Which Type Of Microscope Should Be Used To View A Virus That Is 50 Nm In Size

Abstract

Despite beingness an fantabulous tool for investigating ultrastructure, scanning electron microscopy (SEM) is less frequently used than transmission electron microscopy for microbes such as viruses or bacteria. Here nosotros describe rapid methods that let SEM imaging of fully hydrated, unfixed microbes without using conventional sample preparation methods. We demonstrate improved ultrastructural preservation, with greatly reduced dehydration and shrinkage, for specimens including leaner and viruses such as Ebola virus using infiltration with ionic liquid on conducting filter substrates for SEM.

Introduction

In early on studies, electron microscopy was pivotal in helping to identify the causative agents of infectious diseases^ane. It is nevertheless an important technique that tin help to diagnose pathogens and in testing to identify microorganisms². Traditionally, negative staining for transmission electron microscopy (TEM) has been the "gold standard" for imaging microbial samples, for example in diagnostic virology³. However, negative-stain TEM requires an adequate concentration of bacterial cells or virus particles, since these are adsorbed to a thin back up movie. Thus microbes need to be grown to a high tire and/or concentrated by centrifugation, which is frequently not possible with patient specimens or agents that are not culturable. Equally a result, electron microscopy has historically suffered from depression test sensitivity for many types of microbiological investigations. The detection of agents such as poxviruses or polyoma viruses in patient specimens usually requires a minimum concentration of between 10⁵ to 10⁶ particles/ml for TEM^4,5. Past comparison, the level of detection of viruses using civilisation or nucleic acid testing usually ranges between one and 50 particles per assay⁶. The recent evolution of filtration techniques show that both TEM and SEM identification of viruses can exist carried out with equally piddling as 5000 total particles per sample⁷. Moreover, electron microscopy is useful for identifying the type of microbe present, often to genus, allowing the selection of more than specific tests (for instance primers or specific antibodies) to fully place the agents present. Electron microscopy is thus an platonic "catch all" method giving an "open up view" for situations where a novel or emerging pathogen is being investigated where in that location is no a priori cognition of the type of agent present⁶.

The scanning electron microscope (SEM) can also be useful to reveal morphological features of isolated organisms also as for diagnosis, but difficulty with specimen grooming methods have in the by express the use of SEM for routine microbiology^seven,viii. Nowadays extremely loftier quality polycarbonate filters are available: the optimum pore size can be selected to collect any virus or bacterial species (the pores tin be as pocket-sized equally x nm, less than the smallest viruses). These filters are suitable for surface observation of viruses and bacteria by SEM⁷. 2 main bug occur with obtaining high resolution SEM images of microbes. Firstly, in club to get adequate contrast and to reduce charging for small organic particles such equally bacteria and viruses at magnifications greater than 1000 ten, a conducting surface is needed. Secondly, biological specimens take traditionally needed to be dehydrated for the best imaging functioning in the SEM. If a wet specimen is placed in the microscope, performance under high vacuum conditions tends to dry the specimen out speedily. Both of these factors compromise microscope performance and can reduce contrast and resolution. During SEM observation drying is a problem and ordinarily causes plummet, shrinkage and distortion of the specimen, fifty-fifty after preservation by chemical fixation. Previously, a variety of methods have been developed to dehydrate specimens prior to SEM observation, using solvents, sometimes in conjunction with disquisitional bespeak drying, or by freeze drying. Alternatively, methods to image specimens in the hydrated state take been used, employing "wet-SEM"^{seven,ix,10,11}, environmental SEM¹², or cryo-techniques^13,14. Disquisitional indicate drying permitted conductive blanket of biological specimens, giving reduced charging also as improvements in dissimilarity, simply the specimens suffered from cracking artifacts and shrinkage of up to fifty%, while freeze-drying oft causes baloney and harm due to ice crystal formation¹⁵. Flash freezing or high pressure freezing is often used to reduce ice crystal formation in biological specimens. Prior to SEM observation these frozen hydrated samples can either be cryo-sectioned, or mounted whole on a cryo-stage: in which case ion axle milling can as well be used to investigate interior structure^thirteen,14. Cryo-TEM, a more avant-garde variant of this technique, can also be used to investigate frozen-vitrified samples in the TEM. This requires that specimens be maintained at temperatures beneath ~ −150 °C to remain in an amorphous state and avoid water ice crystal impairment. Cryo-TEM is platonic for the investigation of macromolecular structures including viruses, nonetheless cryo-TEM gives a relatively small field of view, requires a high concentration of virus (~i mg/ml) and most leaner are too thick for loftier resolution TEM imaging in a layer of ice, which tin obscure item^{sixteen,17,xviii}. Specialised "moisture-SEM" or "wet-TEM" specimen holders take also been developed for imaging of fully hydrated samples, but they require highly specialised equipment and piece of work in scanning-manual (STEM) fashion, and so are non directly comparable with SEM. These holders take a liquid chamber or fluid prison cell isolated from the vacuum by one or two electron-transparent windows: the demand for the electron beam to pass through a solid window will ever compromise resolution as compared to SEM, where the specimen surface is illuminated directly^9,ten,11. Some other approach for imaging moisture samples is known as environmental SEM (ESEM), where differential pumping allows the force per unit area around the sample to exist increased to 10–20 torr. Bacteria take been observed by ESEM, but features such as flagellae are non well resolved and drying still occurs at these higher pressures, so that ESEM is more often than not more useful for larger specimens observed below 1000 x magnification¹². The above mentioned techniques, while giving splendid results with the appropriate specimen, are less suited for microbial investigations, including detection and characterisation, where optimum resolution and a large field of view is required. They too require more circuitous, specialised and expensive equipment, such every bit microscopes with field emission illumination sources and/or cryogenic equipment, equally compared to a standard SEM instrument. The method presented here is relatively uncomplicated, quick to perform (~15 minutes) and can be used with whatsoever SEM including those with a standard thermionic tungsten filament. It can be performed on unfixed and fully hydrated specimens. Filtration as well allows investigations of specimens that take a low initial concentration of particles⁷.

