Towards native-state imaging in biological context in the electron microscope

Abstract

Modern cell biology is reliant on light and fluorescence microscopy for analysis of cells, tissues and protein localisation. However, these powerful techniques are ultimately limited in resolution by the wavelength of light. Electron microscopes offer much greater resolution due to the shorter effective wavelength of electrons, allowing direct imaging of sub-cellular architecture. The harsh environment of the electron microscope chamber and the properties of the electron beam have led to complex chemical and mechanical preparation techniques, which distance biological samples from their native state and complicate data interpretation. Here we describe recent advances in sample preparation and instrumentation, which push the boundaries of high-resolution imaging. Cryopreparation, cryoelectron microscopy and environmental scanning electron microscopy strive to image samples in near native state. Advances in correlative microscopy and markers enable high-resolution localisation of proteins. Innovation in microscope design has pushed the boundaries of resolution to atomic scale, whilst automatic acquisition of high-resolution electron microscopy data through large volumes is finally able to place ultrastructure in biological context.

Three-Dimensional Imaging of Hepatic Sinusoids in Mice Using Synchrotron Radiation Micro-Computed Tomography

“…This fixation and staining made simultaneous functional imaging of the liver difficult in our system [23,29]. In addition, in the processes of fixation, postfixation with OsO 4 , and dehydration, the shrinkage and the collapse of morphological structure in the specimen can be induced [33]. However, we could not detect any artifacts in the overall structure of liver tissue when our results were compared with histological images.…”

Intracellular uranium distribution: Comparison of cryogenic fixation versus chemical fixation methods for SIMS analysis

“…This is a quick freezing technic to vitrify the cells water content (Dubochet, ) that immobilizes the sample chemical composition without changing the cell morphology. Other freezing techniques are used in connection with the volume of the sample (Hurbain & Sachse, ; McDonald, ; Mielanczyk et al, ; Vanhecke, Graber, & Studer, ; Weston et al, ). Then the sample is dehydrated at low temperature and embedded in a resin at room temperature (Bell & Safiejko‐Mroczka, ; Lešer et al, ; Weston et al, ).…”

Three-dimensional surface reconstruction and in situ site-specific cutting of the teliospores of Puccinia miscanthi causing leaf rust of the biomass plant Miscanthus sinensis

“…As a relatively novel approach to cross-sectioning of biological structures, including epicuticular waxes (Kim, 2013) and stomata (Kim et al, 2012), FIB-FESEM made possible the in situ site-specific cutting of nonembedded teliospores. In FIB-FESEM, a Ga ion beam, which is inherently destructive, is employed to mill or sputter away the specimen material (Weston et al, 2010). The electron-dense region of the cytoplasm indicated the presence of lipid droplets, which was confirmed using transmission electron microscopy.…”