Fast Electron Microscopy fundamentals

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

chapter 1

Why do we need high throughput microscopy?

In life sciences, electron microscopy (EM) helps biologists to examine cells, cellular processes, and organelle architecture at nanometer-range resolution. Recent improvements in automation and stability have enabled EM to produce data for increasingly large biological projects, where large numbers of samples or large volumes are imaged and analyzed. Still, most EM workflows have issues which limit their power as a biological investigation tool. , The most important issues are the low throughput and the hands-on nature of electron microscopes. 

Due to the limited throughput of EM, microscope operators are forced to strike a compromise between the size or number of samples, the resolution used for imaging and the required imaging time. In addition, operators are often required to babysit the system to ensure consistent imaging quality throughout the project. By removing the bottleneck of throughput, the power of EM as a biological investigation tool increases drastically, since larger or more samples can be imaged automatically, without operator intervention.

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

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chapter 2

What fields benefit from high-throughput microscopy?

Higher throughput for EM is useful in many situations, be it processing multiple samples at the same time or imaging in larger projects, like volume EM or large area imaging.

Volume electron microscopy

Processes in life rarely occur in two dimensions, so three-dimensional EM or volume EM is especially helpful to understand the architecture of tissues or organisms. Most volume EM techniques can produce 3D data by imaging many subsequent sections of a sample, which are then reconstructed into a 3D representation for analysis.

Researchers in fields like cell biology and neurobiology rely heavily on volume EM to answer their research questions. Its high resolution is indispensable to visualize the nanoscale details that define individual cells. This also makes it a useful technique in the connectomics field, where the ability to resolve the intricate structures of neurons at nanometer-range resolution is indispensable to map the interactions between neurons in the brain.

The increased throughput of faster electron microscopes is highly beneficial for volume EM, as larger volumes can be imaged within a smaller amount of time. This opens the way to ever larger projects, like imaging an entire hemisphere or even entire brains for connectomics. At the same time, high-throughput imaging enables comparative studies, which were previously too time-consuming to be feasible.

Application: neurobiology, cell biology, histology, plant biology, biofilm analysis.

Large scale electron imaging

This mode High-throughput electron microscopy is highly useful for large-scale electron microscopy, where high-resolution imaging is performed on large samples ranging from tissues or organs. Large-scale imaging data provides both nanoscale information to analyze subcellular details, but also the context needed to understand the distribution of cell types within tissues or organs.

High-throughput solutions enable a rapid, unbiased method of data collection, which is crucial for large-scale imaging. The throughput facilitates studies with larger sample sizes, comparisons between healthy and diseased material, and screening of drug treatments or mutants, all while retaining nanometer-range resolution.

Application: digital pathology, analysis of tissues, cells, biomaterials, soft matter

High-throughput imaging in facilities

Imaging facilities often handle material from many different researchers, each with a different research question. This presents logistical and biological challenges. Researchers face a high turnaround time of data since data collection is a time-consuming process. At the same time, the slow speed of the EM means that there is a limit to the sample size, which in turn presents a risk of underestimating the heterogeneity of samples.

Automated, high-throughput microscopy enables a fundamental shift in EM: samples can be imaged automatically at high speed, while region of interest (ROI) selection and data analysis can be performed offline (away from the microscope). Since a microscope operator is no longer needed to identify relevant biological structures at the microscope, many or larger samples can be imaged within the same amount of time. Through the increased throughput, it is possible to routinely image many samples while retaining the biological context.

 

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chapter 3

How does FAST-EM enable high-throughput imaging?

FAST-EM’s throughput is achieved by a combination of several technological features. The system is a multibeam scanning electron microscope, capable of imaging with 64 beams in parallel. Combined with highly automated software and robust microscope components, the system sustains throughputs of up to a hundred times higher than a conventional electron microscope.

It is the only high throughput EM where image formation is achieved through a scanning transmission strategy, combined with optical detection. In comparison to typical EM detection modes, where secondary (SE) and backscattered (BSE) electrons are detected, transmission imaging generates higher contrast and superior signal to noise ratios (SNR). This detection setup can be used effectively at dwell times as short as 400 ns, which facilitates a high acquisition speed. Thus, it makes FAST-EM highly usable for high throughput imaging of biological material.

To image signal in transmission mode, samples are placed directly on a scintillator screen, which produces photons when hit by charged particles like electrons. The number of photons depends on the sample: electron dense regions will deflect more electrons away from the scintillator, which in turn produces less light, while electron-lucent regions will produce more light. The differences in light intensity can therefore be used as a mechanism to generate contrast.

The light produced in the scintillator is detected optically. A microscope objective beneath the scintillator projects the light produced by the 64 beamlets onto a fast and highly sensitive silicon photomultiplier (SiPM) array.

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Unique features of FAST-EM
  • High resolution, high-contrast images acquired with transmission electron detection
  • Easy acquisition procedure with continuous operation for a minimum of 3 days
  • Sustained throughput of 100 megapixels per second during routine imaging at 400 ns dwell time
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chapter 4

Sample preparation for high-throughput imaging

For electron microscopy of biological materials, high-quality specimen preparation is essential. The material should be treated to provide electron contrast and resist exposure to the electron beams and the chamber vacuum. The following sample preparation requirements are therefore typically addressed:

  • The sample should provide electron contrast. Biological specimens are usually treated with heavy metal staining agents like osmium tetroxide or uranyl acetate. Heavier elements provide better interaction with electrons, which enhances the visibility of organelles and protein complexes.
  • Ensure the sample withstands vacuum. For biological samples, this is achieved by dehydrating specimens with organic solvents like ethanol or acetone. Afterwards, embed the material in an acrylic or epoxy resin of choice, which helps retain the ultrastructure of the sample.
  • Samples should be sufficiently thin (<150 nm) for imaging. Since FAST-EM images with a transmission approach, electrons need to make it through the sample for detection. Samples embedded in resin can be sectioned using ultramicrotomy, which are then loaded onto a substrate for imaging.
  • Finally, samples need to be conductive to prevent charging by the electron beams. In the FAST-EM workflow, this has been addressed by coating the substrates with a conductive layer, which provides enough charge dissipation for thin sections.

Together, these treatments enable high-quality imaging of biological material. This type of sample preparation is routine for biological electron microscopy projects. Thus, no, or only minimal, alterations are needed to these protocols for material to be compatible with imaging in FAST-EM.

A basic protocol involves 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. Dehydration through a graded ethanol or acetone series
4. Resin embedding of the sample.
5. Ultramicrotome sectioning to a suitable thickness (<150 nm)
6. Placement of sections on substrates.