To produce an electrically conductive surface for SEM, biological specimens are frequently coated using sparse film evaporation or sputtering of carbon or metal in a vacuum coater, which requires prior dehydration of the specimen. This coating process tin can obscure fine ultrastructural details, depending on the thickness of the layer deposited (unremarkably 2–twenty nm). These conventional procedures are difficult to carry out on typical microbiological specimens, which are usually suspensions of minor biological particles in water (<100 nm for most viruses, or in the sub-micrometre size range for many bacteria, fungi and parasites). An additional problem is that the microbes of involvement in patient specimens or environmental samples may be present in relatively depression concentrations, making ascertainment of them on a surface difficult.

In this report we describe methods for concentrating microbial suspensions for SEM observation on pre-coated filter substrates. We testify that, instead of sputter coating, an ionic liquid (1-butyl-iii-methylimidazolium tetrafluoroborate) diluted in water can be used to speedily infiltrate a microbiological SEM sample, forming an electron-lucent conductive surface, which prevents specimen charging and gives adept results with microbial specimens (Fig. 1). Ionic liquids are highly conductive salts that remain in the liquid state at room temperature and take a negligible vapour pressure (≤5 × 10⁻⁹ Torr). Nether the loftier vacuum atmospheric condition of a modern SEM (≤1 × 10⁻⁶ Torr) ionic liquids remain in a liquid state and do not evaporate during performance, while still being conductive^{19,20,21,22,23}. The well-nigh useful ionic liquids for applications in biological SEM have an electrical conductivity of around 100 mScm^−one, are electrochemically stable (having an electrochemical window of effectually 5.8 V), too as being h2o soluble and are hands synthesised²⁴. Ionic liquids with these properties take been previously demonstrated to requite SEM epitome contrast comparable to the use of metal and carbon coating when used with insulating specimens^nineteen,25. They take as well been used for macroscopic imaging of biological specimens, such equally seaweed, tissue cultured cells and condensed chromosomes^20,21,22. Conductive substrates such equally indium-tin oxide, aluminum foil, or metallic coated coverslips have been used to prevent charging²⁰, however, these materials are unsuitable for filtration for SEM investigations of microbes. We discovered that, for optimum results using ionic liquid with subcellular objects such equally viruses or bacterial flagellae, prior coating of polycarbonate filters with aluminium or gold was necessary. We did not detect any specimen drift when using ionic liquid stained biological specimens, since they were well supported by the conducting membrane used during the initial filtration process. The SPI-pore polycarbonate filters are hydrophilic and remain so after metal coating, making them an ideal substrate to work with hydrated biological samples. Ionic liquid staining can besides be performed within a biological safety chiffonier, providing a rapid and safe alternative to sputter coating when working with infectious samples, since vacuum coating equipment can cause aerosols and is non easily independent^20,21,22. We accept elegantly solved the problem of concentrating the sample and preventing charging, past metal coating of the filter substrate itself prior to applying the biological sample (Fig. 2). In the absenteeism of thin film coating of the samples, infiltration with ionic liquids was besides required to avoid charging. The results are comparable with the use of SEM with sputter coating and TEM using negative staining technique (Figs ane and ii: Supplementary Figs S1–S5). Ultra-filtration is an important step since it helps to remove droppings that can obscure the details of viruses or bacteria nowadays in biological samples. In the current report, nosotros demonstrate clear imaging of viruses and bacterial flagellae in uncoated SEM specimens, which previously required dehydration and sputter blanket to achieve, thus extending the resolution and range of microbial samples that can exist investigated with SEM.

