Electron Microscopy Overview

Overview

This lecture by Evan Nogales, a professor of molecular cell biology at UC Berkeley, provides an in-depth introduction to electron microscopy (EM) as a powerful visualization technique in molecular and cellular biology. The lecture explains the fundamental physics behind EM, the main types of electron microscopy, sample preparation methods, and how these techniques are used to study biological structures and processes at high resolution. Examples from Nogales' own research, particularly on microtubules and molecular machines, are used to illustrate key concepts.

Types of Electron Microscopy

• Scanning Electron Microscopy (SEM):

  • Uses focused beams of low-energy electrons to scan the surface of bulky objects.
  • Produces low-resolution, grayscale images that reveal surface details; any color seen in images is artificially added.
  • Useful for visualizing the external morphology of organisms and objects at the micron scale.
  • Example images include crab larvae and dust mites, with scale bars indicating sizes in the hundreds of microns.
  • Magnification can be increased to visualize individual cells, such as mouse oviduct tissue with cilia.

• Transmission Electron Microscopy (TEM):

  • Uses high-energy electrons (hundreds of keV) to image thin samples.
  • Provides much higher resolution than SEM, allowing visualization of internal structures down to the atomic level.
  • Produces projection images of the entire thickness of the sample, not just the surface.
  • Example: TEM images of microtubules, cellular organelles, and protein complexes at the nanometer and even angstrom scale.
  • Capable of achieving atomic resolution in suitable samples, such as nanocrystals of silicon.

Physics and Image Formation in TEM

• Electron Properties and Interactions:

  • Electrons in TEM travel at nearly the speed of light, with energies in the range of hundreds of kilo-electron volts.
  • When electrons pass through a sample:
    • Some pass through without interacting.
    • Elastic scattering: Electrons are deflected by atomic nuclei without losing energy, changing direction and generating useful image contrast.
    • Inelastic scattering: Electrons lose energy to the sample, creating noise in the image and causing radiation damage to biological material.

• Contrast Mechanisms:

  • Amplitude Contrast:
    • Works similarly to x-rays; denser regions of the sample absorb or scatter more electrons and appear darker in the image.
    • Used for imaging sections of cells and tissues, such as plant roots, to reveal internal organelles.
    • Achieved by using an aperture to block elastically scattered electrons, enhancing contrast between dense and less dense regions.
  • Phase Contrast:
    • Essential for high-resolution imaging of proteins and macromolecular complexes.
    • Relies on the interference between scattered and unscattered electron waves, exploiting the wave nature of electrons.
    • Phase shifts in the electron wavefront as it passes through the sample generate contrast, allowing visualization of fine structural details.

• Microscope Structure:

  • The electron gun at the top produces electrons, which travel down a vacuum column.
  • Electromagnetic lenses (condenser, objective, and intermediate lenses) control illumination, focus, and magnification.
  • The sample is placed at the objective lens, often maintained at cryogenic temperatures (liquid nitrogen or helium) to reduce radiation damage.
  • Images can be viewed on a phosphor screen, TV, or recorded on film or CCD cameras.
  • Samples are mounted on tiny metal grids coated with a thin carbon layer, requiring only minute amounts of material.

Sample Preparation Techniques

• Negative Staining:

  • The sample is embedded in a solution containing heavy atoms (commonly uranium salts).
  • The solution is dried to a thin layer, and the sample is introduced into the electron microscope.
  • Heavy atoms provide high contrast, making it easier to visualize the sample.
  • Advantages: Fast, easy, and produces high-contrast images; suitable for beginners.
  • Limitations: Potential for artifacts (e.g., incomplete penetration of stain, collapse of protein structure during drying), and resolution is limited by the grain size of the stain (typically ~15 Å).

• Cryo-Electron Microscopy (Cryo-EM):

  • Samples are rapidly frozen in their aqueous environment, preserving their native, hydrated state.
  • Water is vitrified (becomes amorphous rather than crystalline), preventing damage from the vacuum and maintaining structural integrity.
  • Samples are kept at very low temperatures (liquid nitrogen or helium) during imaging.
  • Advantages: Excellent preservation of native structure, no artifacts from staining or drying, and high resolution is achievable.
  • Limitations: Technically demanding, requires precise handling and rapid freezing, and images have low intrinsic contrast, often requiring computational enhancement.
  • Radiation damage is reduced but not eliminated due to low temperatures slowing the movement of damaging radicals.

