Early results using high-resolution, low-voltage, low-temperature SEM (original) (raw)

Scanning electron microscopy: preparation and imaging for SEM

Methods in molecular biology (Clifton, N.J.), 2012

Scanning electron microscopy (SEM) has been almost universally applied for the surface examination and characterization of both natural and man-made objects. Although an invasive technique, developments in electron microscopy over the years has given the microscopist a much clearer choice in how invasive the technique will be. With the advent of low vacuum SEM in the 1970s (The environmental cold stage, 1970) and environmental SEM in the late 1980s (J Microsc 160(pt. 1):9-19, 1989), it is now possible in some circumstances to examine samples without preparation. However, for the examination of biological tissue and cells it is still advisable to chemically fix, dehydrate, and coat samples for SEM imaging and analysis. This chapter aims to provide an overview of SEM as an imaging tool, and a general introduction to some of the methods applied for the preparation of samples.

Cryomesh™: A New Substrate for Cryo-Electron Microscopy

Microscopy and Microanalysis, 2010

Here we evaluate a new grid substrate developed by ProtoChips Inc. for cryo-transmission electron microscopy. The new grids are fabricated from doped silicon carbide using processes adapted from the semi-conductor industry. A major motivating purpose in the development of these grids was to increase the low-temperature conductivity of the substrate, a characteristic that is thought to affect the appearance of beam-induced movement (BIM) in TEM images of biological specimens. BIM degrades the quality of data, and is especially severe when frozen biological specimens are tilted in the microscope. Our results show that this new substrate does indeed have a significant impact on reducing the appearance and severity of beam-induced movement in TEM images of tilted cryopreserved samples. Furthermore, while we have not been able to ascertain the exact causes underlying the BIM phenomenon, we have evidence that the rigidity and flatness of these grids may play a major role in its reduction. This improvement in the reliability of imaging at tilt has a significant impact on using data collection methods such as random conical tilt or orthogonal tilt reconstruction with cryopreserved samples. Reduction in BIM also has the potential for improving the resolution of 3D cryoreconstructions in general.

The future is cold: cryo-preparation methods for transmission electron microscopy of cells

Biology of the Cell, 2011

Our knowledge of the organization of the cell is linked, to a great extent, to light and electron microscopy. Choosing either photons or electrons for imaging has many consequences on the image obtained, as well as on the experiment required in order to generate the image. One apparent effect on the experimental side is in the sample preparation, which can be quite elaborate for electron microscopy. In recent years, rapid freezing, cryo-preparation and cryoelectron microscopy have been more widely used because they introduce fewer artefacts during preparation when compared with chemical fixation and room temperature processing. In addition, cryo-electron microscopy allows the visualization of the hydrated specimens. In the present review, we give an introduction to the rapid freezing of biological samples and describe the preparation steps. We focus on bulk samples that are too big to be directly viewed under the electron microscope. Furthermore, we discuss the advantages and limitations of freeze substitution and cryo-electron microscopy of vitreous sections and compare their application to the study of bacteria and mammalian cells and to tomography.

Introduction to high-resolution cryo-electron microscopy

Postepy biochemii

For many years two techniques have dominated structural biology - X-ray crystallography and NMR spectroscopy. Traditional cryo-electron microscopy of biological macromolecules produced macromolecular reconstructions at resolution limited to 6-10 Å. Recent development of transmission electron microscopes, in particular the development of direct electron detectors, and continuous improvements in the available software, have led to the "resolution revolution" in cryo-EM. It is now possible to routinely obtain near-atomic-resolution 3D maps of intact biological macromolecules as small as ~100 kDa. Thus, cryo-EM is now becoming the method of choice for structural analysis of many complex assemblies that are unsuitable for structure determination by other methods.

Simple method of thawing cryo-stored samples preserves ultrastructural features in electron microscopy

Histochemistry and Cell Biology

Preservation of ultrastructural features in biological samples for electron microscopy (EM) is a challenging task that is routinely accomplished through chemical fixation or high-pressure freezing coupled to automated freeze substitution (AFS) using specialized devices. However, samples from clinical (e.g. “biobanking” of bulk biopsies) and preclinical (e.g. whole mouse tissues) specimens are often not specifically prepared for ultrastructural analyses but simply immersed in liquid nitrogen before long-term cryo-storage. We demonstrate that ultrastructural features of such samples are insufficiently conserved using AFS and developed a simple, rapid, and effective method for thawing that does not require specific instrumentation. This procedure consists of dry ice-cooled pre-trimming of frozen tissue and aldehyde fixation for 3 h at 37 °C followed by standard embedding steps. Herein investigated tissues comprised human term placentae, clinical lung samples, as well as mouse tissues o...

3D solutions in transmission electron (Cryo) -microscopy in biology

One of the main problems in transmission electron microscopy in the biological field is the tri-dimensionality. This article explains the technical procedures and requirements to prepare biological specimens preserving them closest to their native state to perform 3D reconstruction of the macromolecular complexes and cellular structures in their natural environment. Handbook of instrumental techniques from CCiTUB

Cryo-electron tomography of biological specimens

IEEE Signal Processing Magazine, 2000

C ryo-electron tomography (CET) is an imaging technique capable of visualizing the three-dimensional (3-D) structure of complex viruses, cells, and tissues in a hydrated (and, thus, lifelike) state. With the current resolution of approximately 4-5 nm, CET can resolve supramolecular complexes that are responsible for many cellular functions. The fundamental mathematical principles needed for reconstructing a 3-D volume from a series of two-dimensional (2-D) projections were demonstrated in the early part of the twentieth century, and electron tomography was suggested in the 1960s. Nonetheless, the radiation sensitivity of biological samples meant that automated image acquisition procedures and improved structural preservation were necessary to achieve a practical outcome. These goals have now been realized. Digital signal processing was instrumental in the successful implementation of automated tomography, and it will most certainly play a decisive role in determining the feasibility of future advances. These include the ability to accurately align projection images without the invasive introduction of colloidal particles, faster reconstructions that take into account all frequency information, novel algorithms for efficient denoising and 3-D segmentation, and novel approaches to correlate high-resolution structures obtained by X-ray crystallography with electron density maps. The latter in particular will allow