mPrep/g™ capsules for grid staining and immunolabeling solve multiple challenges faced by microscopists.
Less damage. If you don’t touch a grid – you can’t damage it!
Fewer lost grids. Grids are stored, stained and immuno-labeled in the capsule
Accurate reagent delivery by attaching capsules to pipettors
Efficient and consistent. Prepare multiple grids simultaneously
Always labeled. Documentation for ISO and GLP standards is easy
Pipettor (such as a Gilson P200 volume) to enable reagent pipetting into mPrep/g capsules
mPrep/f couplers to prevent introducing damaging reagents into pipettors
Gilson Pipetman Neo (P200) pipettors bundled with mPrep/g capsules.
mPrep/g Pipettor Kit single-channel
mPrep/g Pipettor Kit eight-channel
mPrep/Bench to seal capsule bottoms during long reagent treatments
mPrep CPD holder when using a Tousimis critical point dryer
Reagent reservoirs to hold reagents for delivery of same reagent into 8 or 12 capsules attached to multi-channel pipettor
Microwell plates to hold reagents for delivery of different or same reagents into 8 or 12 capsules attached to multi-channel pipettor
This brief technical note illustrates automation of immunogold labeling using the mPrep ASP-1000 automated specimen processor. Researchers at the University of Wisconsin prepared C. elegans tissue sections on TEM grids and then used the ASP-1000 to immunolabel an endosomal sorting component with 6 nm gold. The entire 7.5 hour immunolabeling protocol was automatically accomplished. This note also illustrates automated application of 10 nm gold fiducial markers onto thick sections for tomography.
This application note illustrates staining tissue sections on TEM grids using mPrep/g capsules with pipettors and common lab supplies. The protocol shows uranyl acetate and lead citrate staining of kidney and brain sections, although the methods apply to other grid staining procedures. Required supplies and the reagent formulae are listed.
This application note illustrates the preparation of macromolecular specimens for negative stain TEM using mPrep/g capsules, pipettors and Formvar®-filmed TEM grids. The example is a DNA-RecA complex, although the protocol is applicable to many other specimens. The specimen preparation and microscopy was performed in the Department of Biochemistry at the University of Wisconsin – Madison by Mr. Jay Campbell and Dr. Sindhu Chitteni-Pattu, in the laboratory of Dr. Michael M Cox.
This illustrated protocol shows the preparation of nanoparticle specimens for negative stain TEM using Formvar®-filmed grids in mPrep/g capsules. Eight different formulations of ~100 nm drug delivery particles were prepared simultaneously for a pharmaceutical company to evaluate structure and uniformity. This general sample preparation method can be applied to other nano-scale specimens including nano-fibers and viruses.
This application note shows the negative stain TEM preparation of pathogenic Ebola virus in high containment BSL4 labs. Scientists used mPrep/g capsules to eliminate grid handling in the BSL suite. This resulted in easier, safer processing and more reproducible sample preparation. Specimen preparation and microscopy were performed by the US Army Medical Research Institute of Infectious Diseases (USAMRIID) Pathology Division, at Ft. Detrick, MD.
Staining tissue sections on grids requires care to ensure grids are not damaged and stains are consistently applied. Placed in mPrep/g capsules, grids are secured and protected. They may require only two human touches from the microtome to microscope.
Preparing negative stain macromolecular specimens requires handling of fragile filmed-grids. With mPrep/g capsules filmed-grids are secured throughout specimen preparation, which minimizes handling. Controlled and simultaneous delivery of reagents is possible when the capsules are attached to pipettors.
Preparing virus samples requires handling of fragile filmed-grids. This is exceptionally difficult when pathogenic viruses are present, requiring BSL3/4 pressure suits. Using mPrep/g capsules virus suspensions and reagents can be easily handled using pipettors.
Immunolabeling tissue sections on grids requires care to ensure grids are not damaged and immunogold labels are consistently applied. Placed in mPrep/g capsules, grids are secured and protected. With mPrep/g capsules, even IGL may require only two human touches from the microtome to microscope.
For successful 3-D TEM tomographic reconstruction, colloidal gold fiducial markers need to be uniformly and consistently deposited on both grid sides. Using manual droplet methods, this is a hit or miss process. With the ASP-1000, colloidal gold fiducial markers are uniformly and automatically applied to both sides of up to 16 grids at once.
Nanoparticle specimen preparation requires handling of fragile filmed-grids to apply nanoparticle suspensions, stains and rinses. With mPrep/g capsules filmed-grids are safely encapsulated throughout specimen preparation, which minimizes handling. Controlled and simultaneous delivery of reagents is possible when the capsules are attached to pipettors.
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.
