Abstract
As a topographical technique, Atomic Force Microscopy (AFM) needs to establish direct interactions between a given sample and the measurement probe in order to create imaging information. The elucidation of internal features of organisms, tissues and cells by AFM has therefore been a challenging process in the past. To overcome this hindrance, simple and fast embedding, sectioning and dehydration techniques are presented, allowing the easy access to the internal morphology of virtually any organism, tissue or cell by AFM. The study at hand shows the applicability of the proposed protocol to exemplary biological samples, the resolution currently allowed by the approach as well as advantages and shortcomings compared to classical ultrastructural microscopic techniques like electron microscopy. The presented cheap, facile, fast and non-toxic experimental protocol might introduce AFM as a universal tool for the elucidation of internal ultrastructural detail of virtually any given organism, tissue or cell.
Highlights
Our understanding of the detailed structural organization of biological material has ever since mainly been determined by microscopic techniques
The ultra-sectioning, immobilization and dehydration of polyethylene glycol (PEG)-embedded C. elegans individuals yielded samples accessible under ambient conditions by tapping mode Atomic Force Microscopy (AFM), enabling the ultrastructural depiction of typical tissues of this nematode commonly used in life science experiments
In order to obtain fast, facile as well as cheap nanoscopic insight into internal features of organisms, tissues and single cells by atomic force microscopy, a combination of polyethylene glycol embedding, ultra-sectioning, immobilization, dehydration and subsequent data-acquisition in ambient conditions by intermittent contact mode AFM was successfully applied to a variety of representative biological specimens
Summary
Our understanding of the detailed structural organization of biological material has ever since mainly been determined by microscopic techniques. The plethora of our ultrastructural knowledge of organisms, tissues and cells is mostly derived from different electron microscopic approaches such as transmission or scanning electron microscopy. With AFM, the application of vacuum to the sample in order to create imaging information was no longer needed, it became possible to gain ultrastructural data of biological material under physiological conditions[9, 10]. Besides the mentioned advantages of AFM over EM, one striking limitation still caused major problems in the acquisition of ultrastructural data of internal features of biological samples in the past. Overcoming the mentioned problem promises abilities concerning the acquisition of ultrastructural data of biological samples by AFM compared to EM, e.g. the option to access precise z-axis data as well as the viscoelastic properties of a given sample only to name a few
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