Microscope virus

This scanning electron microscope image shows SARS-CoV-2 the round blue objects emerging from the surface of cells cultured in the lab.

Viruses can not be viewed under standard light compound microscopes. A scanning electron microscope SEM scans a sample with a focused electron beam and acquires images with information about the samples' topography and composition. Scanning Electron Microscopes are widely used in nanotechnology, materials research, life sciences, semiconductor, raw materials and industry. A transmission electron microscope TEM uses beams of electrons transmitted through a specimen to form an image.

The specimen is usually an ultra-thin section less than nm thick. A transmission electron microscope captured this image of the SARS-CoV-2 coronavirus, which has a distinctive crown-like appearance. The World Health Organization declared the coronavirus outbreak a global emergency at the end of January. Here is everything we know about the virus , how it spreads, and what you can do to reduce your risk.

Be respectful, keep it civil and stay on topic. These viruses except for poxviruses may take any shape i. Some enveloped viruses have surface projections long enough to be clearly visualized e. Negative stain of an enveloped virus with clear surface projections influenza B virus. Courtesy of Frederick A. Murphy, CDC. Negative stain of an enveloped virus with such short surface projections that they are not often visible in negative stains rubella virus ; the nucleocapsids inside are not morphologically distinct.

Some particles are outlined by the stain, showing the surface of the virus arrow , and some are penetrated by the stain arrowhead allowing visualization of the interior of the virus.

Copyright Wiley-Liss, Inc. Negative stain of an enveloped virus with icosahedral nucleocapsid herpesvirus. The envelope has surface projections that are not readily visualized in clinical material. Courtesy of Erskine L. Palmer, CDC. Enveloped viruses have a nucleocapsid the nucleic acid held together by some structural proteins inside.

It can be spherical icosahedral Fig. Negative stain of a helical like a Slinky nucleocapsid of Nipah virus. Negative stain of a poxvirus particle where the surface is covered by short filaments. Naked viruses cut in thin section may be seen singly or sometimes in paracrystalline arrays Fig. They exit the cell by lysing it and are not usually seen attached to and budding from membranes, as are enveloped viruses. Thin section of a paracrystalline array of a naked DNA virus adenovirus in the nucleus of an infected cell.

Thin section of a naked RNA virus Nodamura virus produced in the cytoplasm, here seen in paracrystalline arrays. Bars, nm. Enveloped viruses are found in association with cellular membranes. They may bud through the nuclear membrane into the cytoplasm e. The location inside the cell and the type of membranes with which the virus is associated can be clues to identification. Thin section of an enveloped DNA virus herpesvirus. Nucleocapsids are produced in the nucleus small arrowheads ; they can bud out through the nuclear membrane large arrowhead to obtain their outer covering, or sometimes they make their way into the cytoplasm naked and then bud into cytoplasmic vesicles or out into extracellular space through the plasma membrane arrow.

Thin section of an RNA virus produced in the cytoplasm. SARS coronavirus particles arrows obtain their envelope by budding through the membranes of the endoplasmic reticulum.

A general rule of thumb for human viruses is that DNA viruses except poxviruses are constructed in the nucleus and RNA viruses are assembled in the cytoplasm. Enveloped DNA viruses can obtain their envelope by budding through the nuclear membrane or by making their way to the cytoplasm and budding into cytoplasmic vesicles or through the plasma membrane.

Naked DNA viruses may be seen in the cytoplasm after the cell's nucleus starts breaking down. If icosohedral viruses are seen in the nucleus, they contain DNA; if icosahedral viruses are seen in the cytoplasm, the next step in identification would be to look in the nuclei of cells to see if they are also present in the nucleus. RNA viruses are not found in the nucleus, with one exception; occasionally, helical paramyxovirus nucleocapsids but not whole enveloped virions may be seen in the nucleus.

Enveloped RNA viruses can obtain their outer membrane by budding into cytoplasmic vesicles or out of the plasma membrane. Many animal viruses conform to these patterns. Exceptions include poxvirus and iridovirus, a DNA virus seen in fish, frogs, and some insects, which multiplies in the cytoplasm. Viruses of lower forms do not conform to this generality of DNA virus replication in the nucleus. EM laboratories can receive different types of specimens for diagnostics. For example, a public health laboratory may deal mostly with viral cultures and human body fluids.

