Benmeradi N., Payre B., Goodman S.L. (2015) Easier and Safer Biological Staining: High Contrast UranyLess Staining of TEM Grids using mPrep/g Capsules. Microsc. Microanal. 21 (Suppl 3), 2015: 721-2. doi: 10.1017/S1431927615004407
This paper demonstrates how a new lanthanide-based heavy metal stain, when used with mPrep/g capsules, provides high contrast tissue section and negative staining with results that are comparable or possibly superior to conventional uranyl acetate staining. Examples show TEM thin section staining of 5 animal tissues and one plant tissue, and a negative stained TEM bacteriophage specimen.
Blancett CD, Fetterer DP, Koistinen KA, Morazzani EM, Monninger MK, Piper AE, Kuehl KA, Kearney BJ, Norris SL, Rossi CA, Glass PJ, Sun MG. (2018) Scanning Transmission Electron Microscopy (STEM) for Accurate Virus Quantification. Defense Technical Information Center, United States Department of Defense.
This comprehensive protocol freely available from the Defense Technical Information Center was developed by Candance D Blancett, et al, and lead by Dr. Mei Sun at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). This protocol demonstrates how to reproducibly and accurately quantify virus concentrations using mPrep/g capsules and pipettors to prepare grids with a mixture of unknown virus and nano-gold particles for large throughput STEM imaging and automated particle counting. The authors report that using mPrep/g capsules (in comparison to droplet grid preparation) was much easier, saved technician time, enabled preparation in biocontainment, provided replicate samples with no increase in effort, and most importantly provided a consistent and accurate method for virus quantification.
Monninger MK, Nguessan CA, Blancett CD, Kuehl KA, Rossi CA, Olschner SP, Williams PL, Goodman SL, Sun MG. (2016) Improved Virus Specimen Preparation for Transmission Electron Microscopy using mPrep/g Capsules: Applications in BSL3-4 Laboratories. Ft. Detrick Science Fair, 5 May 2016.
This poster shows how mPrep/g capsules enable the easy preparation of pathogenic viruses in BSL4 biocontainment. The authors from USAMRIID (US Army Medical Research Institute of Infectious Diseases) prepared Zaire Ebola virus and Chikungunya virus with glutaraldehyde inactivation and negative staining. Samples were also prepared of Ebola nano virus like particles and murine Leukemia virus like particles. Morphology of the viruses and virus like particles were assessed with and without osmium tetroxide vapor inactivation, and with uranyl acetate and phosphotungstic acid negative staining.
Monninger MK, Nguessan CA, Blancett CD, Goodman SL, Sun MG. (2016) Improved Virus Specimen Preparation for Transmission Electron Microscopy using mPrep/g Capsules: Applications in BSL3-4 Laboratories. Microsc. Microanal. 22 (Suppl 3), 2016: 1164. doi:10.1017/S1431927616006668
This paper shows how mPrep/g capsules enables easy and reproducible preparation of highly pathogenic viruses for TEM by eliminating grid handling in BSL3-4 biocontainment suites. Examples are shown of negative stained Zaire Ebola virus and murine leukemia virus-like particles. A pictorial diagrams the preparation protocol inside and outside the biocontainment suite. The authors report that the reduced effort, consistent quality, and simultaneous preparation of multiple grids makes this method equally useful for labs working with non-pathogenic viruses.
Blancett CD, Fetterer DP, Koistinen KA, Morazzani EM, Monninger MK, Piper AE, Kuehl KA, Glass PJ, Sun MG. (2016) Using Scanning Transmission Electron Microscopy (STEM) for Accurate Virus Dosing Quantification. Microsc. Microanal. 22 (Suppl 3), 2016: 1162. doi:10.1017/S1431927616006656
Blancett and co-authors use mPrep/g grid capsules to reproducibly and accurately quantify unknown virus concentrations. Virus suspensions are mixed with a known concentration of nano-gold particles and applied to Formvar-carbon grids using mPrep/g capsules and pipettors. The grids are then imaged with scanning transmission electron microscopy (STEM) followed by automatic image analysis to quantify viral concentration by counting the ratio of viruses to nano-gold particles. They report that mPrep/g preparation improves enumeration accuracy, and that assay results are consistent with virus plaque assays.
Monninger MK, Nguessan CA, Blancett CD, Kuehl KA, Rossi CA, Olschner SP, Williams PL, Goodman SL, Sun MG. (2016) Preparation of viral samples within biocontainment for ultrastructural analysis: Utilization of an innovative processing capsule for negative staining. Journal of Virological Methods 238 (2016) 70–76. doi.org/10.1016/j.jviromet.2016.10.005
This study demonstrates how to prepare Ebola virus, chikungunya virus, murine leukemia virus and virus-like particle in BSL3-4 biocontainment suites using mPrep/g capsules. The study also compares virus inactivation using glutaraldehyde fixation prior to grid preparation and using osmium tetroxide vapor treatment after grid preparation. In addition, uranyl acetate and phosphotungstatic acid negative staining are compared. The authors conclude that mPrep/g capsule preparation saves time and effort and produces more consistent high-quality TEM images than manual droplet grid preparation. The study also concludes that uranyl acetate and phosphotungstatic acid staining provide comparable results, and that osmium tetroxide vapor decontamination is faster than glutaraldehyde decontamination but can reduce image quality.
Blancett CD, Fetterer DP, Koistinen KA, Morazzani EM, Monninger MK, Piper AE, Kuehl KA, Kearney BJ, Norris SL, Rossi CA, Glass PJ, Sun MG. (2017) Accurate virus quantitation using a Scanning Transmission Electron Microscopy (STEM) detector in a scanning electron microscope. J. Virology Methods. 248:136-144.
Blancett and co-authors demonstrate a new method for accurate quantitation of virus particles called STEM-VQ: Scanning Transmission Electron Microscopy - Virus Quantitation. This method uses mPrep/g capsules for virus sample preparation, gold beads for reference counting, high throughput microscopy, and automated software for viral counting. The authors demonstrate high accuracy and reproducibility of the STEM-VQ method in comparison to a standard viral plaque assay and the ViroCyt Virus Counting, while uniquely providing morphology information and the counting of all viruses.
Blancett CD, Monninger MK, Nguessan CA, Kuehl KA, Rossi CA, Olschner SP, Williams PL, Goodman SL,
Sun MG (2017) Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment. J. Visualized Experiments. (125), e56122, doi:10.3791/56122.
This video and print article provides a step-by-step demonstration of how to prepare viruses in BSL3-4 bio-containment suites using mPrep/g capsules beginning with the insertion of Formvar-filmed TEM grids into mPrep/g capsules, through virus decontamination and negative staining. Examples include Ebola and Chikungunya viruses. The authors conclude that, “Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.”