Abstract
Energy dispersive X-ray spectroscopy (EDS) carried out alongside scanning electron microscopy (SEM) is a common technique for elemental analysis. To investigate “wet” biological specimens, complex pre-treatments are required to stabilize them under the high vacuum conditions of high-resolution SEM. These often produce unwanted artifacts. We have previously reported that the polymerization of natural surface substances on organisms by the electron beam of the SEM setup or by plasma irradiation causes a nano-scale layer to form—called a “NanoSuit”—that can act as a barrier and keep organisms alive and hydrated in a field-emission SEM system. In the study reported herein, we examined the suitability of the NanoSuit method for elemental analyses of biological specimens by EDS. We compared experimental results for living Drosophila larvae and Aloe arborescens specimens prepared by the NanoSuit method and by conventional fixation. The NanoSuit method allowed accurate detection of the elemental compositions at high resolution. By contrast, specimens prepared by the conventional fixation method displayed additional EDS signals corresponding to the elements in the chemicals involved in the fixation process. Our results demonstrate that the NanoSuit method is useful for studying hydrous samples via EDS and SEM, particularly in biological sciences.
Highlights
When a sample is irradiated by an electron beam under high vacuum conditions in a scanning electron microscope (SEM), characteristic X-rays are generated
We have previously reported that at atmospheric pressure the surfaces of Drosophila larvae are covered in natural extracellular substances (ECS) (Supplementary movie 1)[3], which suggested that ECS might be components of the NanoSuit
The morphology of Drosophila larvae in scanning electron microscopy (SEM) images acquired under the same vacuum conditions were compared for the following three different sample preparation methods: (1) living specimens underwent electron beam irradiation for 60 min (Fig. 1a); (2) living specimens were placed in the SEM observation chamber for 60 min without concurrent electron beam irradiation (Fig. 1b); and (3) specimens were prepared by conventional fixation methods for SEM (Fig. 1c)
Summary
When a sample is irradiated by an electron beam under high vacuum conditions in a scanning electron microscope (SEM), characteristic X-rays are generated. Biological specimens, because of their high water content require extensive sample preparation to withstand the high vacuum conditions in the SEM and to permit electrical conduction through the biopolymers These conventional preparation methods involve complex pre-treatments of the biological samples such as chemical fixation, dehydration, freeze-drying or critical point drying, and metal c oating[2]. We have shown that plant surface substances act in a similar manner to the animal ECS when exposed to electron beam irradiation[7,8] In these investigations, we found that, when compared to specimens prepared by conventional fixation methods, the surface fine structure of organisms covered with a NanoSuit was intact and well preserved.
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