Marques N, Strong J, Strader T, Hsia R-C (2018) Optimization of Automated Immuno EM for Both Pre- and Post-Embedding Labeling. Microsc. Microanal. 24 (Suppl 1), 2018: 1300. doi:10.1017/S1431927618006980
This proceedings paper and poster presents optimized protocols for ASP-1000 automation of immuno-labeling on grids and for en bloc tissues (pre-embedding). On-grid immuno-labeling is shown with sectioned brain, sectioned HeLa cells, and whole-mount bacteria. En bloc tissue immuno-labeling is shown with mouse brain that is also silver enhanced and then resin embedded using the ASP-1000. The authors discuss how automated labeling fits into the lab workflow by providing a “drastic reduction of hands-on time” from several days to 1-2 hours, by enabling overnight processing, and by eliminating manual specimen and grid transfers that can cause sample damage. Also discussed is that automation increased reproducibility and consistently lowered background labeling noise.
Strader TE, Stewart NR, August BK, Goodman SL (2018) A Versatile All-in-One Automated Processor for Electron Microscopy. Microsc. Microanal. 24 (Suppl 1), 2018: 1122. doi:10.1017/S1431927618006098
This proceedings paper presents how the ASP-1000 provides versatility and a workflow solution for research and clinical electron microscopy labs. The authors show the automated preparation of tissues and cell suspensions from laboratory receipt in aldehyde fixative through epoxy and acrylic resin infiltration as quickly as 45.5 minutes for kidney and 2 hours for other tissues. Additional ASP-1000 applications addressed include serial block face SEM specimen preparation, on-grid and en bloc immuno-labeling, and applying gold fiducials to grids for TEM tomography.
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.”
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.
Frankel EB August BK, Audhya A, Strader TE (2016) Fully-automated Immunogold Labeling of Resin Embedded Specimens and On-grid Deposition of Gold Fiducial Particles. Microsc. Microanal, 22 (Suppl 3), 1010-1. doi 10.1017/S1431927616005894
This proceedings paper and presented poster demonstrates fully automated immunogold labeling of an endosomal sorting complex in a C. elegans 1-cell stage embryo. The authors report that immunogold labeling using the mPrep ASP-1000 was highly reproducible across numerous samples. This work also demonstrates automated application of gold colloids to thick sections on grids to provide fiducial markers for electron tomography. The authors report that gold fiducial particles were adsorbed evenly over the grid surfaces allowing for highly accurate registration of tilt-series images.
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.
Goodman S.L. (2015) Efficient and Documented Preparation of Pharmaceutical Particles for Correlative Microscopy Analyses using mPrep Capsule Processing Microsc. Microanal. 21 (Suppl 3), 2015: 393-4. doi: 10.1017/S1431927615002767
Using mPrep capsules, Goodman prepared two types of pharmaceutical particles for characterization. The mPrep capsules enabled simultaneous and GLP compliant sample preparation. Goodman used mPrep/g capsules to simultaneously prepare 16 grids for TEM from two formulations of siRNA drug delivery nanoparticles with four staining protocols. The grids were then analyzed for morphology and uniformity using TEM. In a separate study he simultaneously embedded four formulations of polymeric drug delivery micro-particles to create eight blocks for cross-sectioning. Thin sections were than analyzed for chemical composition using transmission FTIR and for elemental composition using EDS-SEM.
Benmeradi N, Payre B, Goodman SL (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.
Goodman SL, Wendt KD, Kostrna MS, Radi C (2015) Capsule-Based Processing and Handling of Electron Microscopy Specimens and Grids. Microscopy Today, 23(5): 30-7. doi:10.1017/S155192951500076
Authors from three institutions prepared animal and plant tissues for TEM. They used mPrep/s capsules for fixation through embedding, and mPrep/g capsules for grid staining. Researches assessed reagent consumption and labor efficiency of mPrep processing compared to traditional processing (using vials, embedding molds and droplet staining). The findings included: mPrep/s specimen processing reduced reagent consumption ten-fold and the number of hands-on operations seven-fold. mPrep/g grid staining reduced hands-on operations six-fold.
Jain R, Poyraz AS, Gamliel DP, Valla J, Suib SL, Maric R (2015) Comparative study for low temperature water-gas shift reaction on Pt/ceria catalysts: Role of different ceria supports. Applied Catalysis A: General, 507: 1–13.
Jain, et al used mPrep/g capsules to prepare nanoparticle specimens for high resolution TEM characterization. They tested and characterized different Pt on ceria catalyst using XRD, Raman, BET, SEM and TEM. For TEM imaging, the catalysts were deposited onto carbon filmed 300 mesh TEM grids using mPrep/g capsules.
Jain R, Maric R (2014) Synthesis of nano-Pt onto ceria support as catalyst for water–gas shift reaction by Reactive Spray Deposition Technology. Applied Catalysis A: General, 475: 461–468.
