  
SangYun Lee ◽  
Moosung Lee ◽  
JunTaek Oh ◽  
Su-A Yang ◽  
...  
Keyword(s):  
Refractive Index ◽  
Cell Biology ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Imaging Techniques ◽  
Live Cell ◽  
Label Free ◽  
Essential Information ◽  
Research Fields ◽  
Intrinsic Imaging

AbstractLive cell imaging provides essential information in the investigation of cell biology and related pathophysiology. Refractive index (RI) can serve as intrinsic optical imaging contrast for 3-D label-free and quantitative live cell imaging, and provide invaluable information to understand various dynamics of cells and tissues for the study of numerous fields. Recently significant advances have been made in imaging methods and analysis approaches utilizing RI, which are now being transferred to biological and medical research fields, providing novel approaches to investigate the pathophysiology of cells. To provide insight how RI can be used as an imaging contrast for imaging of biological specimens, here we provide the basic principle of RI-based imaging techniques and summarize recent progress on applications, ranging from microbiology, hematology, infectious diseases, hematology, and histopathology.

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