Application Notes

This application note demonstrates using agar embedding of suspended cells to enable rapid automated preparation with the ASP-1000 Automated processor to fix, post-fix and embed cells in epoxy for TEM.  The example uses yeast, but this method can be readily extended to the preparation of other types of cells in suspension, and for the preparation of other small specimens. By combining agar embedment with ASP-1000 processing, 18 centrifugation and resuspension cycles were eliminated from a typical manual protocol. Once the cells were agar embedded, the total processing time prior to epoxy resin curing was just 1.5 hours.

This application note demonstrates the reproducibility of rapid processing with the ASP-1000. Twenty-four (24) specimens from the same aldehyde perfusion fixed liver were prepared for TEM. Specimens were directly handled only once, when they were orientated in mPrep/s capsules. Automated processing required only 133 minutes from post-fix rinse through osmium fixation and 100% resin infiltration. All 24 samples showed well-preserved ultrastructure homogeneity as verified by TEM imaging.

This application note presents a method for processing mammalian muscle tissue for TEM in just over two hours. Using the ASP-1000 automated specimen processor, specimens were processed from postfixation rinse through infiltration in just 133 minutes. Specimens were handled only once, when they were oriented and inserted into mPrep/s capsules using the mPrep/s Workstation.

This application note presents a fast, fully-automated method of tissue processing. Mammalian kidney was prepared for TEM with the mPrep ASP-1000 programmed for highly efficient rapid-agitation. Reagent processing from postfix rinse through 100% resin infiltration was accomplished in just 45 minutes.

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 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.

This application note illustrates the preparation of polymer specimens for cross-sectional analysis. Polymer drug delivery particles were embedded and cross-sectioned for transmission FTIR spectroscopy and EDS-SEM. The application note also shows how to extend these methods to the preparation of other soft materials including coatings, thin films and fibers.

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 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 application note illustrates mammalian kidney tissue prep for TEM from fixation through embedding. This general protocol applies to other specimens that require embedding for cross-sectioning. Photos show the mPrep capsule reagent exchange process and how mPrep TEM blocks are faced and sectioned while still in capsules. Supplies and the reagent formulae are listed.


Publications

Rapid Automated Serial Block Face SEM Preparation of Brain Tissue. (Soc. Neuroscience 2019)

Effects of Postnatal Hyperoxia Exposure on Cardiac Muscle Examined with 3D Serial Block Face SEM using Rapid Automated Sample Preparation (Microscopy & Microanalysis 2019)

Rapid Automated Preparation for Serial Block Face Scanning Electron Microscopy (Microscopy & Microanalysis 2019)

Automated Heavy Metal Tissue Staining for Serial Block Face Imaging with the ASP-1000 (Microscopy & Microanalysis 2019)

Automated Preparation of Core Needle Biopsy Specimens for TEM Imaging (Microscopy & Microanalysis 2019)

Cryo-Planing Biological Specimens for Scanning Electron Microscopy (Microscopy Today 2019)

Neuraminidase 1-mediated desialylation of the mucin 1 ectodomain releases a decoy receptor that protects against Pseudomonas aeruginosa lung infection (J Biological Chemistry 2019)

Scanning Transmission Electron Microscopy (STEM) for Accurate Virus Quantification (Defense Technical Information Center 2018)

Optimization of Automated Immuno EM for Both Pre- and Post-Embedding Labeling (Microscopy & Microanalysis 2018) 

Automated Rapid Preparation of Tissue Specimens for TEM Pathology (Microscopy & Microanalysis 2018) 

A Versatile All-in-One Automated Processor for Electron Microscopy. Microscopy & Microanalysis 2018)

Sex-Specific Skeletal Muscle Fatigability and Decreased Mitochondrial Oxidative Capacity in Adult Rats Exposed to Postnatal Hyperoxia (Frontiers Physiology 2018)  

Accurate virus quantitation using a Scanning Transmission Electron Microscopy (STEM) detector in a scanning electron microscope (J. Virological Methods 2017)

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment (J Visualized Experiments 2017) 

Rapid Automated en Bloc Staining for SEM of Sections (Microscopy & Microanalysis 2017) 

Fast, Walk-away, Automated Processing of Mammalian Tissue for LM and TEM (Microscopy & Microanalysis 2017) 