Jain et al used mPrep/g capsules to prepare nanoparticle specimens for high resolution TEM characterization. They deposited reactive spray synthesized nano-Pt catalysts onto lacy carbon filmed 300 mesh TEM grids in mPrep/g capsules.
Goodman S. and Kostrna M (2011). Reducing Reagent Consumption and Improving Efficiency of Specimen Fixation and Embedding, Grid Staining and Archiving using mPrep™ Capsule Processing. Microscopy and Microanalysis, 17 (Suppl. 2) , pp 1174-1175. doi:10.1017/S143192761100674X.
This short proceedings paper introduced the mPrep System at Microscopy and Microanalysis 2011. The paper provides an overview of the mPrep System for TEM specimen preparation and TEM grid staining. The System is compared to conventional processing for laboratory efficiency and reagent consumption.
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.
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.
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.
Radi C. (2015) Improving the Reliability, Ease, and Efficiency of Section Staining in a Diagnostic Laboratory with the mPrep/g System for TEM Grid Processing. Microsc. Microanal. 21 (Suppl 3), 2015: 725-6. doi: 10.1017/S1431927615004420
Radi discusses the necessity for efficiently preparing and documenting TEM grids in a clinical diagnostic laboratory setting. He then compares the droplet method to the mPrep system process for uranyl acetate and lead citrate staining of tissue sections . He reports that, “In addition to providing more consistent results, mPrep/g processing was also easier and more efficient.” And notes that “ . . . the time to prepare, store, document and stain each grid took only seconds compared to minutes using the droplet method.”
Strader TE, Goodman SL. (2015) Automated, Programmable Processing of Specimens and Grids with the mPrep ASP-1000. Microsc. Microanal. 21 (Suppl 3), 2015: 11-2. doi: 10.1017/S1431927615000859
Strader and Goodman introduce the mPrep Automated Specimen Processor (ASP), used for simultaneous sample preparation using mPrep capsules. The ASP-1000 prepares TEM and SEM specimens, and stains and labels TEM grids. The paper discusses the ASP-1000 features, including flexibility to efficiently perform routine and complicated tasks. The paper includes pictures of the instrument and examples of heart and skin tissue specimens prepared using mPrep capsule processing.
Vierrether O, TerBush J, Wisner C (2016) Nano-Particle TEM Sample Preparation Primer. Microsc. Microanal. 22 (Suppl 3), 2016: 1914. doi:10.1017/S1431927616010412
This paper and poster provides a guide to selecting grid and support film types for preparing nanoparticles for TEM, and also compares droplet preparation with mPrep ASP-1000 automated processing. The authors concluded that lacey silicon monoxide/Formvar films on 300 mesh grids proved to the be the best for the nanoparticles examined. They also concluded that mPrep automation reduced sample agglomeration thereby allowing for single particle imaging, and this could also be achieved with mPrep/g capsules and manual pipetting.
This video tutorial demonstrates how to use mPrep capsules with multi-channel pipettors and common lab ware to simultaneously deliver reagents to multiple specimens and grids. The video demonstrates TEM fixation and embedding. Reagent processing procedures are similar for all specimen types and for grids with mPrep capsules .
6 minutes 4 seconds
This tutorial demonstrates how to transfer mPrep capsules to a critical point dryer. The transfer is shown with an mPrep CPD holder designed for the Tousimis Samdri. Both mPrep/s and mPrep/g capsules are shown.
4 minutes 7 seconds
This webinar introduces and demonstrates the mPrep System. Webinar covers how the mPrep System works. It discusses the benefits of the mPrep System over current practice. And it addresses multiple TEM and SEM applications: tissues, macromolecules, immuno, nanoparticles, cryo-planing.
A graphical display illustrating how to load grids into mPrep/g capsules, and how to use pipettors to stain or label grids. Use this guide as a lab reference poster.
Instructions for using mPrep/g capsules for staining sections on grids with uranyl acetate and lead citrate.
Protocol for immuno-labeling grids with mPrep/g
A graphical display that illustrates how to easily prepare virus specimens for negative stain TEM in Biological Safety Level 3-4, and other laboratories. Use this guide as a lab reference poster.
Instructions for using mPrep/g capsules for grid handling and processing.
Print this 96-well plate template to organize protocols into columns and rows with a microtiter 96-well plate.
This technical note introduces using mPrep capsule labeling for tracking specimens and grids with a data management system. Topics addressed include mPrep labels, 2D datamatrix barcodes, barcode readers and laboratory information management systems.
How to use the mPrep CPD holder for specimen and grid sample preparation using the critical point method.
Instructions for using the mPrep capsule grid box for storage and labeling of mPrep/g and mPrep/s capsules.