Fast Electron Microscopy fundamentals

Learn everything you need to know about Fast EM imaging and how you can apply it to your research.

chapter 1

Why do we need high throughput microscopy?

Electron microscopy (EM) is a vastly used tool by biologists and life scientists to unveil structures of cells, cellular processes, and organelle architecture at nanoscale resolution. Obtaining images at such high resolution comes with a trade-off, where the field of view becomes limited.

Furthermore, making images at a high resolution is a time-consuming process. For instance, after the first EM implementations in biological sciences, EM functionality and structure evolved together with a researcher demand for more data and image resolution. This demand led to the development of volume EM machines, where image acquisition for big data sets could be obtained in a relatively short time. Various companies took the challenge and started to implement a lot of modifications and to debottleneck image acquisition, high throughput, and process automation to obtain high-resolution images of big datasets in a matter of days and hours.

Read more: optimizing for high sustained throughput in large-scale electron microscopy

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chapter 2

What is the working principle of Fast EM?

High throughput imaging in Delmic’s Fast EM is made possible by several improvements. It is the only volume EM system that is capable of image formation through Scanning Electron Transmission Microscopy (STEM). In this STEM system, image is formed by using an optical STEM concept where samples are placed directly on a carrier – scintillator. The carrier works as a converter of the transmitted electrons into the photons. The number of generated photons depends on the sample thickness. The thicker/denser sample results in the escape of more electrons from the sample surface and the scintillator, thus, resulting in lower light production. Therefore, a contrast in the image is generated by a local difference in density in the sample.

The produced localized cathodoluminescence is further captured, and the image is visualized by using optical microscopy. The cathodoluminescence spots are formed by the 64 individual beamlets that are created from a single field emitter source. These beams scan the sample, and signals are recorded in parallel using a fast and highly sensitive multi-pixel photon counter (MPPC) array.

The detection setup can be used effectively at various dwell times as short as 400 ns that results in high image throughput. In comparison to the EM detection modes, where secondary (SE) and backscattered (BSE) electrons are detected, STEM imaging generates higher contrast and superior signal to noise ratios (SNR). Thus, it makes Fast EM highly usable for imaging of biological material.

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The uniqueness of Fast EM
  • Acquired images with transmission electron detection are of high resolution, nanoscale precision, and high contrast
  • Easy acquisition procedure with continuous operation for at least 3 days
  • High sustained throughput based on 100 ns dwell time, 4 nm pixels:
    • 300 Mpx/s average, 640 Mpx/s peak
    • 8 Gbit/s average, 10.2 Gbit/s peak
chapter 3

How can I apply Fast EM to my research?

Fast EM can be used to explore cell architecture, the interaction of neuronal circuits, and the analysis of any biological material in life-sciences. 

Large volume 3D imaging

This mode can be used to visualize big data sets and reconstruct the 3D structure of the analyzed samples. This function is crucial for biological specimens as biological processes rarely occur in 2D. Thus, there is an increasing interest in techniques to study samples in 3D. Possibility of 3D reconstruction, together with large specimens imaging at nanoscale resolution, facilitated volume EM developments. 

Large volume imaging received a lot of applications in the field of connectomics, where neurons are tracked over vast distances throughout the brain or even the entire body.

Application: neurobiology, cell biology, histology, plant biology, biofilm analysis, and food engineering.

Large scale 2D imaging

This mode used to study big data sets enables an unbiased method of data collection and is more efficient for large samples than manually searching through the section for the structure that supports a hypothesis.

The high throughput feature is also highly suitable for studies where many samples need to be imaged, like to compare mutants or drug treatments. For example, to enable scientific data accuracy and hypothesis probability, hundreds of samples need to be imaged to allow them to achieve a representative sampling size. This results in time-consuming and tedious EM operation for hundreds of samples.

Read more: overcoming the challenges of large-scale electron microscopy

Application: digital pathology, analysis of tissue, cells, biomaterials and soft matter

A literal ‘Fast EM’

To speed up daily facility work.

This mode will enable EM facilities to analyze existing samples faster and decrease the manual workload of the personnel.

 

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chapter 4

Sample preparation for the Fast EM imaging

Without prior treatment, biological specimens are generally incompatible with EM for a variety of reasons. A most common one is samples instability under the electron beam, vacuum exposure, and little contrast of their own. Additionally, the samples are not conductive, so imaging may be distorted by charging. Several treatments are used to render samples suitable for imaging while retaining a morphology that is representative of their native state. This type of sample preparation is common in EM and can be used with minimal adaptations in Delmic’s Fast EM.

A common route to stabilize samples for EM is fixing the material with crosslinking chemicals like aldehydes, removing all water with acetone or ethanol, and embedding in resins like Epon™ or Durcupan™. These resins provide support to the specimen and enable consistent sectioning, which is crucial for large-scale and volume imaging.

To achieve a good contrast, most of the biological specimens are stained with heavy metals (osmium tetroxide, uranyl acetate) to enhance the visibility of organelles, protein complexes, and membranes in the EM. Staining is followed by resin embedding and slicing of the resin block. For the Fast EM technique, samples should be thin enough (<100 nm) for electrons to be transmitted into the underlying scintillator. Ultrathin sections are) are cut from the surface of the resin block with an ultramicrotome and transferred to the scintillator surface.

Typically, sections are coated with a conductive coating after deposition on the substrate. Since the Fast EM substrates come pre-coated with a conductive molybdenum layer, further treatment is not required before loading samples into the microscope.

A basic protocol would involve at least the following steps:

  1. Chemical fixation of the sample with fixatives such as glutaraldehyde and paraformaldehyde.
  2. Staining with heavy metals such as osmium, lead, and uranium
  3. Dehydrate the sample with graded ethanol or acetone series
  4. Resin embedding of the sample.
  5. Thinly slice embedded sample with an ultramicrotome.
  6. Place sections on the scintillator

These abovementioned steps are the standard practices in biological SEM/TEM sample preparation. Thus, no costly equipment is required.

It is important to wear protective clothing and work in the fume hood because chemicals used are toxic.