Achieving and Enhancing Resolution

• Challenges with Biological Samples:

  • Biological materials are highly sensitive to radiation and have low intrinsic contrast because their constituent atoms (carbon, nitrogen, oxygen) scatter electrons similarly to water.
  • High-resolution imaging is limited by the need to use low electron doses to avoid damaging the sample, resulting in noisy images.

• Technological Solutions:

  • State-of-the-art TEMs use energy filters to remove inelastically scattered electrons, reducing noise.
  • Samples are maintained at cryogenic temperatures (liquid nitrogen or helium) to minimize radiation damage and sample movement.
  • Atomic resolution is possible for robust, radiation-resistant samples (e.g., nanocrystals), but biological samples require special preparation and imaging strategies.

• Resolution in Practice:

  • Negative staining provides high contrast but limited resolution due to graininess and potential artifacts.
  • Cryo-EM allows for much higher resolution, even approaching atomic detail in favorable cases, but requires advanced instrumentation and computational processing.

Image Processing and 3D Reconstruction

• From 2D Images to 3D Structures:

  • TEM images are 2D projections of 3D objects, often noisy due to low electron doses.
  • To reconstruct 3D structures, multiple images of the same object in different orientations are collected, aligned, and averaged to enhance signal and reduce noise.
  • The process of combining these images to recover the 3D structure is called reconstruction.

• Types of Samples and Reconstruction Methods:

  • 2D Crystals: Proteins arranged in a single plane; require tilting the sample to obtain different views for 3D reconstruction. Computational processing is straightforward, and high resolution can be achieved.
  • Helical Arrangements: Molecules organized in helices (e.g., microtubules); different orientations are inherent in the structure, allowing 3D reconstruction from a single image. Medium to high resolution is possible.
  • Single Particles: Most common for biological samples; individual particles are randomly oriented on the grid. No tilting is needed, but computational processing is intensive. Resolution depends on sample symmetry and stability.
  • Electron Tomography: Used for unique, non-repetitive objects like organelles or cells. Multiple images are taken by tilting the same object, and 3D reconstruction is achieved by back projection. Interpretation is challenging due to complexity and noise.

• Example: Microtubule Structure and Dynamics:

  • Microtubules are studied using helical reconstruction and 2D crystal methods to reveal atomic details and understand assembly/disassembly processes.
  • Cryo-EM has been used to capture structural intermediates during microtubule polymerization and depolymerization, providing insights into cellular dynamics and the effects of drugs like taxol.

• Example: Single Particle Analysis of the Exosome:

  • Individual images of the exosome complex are picked, aligned, classified, and averaged to enhance structural details.
  • 3D reconstruction reveals the arrangement of subunits and functional regions, often combined with atomic models from crystallography in hybrid approaches.

• Example: Electron Tomography of Septin Filaments:

  • Tomography is used to study the organization of septin filaments in yeast cells during cell division.
  • Multiple tilted images are combined to reconstruct the 3D arrangement of filaments, membranes, and organelles, with segmentation used to simplify and interpret complex data.

Key Terms & Definitions

• Electron Microscopy (EM): Visualization technique using electron beams to image biological structures at high resolution.
• Scanning Electron Microscopy (SEM): Method for imaging surfaces of bulky samples.
• Transmission Electron Microscopy (TEM): Method for imaging thin samples at high resolution, revealing internal structures.
• Amplitude Contrast: Image contrast resulting from differences in electron absorption or scattering by dense regions.
• Phase Contrast: Image contrast generated by interference between scattered and unscattered electron waves.
• Negative Staining: Sample preparation using heavy metal stains to enhance contrast.
• Cryo-EM: Technique involving rapid freezing of samples to preserve their hydrated state for imaging.
• Vitrification: Rapid freezing that prevents ice crystal formation, maintaining sample structure.
• Helical Reconstruction: Computational method for reconstructing 3D structures from helical arrangements.
• Single Particle Analysis: Technique for reconstructing 3D structures from many images of randomly oriented particles.
• Electron Tomography: Method for reconstructing 3D structures from multiple tilted images of a single object.
• Segmentation: Process of simplifying complex tomographic data by tracing and highlighting specific structures.

Action Items / Next Steps

• Review the differences between SEM and TEM, focusing on their principles, applications, and image types.
• Study the physics of electron interactions and how amplitude and phase contrast are generated in TEM.
• Understand the advantages and limitations of negative staining and cryo-EM for sample preparation.
• Explore the methods for 3D reconstruction from 2D EM images, including single particle analysis, helical reconstruction, and electron tomography.
• Familiarize yourself with key terminology and concepts related to electron microscopy and image processing.