Imaging internal features of whole, unfixed bacteria

Abstract

Wet scanning-transmission electron microscopy (STEM) is a technique that allows high-resolution transmission imaging of biological samples in a hydrated state, with minimal sample preparation. However, it has barely been used for the study of bacterial cells. In this study, we present an analysis of the advantages and disadvantages of wet STEM compared with standard transmission electron microscopy (TEM). To investigate the potential applications of wet STEM, we studied the growth of polyhydroxyalkanoate and triacylglycerol carbon storage inclusions. These were easily visible inside cells, even in the early stages of accumulation. Although TEM produces higher resolution images, wet STEM is useful when preservation of the sample is important or when studying the relative sizes of different features, since samples do not need to be sectioned. Furthermore, under carefully selected conditions, it may be possible to maintain cell viability, enabling new types of experiments to be carried out. To our knowledge, internal features of bacterial cells have not been imaged previously by this technique.

The Condensation of Water on Adsorbed Viruses.

“…We estimate the resolution for our specimens in ESEM to be 10− 15 nm, while the wet-STEM was able to reach a resolution of 5 nm. 19,20 This range of values is comparable to those reported in the literature. 19,20 The samples were virions adsorbed on oxidized or functionalized silicon wafers, gold substrates, and on carbon films, the standard substrate for TEM.…”

“…19,20 This range of values is comparable to those reported in the literature. 19,20 The samples were virions adsorbed on oxidized or functionalized silicon wafers, gold substrates, and on carbon films, the standard substrate for TEM. However, the presence of water required special strategies to avoid sample damage, which can occur while recording a single EM image frame (see Supporting Information, Figure SI 2).…”

Fast, inexpensive, and reliable HPLC method to determine monomer fractions in poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

“…There are a number of analytical methods in literature for determining the monomer content of PHAs (Koller and Rodríguez-Contreras 2015 ; Tan et al 2014 ). Some methods, such as analysis with a transmission electron microscope (TEM) (Thomson et al 2011 ) or Nile red staining (Gorenflo et al 1999 ; Wu et al 2003 ; Pick and Rachutin-Zalogin 2012 ; Zuriani et al 2013 ), focus on the detection of the intracellular granules that contain the PHAs. Beside using harmful chemicals for TEM fixation, absolute quantification after staining with Nile red is often a problem.…”

Techniques for tracing PHA‐producing organisms and for qualitative and quantitative analysis of intra‐ and extracellular PHA

“…In addition, fewer artifacts are generated during “wet STEM” than normally reported for STEM preparation. As a drawback, “wet STEM” is reported to have reduced resolution .…”