Sample Preparation for Scanning Electron Microscopy: The Surprising Case of Freeze Drying from Tertiary Butanol

Introduction For scanning electron microscopy, our ability to examine wet specimens continually improves thanks to advances in instrumentation; nevertheless, drying wet samples remains indispensable, particularly for high resolution or for a sample that needs to be imaged repeatedly. In many laboratories, the standard method for drying is critical-point drying. In this method, samples are dehydrated with ethanol and then infiltrated with cold liquid carbon dioxide, which is then heated up under pressure to pass through its critical point, forcing the carbon dioxide to undergo a transition from a super-critical liquid to gas. In doing so, the solvent molecules enter the gas phase more or less simultaneously throughout the sample, avoiding the formation of a liquid–gas interface and the accompanying large surface tension forces. Despite the widespread use of critical-point drying, it is not ubiquitous. In Japan, the standard method for drying wet samples is to freeze-dry them from tertiary butanol (t-butanol). In this method, samples are dehydrated with ethanol and then infiltrated with t-butanol. Infiltrated samples are frozen and then put under a vacuum, which sublimates the frozen alcohol, meaning it goes from the solid to the gas phase and thereby avoids a liquid–gas interface and the concomitant surface tension. Because t-butanol freezes at about 25°C, freezing and sublimation are readily accomplished. Freeze-drying from t-butanol gained popularity in Japan following the publication in 1988 of a paper by Inoué and Osatake [1]. These authors examined mouse tissues (pancreas, diaphragm, ciliated trachea, and red blood cells) and concluded that micrographs obtained from the t-butanol method were as good as, and sometimes better than, those obtained from critical-point drying. Attesting to the impact of this paper, when checked in September 2013 on the Web of Science, it had been cited more than 260 times, mainly by Japanese laboratories, in which this method is applied to diverse kinds of wet sample. Despite widespread use in Japan, freeze-drying from t-butanol appears to be little used elsewhere. For example, two recent comprehensive reviews of biological sample preparation for scanning electron microscopy omit the method altogether [2, 3]. Frankly, it is puzzling that freeze-drying from t-butanol could be standard operating procedure in one country and almost unheard of in another. To explore the use of t-butanol firsthand, we built an apparatus for freeze-drying, and we prepared samples for imaging with scanning electron microscopy. We examined sections of a plant seedling stem and the glans penis of a bat. Here, we show that these samples freeze-dried from t-butanol look as good as, or better than, those prepared by critical-point drying.