McClain and coauthors prepared whole planarian flatworms for serial SEM imaging by developing a rapid automated version of the serial block face SEM protocol developed at the National Center for Microscopy and Imaging Research (NCMIR), which included osmium- thiocarbohydrazide–osmium and lead aspartate staining. With the McClain method, similar results were achieved in just 1.5 days by using rapid automated reagent exchanges with the ASP, compared to 7 days of manual reagent exchanges with the NCMIR protocol. This poster presentation at Microscopy and Microanalysis 2017 also received the 2nd place Diatome Award.

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.

McClain simultaneously prepared 16 tissue specimens for TEM using 16 protocols to establish an optimal fixation and embedding protocol. This study varied buffer molarity, en bloc stains, and embedding resins. Reagent processing was conducted simultaneously using multi-channel pipettes and microtiter plates. The author reported, “The mPrep System facilitated the controlled and consistent testing of many conditions, which greatly increased the chances of obtaining optimized specimen preservation in one processing run.”

This work used the mPrep ASP-1000 and mPrep/s capsules to prepare rat gastrocnemius muscle for TEM in order to examine mitochondrial ultrastructure in a study that examines postnatal hyperoxia as a model for premature birth effects on aerobic capacity.

McClain developed a TEM tissue fixation and embedding protocol that utilized high speed agitation on the ASP-1000 to prepare planarian worm specimens for resin curing that required only 3 hours. The results were comparable to a 7-day embedding protocol that was previously used to prepare these worm specimens.

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.

Stewart et al. provides a method for the easy embedding of tissue in LR White resin for TEM immuno-labeling and related applications. The method enables the reliable location of non-osmium treated tissue after embedding, non-messy specimen orientation prior to resin infiltration, minimal specimen handling, and automated reagent processing with the ASP-1000 in less than 45 minutes prior to resin curing.

Stewart and coauthors demonstrate the automated preparation of kidney, liver, skeletal and cardiac muscle using the mPrep ASP-1000. Reagent processing of kidney was completed in just 45 minutes and liver and muscle in only 2 hours, prior to resin curing. The method provided easy non-messy specimen orientation prior to resin infiltration, and specimens that were only directly handled once - when they were oriented and inserted into labeled mPrep/s capsules.

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.

This proceedings paper and presented poster shows how the ASP-1000 can accelerate the preparation of TEM pathology tissue specimens from 1-2 days to several hours with conventional chemical reagents. The poster provides automation protocols for the ASP-1000 that accelerate diffusion of processing reagents into specimens by directed fluid flow that is repeated as often as 600 times in just 5 minutes. Examples are provided for multiple tissues including kidney, skeletal muscle, cardiac muscle, liver and brain. The poster also addresses how the automated preparation and mPrep/s specimen capsules and Workstation provide an efficient workflow from specimen acquisition through microtomy that also provides traceability.

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.

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.

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 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.

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.

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.

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.

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.

This comprehensive protocol freely available from the Defense Technical Information Center demonstrates how to reproducibly and accurately quantify virus concentrations: mPrep/g capsules are used to prepare grids with a mixture of unknown virus and nano-gold particles. Virus counts are obtained using large throughput STEM imaging with automated particle counting. The authors report that this mPrep/g capsule method (in comparison to droplet grid preparation) was much easier, saved technician time, enabled preparation in biocontainment, and provided replicate samples with no increase in effort. And, most importantly, provided a consistent and accurate method for virus quantification.

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.

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 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.

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 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.

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.”

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.

Stewart et al. provides a method for the easy embedding of tissue in LR White resin for TEM immuno-labeling and related applications. The method enables the reliable location of non-osmium treated tissue after embedding, non-messy specimen orientation prior to resin infiltration, minimal specimen handling, and automated reagent processing with the ASP-1000 in less than 45 minutes prior to resin curing.

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.

In this study of Pseudomonas aeruginosa lung infections, Lillihoj and co-authors used an mPrep ASP-1000 to perform automated immunogold labeling of MUC1-ED receptors on Pseudomonas aeruginosa bacteria adherent to Pioloform-coated grids. The automated ASP-1000 protocol included a dozen sequential reagent steps over ~12-hours which included primary labeling of MUC1-ED receptors with anti-MUC1-ED antibodies (or control antibodies) followed by 10 nm gold labels conjugated to goat-anti mouse IgG secondary antibodies.

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.

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.

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.

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.

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.