Cryo-scanning electron microscopy of plant samples without metal coating, utilizing bulk conductivity

A classical problem in scanning electron microscopy is the alteration of specimen surfaces due to sample preparation. Simplified techniques for sample preparation with minimized surface alteration are therefore desirable. For fresh (hydrated) biological material, cryo-SEM after preceding shock-freezing and metal-coating is favoured when the prevention of drying artifacts has priority. A detailed knowledge of the sample properties and behaviour can facilitate new ways to optimize or simplify preparation and microscopy. Above ca.-100oC, ice has a measurable electrical conductivity due to its proton-conducting properties, which is sufficient to prevent frozen hydrated samples from charging during scanning electron microscopy, at least at low beam intensities. The examination of frozen samples without metal coating enhances the flexibility and versatility of cryo-SEM and simplifies sample preparations. We present some applications for the study of botanical samples, including compositional contrast imaging using backscattered electrons (BSE), and the study of wetting phenomena. The capabilities of simplified procedures are here demonstrated, particularly in combination with slow-freezing and the use of suitable test liquids for certain applications. The techniques can be easily adopted for conventional SEM setups. Corresponding author details Hans-Jürgen Ensikat, Nees Institute for Biodiversity of Plants,