Preparing LR White Embedded Tissue with mPrep/s Specimen Capsules (Microscopy & Microanalysis 2017) 

Using Scanning Transmission Electron Microscopy (STEM) for Accurate Virus Dosing Quantification (Microscopy & Microanalysis 2016) 

Fully-automated Immunogold Labeling of Resin Embedded Specimens and On-grid Deposition of Gold Fiducial Particles (Microscopy & Microanalysis 2016) 

3D Cell Culture and Microscopy in a Capsule with Scaffolds, Tumors & Stem Cells (Microscopy & Microanalysis 2016)

Application of a New Miniature Bioreactor System to Generate and Test Artificial Tumor and Normal Breast Ductal Tissues Using MCF-7/MCF10A Cells (Experimental Biology 2016) 

Bringing Speed and Automation Together with the ASP1000 (Microscopy & Microanalysis 2016) 

Improved Virus Specimen Preparation for Transmission Electron Microscopy using mPrep/g Capsules: Applications in BSL3-4 Laboratories (Microscopy & Microanalysis 2016) 

Improved Virus Specimen Preparation for Transmission Electron Microscopy using mPrep/g Capsules: Applications in BSL3-4 Laboratories (Ft. Detrick Science Fair 2016)

Preparation of viral samples within biocontainment for ultrastructural analysis: Utilization of an innovative processing capsule for negative staining (J Virological Methods 2016)

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.

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 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.

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.

McClain M. (2014) High Throughput Multi Parameter TEM Chemical Processing Protocol Development with the mPrep-s Capsule System: Schmidtea mediterranea. Microsc. Microanal. 20 (Suppl 3), 2014: 1288-9.   doi:10.1017/S1431927614008174

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.”

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.

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.

Plumley WC. (2014) Cryo-Planing of Small Biological Specimens for SEM Using mPrep Capsules. Microsc. Microanal. 20 (Suppl 3), 2014: 1442-3. doi:10.1017/S1431927614008940

Plumley developed a fast and easy cryo-planing method to prepare animal and plant tissues for SEM using the mPrep System. Cryo-planing (also call cryo-facing) provides a unique 3D view of internal and surface features by cutting a cross-section through frozen tissue. Cryo-planing has not been widely used due to the difficulty of handling specimens throughout the tissue preparation process. Plumley used the pinch holding function of mPrep/s screens with modified mPrep/s capsules to hold and orient tissue during this process of rapid freezing, cryo-microtomy, freeze-drying and SEM mounting. He reported that this procedure “enabled easier, faster preparation of multiple small samples, while maintaining the excellent morphology and sectioned view… that cryo-planing is best known for…”

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.


Videos

Specimen and Grid Reagent Processing for TEM and SEM

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

Using the mPrep Workstation for Oriented and Unoriented Specimens

The tutorial starts by introducing the mPrep Workstation. Then it demonstrates how to load and orient specimens into mPrep capsules. Three methods are shown: pinch, compression and cup.

5 minutes 19 seconds

Preparing Embedded TEM Specimens for Sectioning

This video tutorial shows how to trim and face an embedded block inside an mPrep/s capsule. The capsule is clamped into a microtome chuck for trimming, facing and sectioning.

1 minutes 17 seconds

Critical Point Dry (CPD) Sample Prep

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

Webinar: TEM & SEM Sample Preparation with mPrep Capsules

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.
45 minutes

WEBINAR: Walk-away Biological Specimen Prep for the 21st Century Lab

This 18-minute webinar (broadcast 25 September 2019) presents how the ASP-1000 automates the preparation of biological specimens for TEM and SBEM (serial block face SEM) to provide walk-away convenience, reduced processing time, and easy clean-up. The webinar also discusses how the ASP-1000 provides pre-programmed protocols for preparing multiple tissues and for immunolabeling, and how users can easily modify existing or create new protocols.


Instructions

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.

A single page graphical display of the three steps to insert specimens into mPrep/s capsules using the mPrep/s insertion tool. Use this guide as a lab reference poster.

A single page graphical display of the three steps to orient and load specimens into mPrep/s capsules. Both compression and pinch methods are shown. Use this guide as a lab reference poster.

Instructions for using mPrep/s capsules for specimen 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.