Hospital EM laboratories can monitor polyomavirus excretion in urine of bone marrow and kidney transplant patients by negative-staining EM as an indication of polyomavirus infection 39 , 40 , 61 , 82 , 83 , 88 and can distinguish it from adenovirus or other virus infections. EM results from fluid specimens are routinely reported to the attending physician in 1 to 2 h, and therapy can be immediately initiated e.

Also, numerous stool specimens from patients with gastroenteritis are received, as enteric viruses do not readily grow in cultures customarily maintained by virology laboratories. In bronchoalveolar lavage specimens, the yield of viruses from the fluid phase is generally low; for this reason, the exfoliated cells are pelleted out and embedded for thin sectioning.

Other samples received in a hospital setting include biopsy tissues, cells pelleted from fluids, and occasionally tissue cultures inoculated with clinical material. The turnaround time for thin-sectioned specimens is usually around 24 to 36 h, unless a rapid procedure is used in emergency situations.

In addition to detecting or identifying viruses, EM can sometimes elaborate ultrastructural differences in the morphologies of similar viruses. For example, differences in Marburg and Ebola viruses, both of which are in the filovirus family, have been demonstrated Marburg virus virions are shorter than those of Ebola virus, and their surface spikes differ. Further, those authors showed some similarities between nascent filovirus inclusions and proviral inclusions of paramyxoviruses. Beyond simply detecting viruses in clinical specimens, the study of virus effects on cells and tissues provides important information on which cells and organ systems are involved and how viruses cause disease.

For example, EM of monkeypox virus infection in cynomolgus monkeys determined that death was due to fibrinonecrotic bronchopneumonia and that systemic dissemination was via a monocytic cell-associated viremia. Guarnieri-like bodies were seen in epithelium of the oral mucosa, intestinal mucosa, and skin In a different example, a virus that causes hepatitis and splenomegaly in chickens was shown by EM to be a nonenveloped particle of 30 to 35 nm, similar to hepatitis E virus Genetic studies then indicated that this virus is related to but distinct from human hepatitis E virus.

This information is useful in comparison of the disease in animal models to that in humans. Besides clinical diagnostic use, EM is important in the study of ultrastructural features of viruses, which in turn is useful in elucidating the function of various viral components. Proteins on the surface of viruses are responsible for their attachment to and entry into cells 27 as well as for their ability to elicit an immune response Proteins in poxvirus have been altered genetically, and the effect of the alteration on the location inside virus factories in the cells has been studied by thin sectioning 53 ; detecting the association of these proteins with other proteins contributes to knowledge of antigenicity and hence vaccine production.

In a different example, a matrix protein was shown to be important in the transport of Ebola virus nucleocapsid proteins to the cell surface and their incorporation into enveloped virions. The budding mechanism of this virus has been studied by EM in order to shed light on ways to prevent budding with antiviral drugs Negative staining of subviral particles can elucidate the function of different proteins in constructing the capsid and holding it together. Scaffolding proteins, produced by isolating them on gels or by culturing viruses in the presence of inhibitors, were examined by EM, which showed how the portal complex, the ring-shaped structure at the vertices of herpesviruses, is assembled Earlier studies of reovirus proteins introduced into cells by poxvirus vectors, individually and in various combinations, found particles resembling reovirus cores by negative staining Cryo-EM and tomography have shown the viral structure of non-chemically fixed virus and provided three-dimensional information 59 , 77 , 86 , 91 Fig.

Even enveloped viruses with considerable pleomorphism, as well as their relationship to subcellular organelles, can be studied. A cell receptor for adenovirus has been demonstrated by cryo-EM, and this has implications in gene therapy Electron tomography. A Simian immunodeficiency virus viewed frozen hydrated and unstained in a cryo kV transmission electron microscope; glycoprotein spikes and the internal core are visible.

B Four 1-nm-thick slices from a tomogram. C Computer-generated three-dimensional reconstruction of one viral particle seen in panel B. Bars, 50 nm. Reprinted from reference Scanning EM can add valuable information concerning the exterior of infected cells. As another example, the three-dimensional appearance of the SARS coronavirus elucidated the trimeric structure of the to nm surface spikes Knowing how viruses attach to cellular structures and egress from the host sheds light on compounds that can prevent these processes.

