Evaporation-Controlled Automated Embedding and Polymerization


Jan L.M. Leunissen1,3 , Stacie Kirsch and Al Coritz2


1. Aurion ImmunoGold Reagents, Wageningen, The Netherlands 2. Electron Microscopy Sciences, Hatfield PA, USA


3. Microbiology and Immunology Dept, University of Otago, Dunedin, New Zealand


Resin embedding is traditionally performed via sequential applications of a graded series of solvent/resin mixtures [1]. The procedures are time consuming (see also [2]) and the chemicals used can pose health hazards. In addition there is an amount of waste, usually containing both solvent and resin and often larger in volume than the polymerized specimens. When processing large numbers of samples there is also hands-on time to consider. Finally there is the risk of losing specimen material especially in the exchange steps with high viscosity resin.


We have looked into alternatives for the classic embedding procedures, aiming to improve on the drawbacks described above. We describe an approach and an instrument that


(i) automates embedding from the first solvent/resin step up to pure resin,


(ii) reduces solvent and resin use with no waste, except what is left in the mixing vials,


(iii) minimises exposure to hazardous and irritating chemicals, (iv) reduces hands-on time to the bare minimum, (v) facilitates the processing of up to 52 samples in one instrument run and (vi) prevents any specimen loss.


These goals were achieved by eliminating the step-procedure and removing solvent by making use of its volatility. Common solvents for dehydration and embedding are highly volatile whereas most resin components do not evaporate readily. Evaporation depends on the kinetic energy of solvent molecules, the degree to which solvent is held back by the resin and how easily molecules can stay in the gas phase (saturation pressure). Temperature plays the most prominent role, next to vacuum pressure and the concentration of ‘solvent’ gas molecules in the gas phase over the samples. Evaporated solvent needs to be removed or diluted by flushing air over the liquid surface at set intervals.


To be able to have full control over temperature, pressure and flushing with fresh air, an instrument was developed that achieves these goals, the EMS Poly III. At the heart of the instrument is a specimen chamber that is temperature controlled over a range of 10°C - 70°C. The pressure in the chamber can be reduced from ambient pressure to a controlled level as low as 10kPa with a built-in vacuum pump. The instrument chamber accepts up to 52 BEEM specimen vials. Preset and user-adjustable programs coordinate temperature, pressure and pressure-increase-induced flushing. This results in an efficient and reproducible removal of solvent from the specimens. Initial steps aiming at bulk removal of solvent are followed by steps for the thorough removal of trace amounts.


In practice: the instrument can be loaded with specimens in e.g. acetone/resin by the end of a workday The next morning the vials are ready for polymerization once they have been topped up with pure resin to compensate for the removed solvent


The efficacy of the approach was evaluated by embedding yeast cells (see micrograph at right) in epoxy resin, using acetone as the solvent according to protocol. This leaves the specimens covered in a thin layer of resin by the end of the embedding run.


Yeast cells were fixed with glutaraldehyde in cacodylate buffer, washed in distilled water and postfixed with 1% KMnO4 in distilled water. After washing in distilled water the cells were dehydrated in a graded series of acetone/water mixtures: 50% - 70% - 80% - 90% - 100% - 2x100% absolute. Each step lasted 10 min. Cells were then resuspended in resin/solvent 1:3 (v/v) in BEEM capsules.


Once the BEEM vials had been transferred into the instrument chamber, the embedding program was started. Upon completion it was followed by the polymerization program, once the BEEM vials had been topped up with pure resin.


References:


[1] J. J. Bozzola, L.D. Russell “Electron Microscopy 2nd edition”, ed. Jones & Bartlett Publishers, Boston. [2] K. L. McDonald, Microsc. Microanal. 20 (2014) 152–163.


Acknowledgement: TEM image courtesy of OCEM, University of Otago


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