The images of leaner stained with ionic liquid had a smoother surface topography than those that were dehydrated and sputter coated. Size measurements show that the dehydrated specimens shrank by approximately 10–xx per cent (Table one). Nosotros interpret the surface item on the dehydrated, sputter coated bacteria every bit wrinkling of the prison cell wall due to shrinkage, rather than ascertainment of additional features that are nowadays in vivo: these wrinkles are thus likely to exist artefacts due to drying. Bacterial flagellae were besides clearly visible with ionic liquid treatment on the conducting substrates (Fig. 1, Supplementary Fig. S3). These results were comparable to those nosotros observed with SEM-sputter coating and TEM-negative staining.

Table ane Width measurements of microbes.

Full size tabular array

Ionic liquid techniques tin also exist used safely with infectious pathogens, in a biologically contained SEM enclosure, allowing the characterisation of novel infectious agents in a status closer to their hydrated "native state" than by conventional sample preparation techniques⁸. In the case of this investigation our conventional protocol involved dehydration in ethanol serial, followed by air drying and metal blanket earlier SEM imaging. For the ionic liquid protocol, the biological sample had a drop of a 2.5% aqueous solution of 1-butyl-3-methylimidazolium tetrafluoroborate placed directly on it. After blotting to remove backlog fluid the moisture sample was then placed directly in the SEM. When dealing with infectious samples, an additional aldehyde fixation footstep is needed for the conventional procedure to avoid the risk of infectious aerosols that could be generated during the sputter coating process. This fixation pace is not required with the ionic liquid technique, since the sample can be processed in a biological containment hood and then be placed direct in a SEM in a biologically contained SEM enclosure⁸, for imaging in an unfixed, hydrated state, which is much closer to the native state of the organism. Microscopy complements conventional diagnostic tests that tin miss novel or variant strains^3,26,27,28 and can rapidly identify the type of organism present, guiding the pick of more than specific tests²⁹. All the same, electron microscopy usually requires a minimum concentration of particles for reliable identification of microbes. For viruses, this is between 10⁵ to x⁶ virus particles/mL^4,five. With filtration techniques, both TEM and SEM of viruses can be carried out with every bit niggling as 5000 particles per sample⁷.

Ionic liquid staining on pre-coated filters is widely applicable to any biological sample which can benefit from filtration to concentrate particles of interest. The use of metallic coated filters with more than one type of coating on different areas allows selection of whichever of these coatings gives the optimum results for observing a particular specimen or specific features and helps to salve time (Fig. 2a,e–k). For example, after ionic liquid staining bacterial flagellae appeared brighter against the Al-coated substrate, while on the Au-coated expanse of the filter the contrast is reversed and the flagellae appeared darker (Fig. 2h–k). Similarly the images of Ebola virus and Leptospira biflexa showed practiced quality topographic detail when imaged with an aluminum coated filter, but the biological material had less detail and appeared as a dark apartment silhouette when imaged on gold coated filters (Supplementary Figs S4 and S5). We propose that this is due to the higher secondary electron emission bespeak from Au as compared to Al. In this investigation we collected SEM images with the secondary electron detector, which is virtually commonly used for routine imaging with biological specimens. In SEM, the secondary electron emission coefficient (δ) is relatively constant regardless of atomic number. Yet an exception is with Au, for which δ is about twice that of Al and many other elements. The value of δ is also affected by beam energy: at twenty kV, δ is 0.1 for Al and 0.two for Au^thirty. By measuring the intensity in secondary electron images taken at 4 kV with both Al and Au in the same image (Fig. 2f), nosotros calculated the indicate from the Au to be 2.1 times the intensity of the Al, which is close to that expected theoretically. With the images recorded of specimens on the gold coated filters, we interpret the results as producing also much contrast from the background substrate, which tended to obscure fine details such equally flagellae which appear as a "silhouettes" on a bright background. Still, the ionic liquid infiltrated microbes and the aluminum coated filter, take similar emission coefficients, thus the contrast is largely due to the topography rather than the differences in fabric composition, allowing more than fine details to be seen.