The effects of a mutation event or antiviral agent on virus production can be detected and monitored by EM The more we know about virus replication, the more insight we have about methods for preventing replication by using drugs that alter these processes and for designing vaccines that will produce an effective immune response.

Another important use of EM is the identification of an unknown virus that has been isolated in tissue culture. Viral culture has been one of the gold standards for identification of unknown viruses; this allows for amplification of the virus to a titer that is detectable by EM, i. Even with many molecular tests available, EM is still important in cases where no probes are available.

Sometimes endogenous virus or contaminants can confuse the diagnosis of clinical inoculates. These extraneous viruses can prevent the growth of inoculated cultures or confuse diagnosis by producing cytopathogenic effects of their own.

For example, cultured monkey kidney cells occasionally are contaminated with simian virus 40, a polyomavirus, and if suspected, it may be detected by immunostaining. If not, simian virus 40, retroviruses, and others can be identified by thin sectioning of the culture and sometimes by negative staining of the culture medium 14 , 18 , 76 , Additionally, mycoplasmas may contaminate cell cultures and result in confusing cytopathogenic effects; they can be seen by EM around the outside of cells cut in thin section 38 , Because of its rapid turnaround time and its ability to detect the unknown and unsuspected organism, EM is on the front line in surveillance of agents in new outbreaks.

Guidelines for participation by EM laboratories have been mentioned above. In fluid samples, the concentration of viruses has to be high for detection. Liquids should routinely be ultracentrifuged, after first clarifying them at low speed, to pellet and concentrate viruses. Other concentration methods, including ultrafiltration, agar diffusion, pseudoreplica technique, and immunoaggregation have been reviewed 35 , In tissues, if the pathology is focal, a small biopsy specimen or a single EM block may miss the area of infection.

Embedding and cutting semithin sections of multiple EM blocks to examine for tissue pathology is one way to cover more area. It is also possible to examine large 1- to 2-cm tissue slicer sections of wet tissue by confocal microscopy to select areas of unusual tissue morphology inflammation, giant cells, necrosis, etc. Sample preparation is important in preserving recognizable viruses.

Sometimes specimens that are sent for EM examination have not been properly shipped or preserved, and this may cause distortion of viruses and cellular ultrastructure that may be confusing. Drying fluid specimens prior to negative staining distorts viruses.

In one report, parapoxvirus was mistaken for orthopoxvirus until another sample prepared from wet material was examined. This has implications in the surveillance of smallpox virus as a potential bioterrorist agent However, frequently, suboptimal specimen preservation can yield diagnostic results.

Some viruses e. Thus, the microscopist should be aware of the shipping and storage conditions and consider them in sample examination. Finally, there are numerous confusing cell organelles and artifacts that may be mistaken for viruses. Examples of cellular components and the viruses with which they can be confused include perichromatin granules parvovirus , improperly fixed chromatin paramyxovirus nucleocapsids , nuclear pores and neurosecretory granules herpesvirus , melanosomes poxvirus , and cilia influenza virus.

Many other examples have been described in the literature 17 , 56 , EM is still on the forefront of virus identification, particularly in cases where agents are unknown or unsuspected. It is a valuable technique in the surveillance of emerging diseases and potential bioterrorism agents. Finally, methods for treatment of or vaccination against viral diseases are being investigated through ultrastructural studies that elucidate both viral makeup and the relationship of viruses to the cells they infect.

We are grateful to David Howell and Sherif Zaki for critically reviewing the manuscript. We thank Sriram Subramaniam for providing Fig. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. Cynthia S. She has worked with several emerging viral diseases, such as those caused by hantavirus, Ebola virus, Nipah virus, and SARS coronavirus.

Her interests include diagnostic virology and the study of the replication and morphogenesis of viruses. She presently serves as secretary of the Southeastern Microscopy Society. Sara E. She is currently Professor of Pathology at Duke University Medical Center and Director of the Electron Microscopy Laboratory, where her facility maintains four electron microscopes three transmission electron microscopes and one scanning electron microscope with six highly skilled EM technologists.

Additionally, she has hosted scientists visiting from around the world to learn virology in her laboratory. Along with the coauthor of this paper, she coteaches a course at the CDC on diagnosing viruses by EM, particularly agents of potential bioterrorism. She served as president of the Microscopy Society of America. National Center for Biotechnology Information , U.

