Liquid phase electron microscopy of biological specimens
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LP-EM technology
Conventional electron microscopy requires biological samples to be in a fully solid state to withstand the high vacuum in the chamber of the electron microscope, which is usually achieved by either fixation, dehydration, and embedding or rapid freezing of hydrated specimens. However, life happens in water. As such, samples would intuitively be studied in their native liquid environment with electron microscopy, as is done with light microscopy. The desire to image biological specimens in their natural, liquid state was already expressed at the onset of electron microscopy in the 1940s, but liquid phase electron microscopy (LP-EM) with nanometer resolution has become available to the broad electron microscopy community only in the recent decade.1,2 Since then, the research community has observed an increase in publications on different biological samples imaged in liquid with transmission electron microscopy (TEM), and scanning TEM (STEM), in addition to several innovations in scanning electron microscopy (SEM).1,3–6 Here, a brief overview of current possibilities in LP-EM of biological samples is presented. Due to the constraints of this article, it is impossible to provide a comprehensive overview, so only highlights are given. We will discuss potential future developments of the field, and aim to answer the questions regarding the benefits and unique information that may be obtained from LP-EM.
Two different technological principles for LP-EM now exist since the early days of electron microscopy:7,8 open environment systems maintaining a liquid layer directly in the specimen chamber of the electron microscope, and closed systems in which the liquid is separated from the vacuum by at least one thin membrane through which the electron beam propagates. Variable pressure, also known as environmental SEM, has been available to the broad community the longest,9,10 but could only achieve limited resolutions for samples in liquid. This was finally improved in the mid 2000s by using STEM detection11 providing nanometer resolution on materials embedded in a thin liquid layer due to efficient detection in transmission mode.12 A liquid capsule with a polymer membrane was used in SEM in the early 2000s, though this method also had limited resolution.13 The ability to image samples fully embedded in liquid with nanometer resolution became available in late 2000s,14 using closed systems with silicon nitride membranes.15 The achievable resolution of LP-EM for nanoparticle-labeled proteins in a whole eukaryotic cell using a liquid flow specimen holder2 reached ∼4 nm, a similar resolution to that achieved for imaging labeled bacteria in a static liquid enclosure (no flow).16 In 2010, a third principle was introduced, atmospheric SEM (ASEM). ASEM consists of a modified cell culture
Diana B. Peckys, Department of Biophysics, Saarland University, Germany; [email protected] Elena Macías-Sánchez, Radboud University Medical Center, The Netherlands; [email protected] Niels de Jonge, INM–Leibniz In
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