Electron Microscopy

Basic Principles

Electron microscopy (EM) works in a similar fashion to normal light microscopy, where a sample is imaged based on its interaction with visual light followed by magnification of the image with a series of lenses. In EM we use electrons instead of light, magnetic lenses instead of optical solutions, and the electrons interact based charge interactions with the target nuclei.

Why do we need electrons?+-

Any source used for imaging a sample can only resolve an object that is bigger than half of the respective wavelength. When we look at the range of visible light we see that the resolution is limited to 200nm (based on the wavelength range from 380nm - 700nm (Ultraviolet to infrared). If we want to go smaller in size we can exchange the imaging source used. Electrons used in EM have a typical wavelength of around 2pm - 4pm (0.002nm - 0.004nm), thus the wavelength is significantly smaller, which allows us to image drastically smaller objects.

What imaging method do we use?+-

In our lab we use transmission electron microscopy. This means we detect electrons that interacted and pass through the sample. This is only possible if the sample is thin (max 150nm) enough for the electron to penetrate.

Are there some disadvantages?+-

Like with every method there are some disadvantages. One of the main constraints comes from the sample source which is used in our research. Biological samples mainly consist of Carbon, Hydrogen, Nitrogen, Oxygen, Phosphorus and Sulfur. All of these elements have a relatively small mass which makes interaction between electrons and sample less likely. The result are noisy images that need to be processed after image acquisition.

Another constraint comes from the high potential energy of individual electrons. This will eventually cause radiation damage and drift that limits the quality of the micrographs.

Negative Stain EM

To increase the interaction between electrons and a biological sample we make use of negative staining. This means that we coat the sample with heavy metals (e.g. Uranyl Acetate) before imaging. Heavy metals have a higher mass in comparison to elements found normally in biology. As a consequence the electrons interact stronger in a negative stained sample which will lead to an increase in contrast in the final image.

Cryo-EM

Cryo-electron microscopy is used to determine the high-resolution three-dimensional (3D) structure of isolated protein complexes. Samples are flash frozen in a thin layer of vitreous ice and imaged at cryogenic temperatures by transmission electron microscopy (TEM), e.g., using the TFS Titan Krios microscope. The direct electron detectors (DEDs) of these instruments greatly increase the achievable image contrast, and allow ‘movie mode’ imaging. In this mode, a series of dose-fractionated images (i.e., movies that are containing 40 to 100 frames) of suitable  sample regions are recorded. The movie mode data collection allows sample drift to be detected and corrected. After the required alignment, the individual frames are merged to increase image contrast, i.e., to improve the overall signal-to-noise ratio.

The Falcon4 detectors employed allow recording image data as a series of X/Y electron impact coordinates, together with the time point of the arrival of the electron. Such "electron event recordings" can later be interpreted by software and transformed into movies of variable pixel resolution and a variable number of frames for a movie.

Each sample region imaged contains many protein complexes oriented in different ways. Their images, i.e., projections in 2 dimensional space (termed 2D projections) are matched, classified and reconstructed in a procedure called ‘single particle analysis’ to obtain the 3D structure of the complex. Alpha helices and beta sheets can be visualized by this method, and, since the introduction of DEDs, atomic resolution is frequently achieved.

Electron microscopy in Life Sciences can be grouped into several categories:

  1. Transmission Electron Microscopy (TEM) can be used with room temperature samples to perform first low-resolution studies of the samples.

  2. Cryo-Electron Microscopy (Cryo-EM) can be used with frozen samples to obtain atomic resolution structures of proteins.

  3. Cryo-Electron Tomography (Cryo-ET) provides 3D volume analysis of sections of cells or organelles or bacteria, and cryo-ET can also be used on isolated particles.

  4. Correlative Light and Electron Microscopy (CLEM) is a method that combines the analytic possibilities of fluorescense light microscopy with the high-resolution structural analysis of the electron microscope.

 

There are several excellent online courses about cryo-EM available.

  1. We recommend subscribing to the 3DEM email list. It has an impressive archive of earlier emails.
  2. The CCPEM email list is about single particle cryo-EM. It is also worth subscribing to.
  3. Grant Jensen's online class „Getting started in cryo-EM".  This 14-hour online class covers the fundamental principles underlying cryo-electron microscopy starting with the basic anatomy of electron microscopes, an introduction to Fourier transforms, and the principles of image formation. Building upon that foundation, the class then covers the sample preparation issues, data collection strategies, and basic image processing workflows for all 3 basic modalities of modern cryo-EM: tomography, single particle analysis, and 2-D crystallography.
  4. Lecture series on EM given at the MRC Laboratory of Molecular Biology - LMB
  5. Lecture series by the SBGrid consortium
  6. Lectures on various structural biology software including cryo-EM packages hosted by SBGrid consortium
  7. Lectures from workshops organized by the National Resource for Automated Molecular Microscopy (NRAMM)
  8. Eva Nogales' iBioSeminar on cryo-EM