Pre-coating of the filters with metal did non impact the pore sizes, or the filtration capacity of the filters (Fig. two). The entire ionic liquid staining protocol can exist carried out on a standard laboratory bench in almost 15 minutes and fits inside a biosafety cabinet (Fig. 2c,d). In sputter coating for SEM, too sparse a layer causes poor conduction and charging, while likewise thick a layer obscures fine details. The thickness used for pre-coating the substrates tin can be much greater than sputter coatings typically used for biological specimens, to ensure proficient conductivity, so long every bit the filter pores are not blocked. (Fig. 2e–k, Supplementary Fig. S2).

In this investigation we used coatings of xviii and 27 nm for Al and Au respectively, since these thicknesses were establish to be sufficient to prevent charging, as compared to uncoated filters (Supplementary Fig. S2). Substrates with these minimum thicknesses were easily selected since they were visible as a shiny metallic coating. When coatings of less than 27 nm for Au or 18 nm for the Al were nowadays, they had an opaque or apartment-white advent (Fig. ii). With ionic liquid staining using these metallic coated filters, nosotros were able to visualize the fine structural details of leaner, such every bit the flagellae of Salmonella, which are 20 nm in diameter, by SEM (Fig. 2j,m, Supplementary Fig. S3).

The results obtained with filtration and uncomplicated ionic liquid infiltration for SEM are very comparable in quality with those from conventional sputter blanket in SEM and negative staining in TEM for a multifariousness of bacterial and viral specimens, including Leptospira, Salmonella, vaccinia virus and Ebola virus (Fig. one, Supplementary Fig. S3–S5)^7,16,31,32. We found that at that place was much less shrinkage of the ionic liquid infiltrated bacteria and viruses as compared to both the dehydrated sputter coated SEM preparations and the TEM negative stained images (Table 1). In all cases the dimensions of the dehydrated SEM sputter-coated and negative-stained TEM microbes were from 9.nine% to 18.9% smaller than the ionic liquid treated specimens (Table 1). In a previous investigation we imaged frozen-vitrified Ebola virus by cryo-electron microscopy the bore of the Ebola virus was measured every bit 96–98 nm¹⁶ which is very like to the value of 98.5 ± 10.2 nm for the diameter measured of the same specimens treated with ionic liquid in the present study. This farther demonstrates that the volumes of the ionic liquid infiltrated samples are comparable to those measured nether frozen hydrated conditions and closely reflect the fully hydrated native country of Ebola virus. For a bacilliform structure, a 10 per cent reduction in dimensions is equivalent to a 27% reduction in book due to dehydration, though collapse and flattening of the cylindrical shape would imply an fifty-fifty greater caste of water loss. It is clear from that this flattening and collapse due to dehydration is present to some extent in all of the images of sputter coated viruses and bacteria (Fig. 1).

Although the images appear relatively similar, gold sputter coating appears to give slightly more dissimilarity than ionic liquid. Some other observable difference is less of surface roughness of the bacterial cell walls in the ionic liquid stained images. This can be seen in the images of Salmonella (Fig. 1, Supplementary Fig. S3). In these images at that place is a clear textured and wrinkled appearance on the surface of the sputter coated bacterial cells and a smooth appearance on the prison cell walls of the ionic liquid infiltrated preparations. Nosotros propose that this observed difference is largely the effect of loss of cell turgor due to dehydration and book loss in the sputter-coated specimens and thus the wrinkles may actually be an antiquity or feature that is accentuated by dehydration. Supporting evidence for this comes from the fact that other fine structures, such as flagellae, are clearly visible (and of similar appearance) in both the sputter-coated and ionic liquid treated specimens. Thus, the results of previous studies using sputter coating of bacteria may have to exist charily re-interpreted in the calorie-free of possible aridity effects.

The ionic liquid procedure presented in this investigation is rapid and reproducible since specimen filters tin can be prepared in accelerate. As the ionic liquid has a very low vapour pressure, an added benefit is that drying artifacts such every bit shrinkage, wrinkling or corking that can occur during SEM observation are avoided (Table one, Supplementary Fig. S3). In the future, we anticipate the development of a variety of different types of filter coatings to further improve SEM techniques using ionic liquid staining for biological specimens in the nanometre size range.

