Capsule-Based Processing and Handling of Electron Microscopy Specimens and Grids


Steven L. Goodman , 1 * Kristy D. Wendt , 1, 2 Michael S. Kostrna , 3 and Craig Radi 4 1 Microscopy Innovations LLC , 213 Air Park Road , Suite 101 , Marshfi eld , WI 54449 2 Laboratory for Optical and Computational Instrumentation , University of Wisconsin , 1675 Observatory Drive , Madison ,


WI , 53706 3 Electron Microscopy Training Program, Madison College. 1701 Wright Street , Madison , WI 53704 4 Wisconsin Veterinary Diagnostic Laboratory , 445 Easterday Lane , Madison , WI 53706


* steven.goodman@microscopyinnovations.com Introduction


It is a testament to the brilliance of the earliest biological electron microscopists that half a century later, most cell and tissue specimens for transmission electron microscopy (TEM) are still prepared using chemical fi xation, polymer embedding, microtomy, and heavy-metal staining protocols based on those developed by Palade, Porter, Fullam, Claude, and others in the 1950s–1970s [ 1 – 5 ]. T ese methods remain in use today because they provide good fi xation and electron contrast, and they enable thin sectioning. To perform these protocols, biological specimens are placed into vials fi lled with each chemical reagent, followed by manually exchanging reagents using trans- fer pipettes. Similarly, grids are stained by manually moving them between reagent droplets or dishes. However, why these fl uid-handling methods have remained largely unchanged in the modern electron microscopy lab is surprising because fl uid handing in life science laboratories was transformed during the same decades as TEM revolutionized biology. Two leading advancements in fl uid handling were the introduction of the variable-volume pipetter by Gilson [ 6 ] in 1972 and the mass- produced micro-well plate (microtiter plate) invented by Cooke [ 7 – 8 ] and commercialized about 1966 [ 9 ]. T e importance of fl uid handling in electron microscopy is considerable. Typical TEM specimen preparation protocols comprise 20 or more fi xative, rinse, dehydrant, and resin exchanges. T us, the process of preparing only 5 diff erent tissue samples entails over 200 discrete vial-fi lling and emptying steps. Not only is this tedious, but specimens can be damaged by contact with the container, by being accidentally aspirated into transfer pipettes, by air exposure when specimens fl oat or stick to the vial sides, and when transfers between reagents take too long. Specimens may even be lost because many tissues are diffi cult to see when immersed or when they stick to the sides of vials. It is also diffi cult to maintain consistent timing when several specimens are being prepared, thus reducing experi- mental reproducibility. Although automatic tissue processers ease this tedium and equalize reagent timing, most such devices require cumbersome manual specimen transfers from porous baskets into embedding molds. More importantly, automatic processors are not able to simultaneously prepare a group of specimens with diff erent staining and fi xation protocols as commonly required in research.


Manual processing can be wasteful of reagent because common lab vessels (scintillation vials, microfuge tubes) require at least 1–2 ml of reagent simply to keep specimens


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immersed, yet only 7–10 times the volume of a specimen is suffi cient for each reagent step [ 10 ]. Given that biological tis- sue TEM specimens are typically 1 mm 3 , specimen volume is thus about 1 μ l, and therefore only 7–10 μ l of reagent is actu- ally required. T us, reagent volumes consumed in vial process- ing easily exceed 100 times that required for suffi cient reaction.


For most labs the process for staining sections on grids with heavy metals (or immuno-labels) has also not changed [ 11 – 12 ]. Grids are extensively handled using forceps to transfer them into and out of grid boxes, onto and between stain droplets or other staining apparatuses, back into grid boxes, and eventually onto the TEM stage. Because grids are fragile, it is challenging to not damage, drop, or lose them. Also, because individual grids are not labeled, they can easily be mixed up. It is also diffi cult to obtain identical timing when multiple grids are being prepared, thus reducing experimental reproducibility. T is article describes a new capsule-based processing system for preparing and handling EM specimens and TEM grids. It is based on two types of microliter-volume, purpose-built vessels called mPrep™ capsules. Each mPrep/s capsule (micro- Preparation for specimens) entraps an individual specimen, while each mPrep/g capsule (micro-Preparation for grids) holds one or two TEM grids. Both mPrep capsule types connect to standard and widely used adjustable-volume laboratory pipettors in the same manner as pipette tips. T is provides for effi cient delivery of reagents directly to the specimens or grids entrapped in the capsules. Once a specimen or grid is inserted into an mPrep capsule, it may never require removal except to place grids into the microscope. T is capsule system eliminates almost all specimen and grid handling, greatly reducing the potential for damage, loss, or misidentifi cation because each capsule is readily labeled.


Materials and Methods


Several mammalian and one plant specimen were prepared and examined. Tissues from laboratory rats ( Rattus norvegicus ), Yucatan mini-pigs, and Dieff enbachia (dumb cane) plant tissue were prepared using mPrep/s specimen capsule processing followed by section staining on grids using mPrep/g capsules. Sections of vampire bat brain ( Desmondontidae ) were stained using mPrep/g capsule processing from specimens provided aſt er fi xation and embedding using conventional methods. Specimen processing . Rat and pig tissues were prepared from fresh specimens provided from other research investigations.


doi: 10.1017/S1551929515000760 www.microscopy-today.com • 2015 September


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