Ultrastructure of hydrated proteoglycans using a pulsed plasma source

During the past 10 years, attempts have been made to examine hydrated biological specimens by using wet chambers (at ambient temperature) [1-3] or cold stages (at -30 degrees C and below) during electron microscopic examination. Obtaining sufficient contrast of unstained hydrated biological samples has proven a considerable problem using both of these methods. Many fragile biological specimens, when examined hydrated, frozen or dried, are severely damaged by the electron beam and cannot be imaged by conventional scanning or transmission electron microscopy. In order to increase specimen contrast and eliminate electron beam induced trauma to the specimen, we have developed a wet-cell [4], which when used in concert with a pulsed plasma soft X-ray source, provides high contrast contact replicas of totally hydrated, unstained biological specimens. Although it has been postulated that hydrated unstained samples can be imaged by soft X-ray contact microscopy [5-7], to date there has been little success due to cell movement or degradation of the wet sample during the long exposure period necessary for an adequate imaging dose [8]. With the pulsed plasma source described in this study we have been able to use exposure times of approximately 40-60 ns while maintaining the sample in its hydrated state at atmospheric pressure. The resultant contact replicas exhibit good contrast and better than 30 nm spatial resolution when examined by conventional scanning electron microscopy.