Methods

Bacterial growth

Salmonella Senftenberg (kindly provided by Dr. George Golding, National Microbiology Laboratory) strains were grown overnight in 3 mL LB broth at 37 °C with shaking. Fifty μL of bacteria were then sub cultured into 3 mL LB broth and grown at 37 °C with shaking for four hours to the gauge mid-log stage of growth. The bacteria were fixed at 1:1 v/five in 1% paraformaldehyde and 2% gluteraldehyde for 1 hour at room temperature. Leptospira biflexa serovar Patoc (kindly provided by Dr. Robbin Lindsay, National Microbiology Laboratory) was grown in Ellinghausen and McCullough media modified by Johnson and Harris (EMJH) (Regal Tropical Institute, The Netherlands). The inoculate culture was placed at 30 °C for 14 days and and then stored at room temperature in low calorie-free.

Growth and Purification of Modified vaccinia Ankara Virus

Babe hamster kidney fibroblast cells (BHK-21:ATCC) were grown to 80% confluence in high-glucose Dulbecco's Modified Hawkeye'due south Medium supplemented with 10% fetal bovine serum and 1X Penicillin-Streptomycin. The BHK-21 cells were infected with 1 mL of Modified vaccinia Ankara (MVA) virus (kindly provided by Dr. Jingxin Cao, National Microbiology Laboratory) and incubated for 48 hours at 37 °C with five% CO₂. The infected cells underwent 3 freeze-thaw cycles in the presence of the growth media, alternating between −80 °C and room temperature. MVA was collected in the supernatant subsequently removing cells and debris by centrifugation at 3000 × yard for three min.

Growth and Purification of Ebola virus

Zaire Ebola virus (kindly provided by Dr. Steven Jones) was propagated, purified and rendered non-infectious as previously described^xvi,33.

Golden Coated Sample Training for SEM Imaging

Unless otherwise stated sample training was performed in a class Two biosafety cabinet. All samples were passed through SPI-pore polycarbonate track etch filters (SPI Supplies, West Chester, PA, Usa) held inside a xiii mm Swinnex® filter unit (Millipore, Billerica, MA, USA). For bacterial sample preparation, 0.08 μm pore size filters were used, while the 0.05 μm pore size was used for MVA and Ebola virus preparations. Filters were start wetted using ii mL Dulbecco's phosphate buffered saline (DPBS) in a three mL Luer-Lok™ syringe. To load the sample onto the filter, 100 μL of sample was added to 5 mL DPBS in a v mL syringe. After attaching the syringe to the filter holder, the sample was filtered through the filter using a Legato 200 syringe pump (KD Scientific, Holliston, MA, USA) at a rate of 1000 μL/min. In some cases the concentration of the sample was too loftier which would overload the filter and fluid could non flow through. If such an event occurred the sample was diluted one:5 or 1:10 until fluid could menstruation freely through the filter. The sample filters were washed using iii mL syringes containing ii mL each of l%, 70%, 85%, 95% and 100% ethanol in increasing concentration. Following the last wash the filter was removed from the filter unit of measurement and immune to air dry out for 30 min. The filter was trimmed, cutting in half and placed on a 9 mm carbon disc (SPI Supplies) and mounted on a 3/8" aluminum stub (SPI Supplies). Flash Dry silvery paint (SPI Supplies) was used on each of the four corners of the filter to create a contact between the filter and the stub. The samples were removed from the biosafety chiffonier and sputtered with aureate using a Quorum Q150R Due south (Quorum Technologies, E Sussex, Uk) containing a 0.i mm gold target. The sample was pumped down, purged with argon and sputtered with gold for 120 sec on a rotating stage.

Training of Metal Coated Polycarbonate Filters

For a filter with a single type of metallic coating the following procedure was used (Supplementary Fig. S1a). For the forts stride either ii cm of 0.2 mm diameter Al wire or three cm of 0.ii mm Au wire was wrapped around the tungsten filament. SPI-pore polycarbonate track etch filters were placed on a filter newspaper in a drinking glass dish and secured from movement with metal washers. The dish was then placed in a Turbo Carbon Coater (Agar Scientific Ltd, Stansted, Essex, United kingdom of great britain and northern ireland). One time nether vacuum, the voltage was gradually increased until the Al or Au wire had completely evaporated. The 2 cm of aluminum wire would create a metal moving picture approximately 18 nm thick and the 3 cm of Au would create a metal pic approximately 27 nm thick. To produce a filter with two types of metal on it the post-obit variant of the to a higher place procedure was used (Supplementary Fig. S1b). The filter was placed on elevation of the filter paper on the drinking glass dish and a razor blade was placed straight over 1 half of the filter. I of the above metals (Au or Al) was evaporated as described above. Post-obit this, the razor blade as removed and a 2d razor blade was used to embrace the region of the filter where the previous metallic was evaporated and the remaining surface area of the filter was then coated with the second metallic.