Journal List Clin Microbiol Rev v. Clin Microbiol Rev. Miller 2. Author information Copyright and License information Disclaimer. Phone: Fax: E-mail: vog. This article has been cited by other articles in PMC. Abstract Summary: Electron microscopy, considered by some to be an old technique, is still on the forefront of both clinical viral diagnoses and viral ultrastructure and pathogenesis studies. Negative Staining Negative staining is a rapid procedure used for viewing small particles, such as viruses, in fluids.

Thin Sectioning Thin sectioning is used for cells and tissues because they are too thick for the electron beam to penetrate whole. Immunolocalization Studies with viral antibodies and gold-labeled secondary antibodies have demonstrated the location inside cells of various viral proteins, which sheds light on how the proteins are assimilated.

Bioterrorism Agents EM is instrumental in the detection of poxviruses in clinical samples and can be used to differentiate variola virus, the causative agent of smallpox, from varicella-zoster virus, a herpesvirus that is the causative agent of chicken pox and shingles.

Cryo-EM and Tomography Rapid freezing of virus suspensions and examination of the vitrified samples permits examination of non-chemically altered structures 1. Appearance in negative stains. Open in a separate window. Appearance in thin sections. Diagnostics EM laboratories can receive different types of specimens for diagnostics. Pathology Beyond simply detecting viruses in clinical specimens, the study of virus effects on cells and tissues provides important information on which cells and organ systems are involved and how viruses cause disease.

Use in Detection of Viruses in Tissue Culture Another important use of EM is the identification of an unknown virus that has been isolated in tissue culture. Bioterrorism and Emerging Disease Surveillance Because of its rapid turnaround time and its ability to detect the unknown and unsuspected organism, EM is on the front line in surveillance of agents in new outbreaks.

Caveats In fluid samples, the concentration of viruses has to be high for detection. Acknowledgments We are grateful to David Howell and Sherif Zaki for critically reviewing the manuscript.

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Preliminary studies on the aetiological agent. Lancet i : Briquet, S. Immunolocalization studies of an antisense protein in HIVinfected cells and viral particles.

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Malaysia 62 : Cossart, Y. Field, B. Cant, and D. Immunofluorescence is one method of diagnosing and quantifying certain viral infections. The advancement of fluorescence labeling methods and microscopic instrumentation opens further possibilities to perform more refined studies on host-virus interactions, virus spreading, and virus replication, e.

Increased sensitivity, better resolution, and higher automation of those microscope systems provide the basis for screening applications that allow researchers to get a wealth of information on virus infected cells , e. Electron microscopy is often used to examine the ultrastructure and to identify certain viruses.

In particular, the correlation between light and electron microscopy CLEM can provide unique insights into the interactions between virus and host. For observation and maintenance purposes in cell culture, inverted light microscopes with a small footprint, LED fluorescence option, good ergonomics and high-quality optics for reliable digital documentation are essential tools.

Immunofluorescence opens up the rapid detection of viral agents with direct DFA or indirect fluorescence antibody IFA tests, including antibody test kits against herpes simplex HSV , Influenza A, other respiratory viruses and enteroviruses.

Automated boxed microscopes with integrated calibration, environmental control and fluorescence options are ideal for lab environments with high throughput demands, enabling fully automated 2D and 3D screening of cell cultures and tissues. Confocal microscopes give the virologist the option to investigate the details of cellular invasion in greater detail and prepare the respective sample for further investigation with immune electron microscopy.

Recent developments in scanning electron microscopy SEM have shown to meet the resolution and image quality requirements for virus studies.

The large field of view imaging mode in combination with correlative light microscopy and automated workflows saves valuable time in finding relevant viral spots and provides fast results , even in 3D. Examine unstained cells in phase contrast and GFP-labeled cells in fluorescence contrast quickly and efficiently. The inverted microscope is especially perfect for universal tasks in clinical routine such as observation and maintenance of your lab cell culture. With the integrated camera of Primovert HDcam you use your iPad and the free imaging App Labscope to discuss the monitor image together in the team.

Snap microscope images, annotate and create reports, and share them easily and wirelessly. A1 Choose from all standard contrasting techniques, including Differential Interference Contrast DIC , to investigate your cell cultures. Axio Vert.