Ionic Liquid Sample Preparation for SEM

The ionic liquid ane-butyl-iii-methylimidazolium tetrafluoroborate was diluted to a final concentration of two.5% in distilled water and placed at 40 °C for 30 min to reduce viscosity⁹. The metal-coated filters were placed inside the filter unit and wetted using two mL DPBS. A total of 100 μL of sample was added to v mL DPBS and filtered through the coated filter using the Legato filter pump. Subsequently the sample was done with 5 mL distilled water the filter was removed from the filter associates and excess liquid was wicked off with tissue newspaper. The filter was immediately trimmed, placed on a carbon disc mounted on an aluminum stub. The four corners of the filter were painted to the aluminum stub using Flash Dry argent paint. A total of 50 μL of ionic liquid was pipetted onto the sample and the excess was removed after sixty sec by blotting with filter paper. The samples were then viewed by SEM.

SEM Sample Imaging

All samples were imaged in a JCM-5700 Scanning Electron Microscope (JEOL U.s.a., Peabody, MA, USA) contained inside a mobile biological containment enclosure (Dycor Technologies Ltd, Edmonton, AB, Canada)⁸. Gold coated specimens were imaged under high vacuum at 6 kV, with an viii mm working altitude and a 30 μm objective lens discontinuity. Images were collected using the secondary electron detector, the acquisition time per image was 160 sec and each image was 2560 × 1920 pixels. Images of ionic liquid stained samples were obtained using the above noted settings with the exception that the acceleration voltage was adjusted to 4 kV. SEM images were recorded at magnifications ranging from 3,000 x to xx,000x.

TEM Sample Preparation and Imaging

Samples were adsorbed for ane min to a formvar pic on a carbon-coated 400-mesh copper filigree. The adsorbed samples were washed 3X in distilled water and negatively contrasted with 2% methylamine tungstate (Nano-W; Nanoprobes, Yaphank, NY, United states). Imaging was performed at 200 kV using a FEI Tecnai twenty manual electron microscope (FEI Company, Hillsboro, OR, Usa). Digital images of the specimens were acquired using an AMT Reward XR 12 CCD photographic camera (AMT, Danvers, MA, U.s.). TEM images were recorded at magnifications of three,500 10 to nineteen,000x.

Image processing: length measurements

Ebola virus, Salmonella Senftenberg and Leptospira biflexa diameter measurements were made using the Epitome J software package³⁴ using the straight line tool and the analyze/measure out function. Length measurements were calibrated using the calibration bars on the image and the clarify/set scale function in Epitome J. For this analysis, diameter measurements only were made, since the leaner and viruses have varying lengths, but relatively constant diameters. Measurements were collated and analysed using MS Excel.

Additional Information

How to cite this commodity: Golding, C. G. et al. The scanning electron microscope in microbiology and diagnosis of infectious disease. Sci. Rep. 6, 26516; doi: 10.1038/srep26516 (2016).

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Acknowledgements

We would like to thank Dr. George Golding, Dr. Jingxin Cao, Dr. Robbin Lindsay and Dr. Steven Jones (all from the National Microbiology Laboratory, Winnipeg, MB, Canada) for providing us with the Salmonella Senftenberg, Modified vaccinia Ankara, Leptospira biflexa and Ebola virus, respectively.

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Viral Diseases Division, National Microbiology Laboratory, Public Wellness Agency of Canada, Winnipeg, Manitoba, Canada

Christine G. Golding, Lindsey L. Lamboo, Daniel R. Beniac & Timothy F. Booth
Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada

Lindsey Fifty. Lamboo & Timothy F. Berth

Contributions

C.G.1000., D.R.B. and T.F.B designed the experiments. C.G.G. and Fifty.L.50. conducted the experiments. C.G.One thousand., D.R.B. and T.F.B. wrote the manuscript.

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The authors declare no competing financial interests.

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Golding, C., Lamboo, L., Beniac, D. et al. The scanning electron microscope in microbiology and diagnosis of infectious disease. Sci Rep 6, 26516 (2016). https://doi.org/x.1038/srep26516

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Received: 11 Feb 2016
Accepted: 03 May 2016
Published: 23 May 2016
DOI : https://doi.org/10.1038/srep26516

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