Scanning Electron Microscopy (SEM) | Working Principles and application of SEM in biology

Summary

This video introduces the concept of Scanning Electron Microscopy (SEM), explaining its high-resolution capabilities for imaging biological and non-biological samples. Unlike light microscopes, SEM uses electron beams, offering better resolution due to shorter wavelengths. The video covers the main components of SEM, including the electron gun, electromagnetic lenses, and detectors, which work together to create detailed surface images. It also discusses the role of accelerating voltage in penetration depth and surface resolution. The video touches on the advantages and limitations of SEM, including its suitability for surface topology but not for internal cellular details.

Takeaways

• 😀 Scanning Electron Microscopes (SEM) are used to obtain high-resolution images and detailed surface information of samples, both biological and non-biological.
• 😀 SEM uses a focused beam of electrons instead of light, which enables much higher resolution compared to light microscopes (less than a nanometer vs. 200 nm).
• 😀 The resolution limit of a light microscope is around 200 nanometers, while SEM's resolution can be much better due to the shorter wavelength of electrons.
• 😀 SEM's resolution is a function of the accelerating voltage; higher voltage reduces wavelength, improving resolution.
• 😀 SEM consists of several key components: the electron gun (electron source), electromagnetic lenses (for focusing), deflection coils (for scanning), and detectors (for capturing emitted electrons).
• 😀 SEM uses secondary electrons, back-scattered electrons, and X-rays for imaging and elemental analysis. Each type provides different information about the sample's surface and composition.
• 😀 The SEM system works in a vacuum chamber, where samples are mounted and analyzed. The specimen can be tilted to obtain optimal imaging angles.
• 😀 The acceleration voltage affects both the surface resolution and penetration depth of the electron beam: higher voltage increases depth but reduces surface detail.
• 😀 Samples, especially biological ones, must be coated with conductive materials (e.g., gold, platinum) before imaging, as SEM requires conductive surfaces.
• 😀 SEM is best for imaging surface topology, but is not suited for examining internal structures of cells; for that, Transmission Electron Microscopy (TEM) is preferred.
• 😀 SEM is widely used across various fields such as nanomaterials, biology, and condensed matter physics, but it requires expensive equipment, skilled operation, and cannot image live cells.

Q & A

What is the main principle behind the operation of a Scanning Electron Microscope (SEM)?

The main principle behind SEM is that it uses a focused beam of electrons instead of light to scan the surface of a specimen, producing high-resolution images and detailed surface topology information.

How does SEM achieve better resolution compared to light microscopes?

SEM achieves better resolution due to the shorter wavelength of electrons compared to light. The wavelength of light is much longer, limiting light microscopes to a resolution of around 200 nanometers, while SEM can achieve resolutions down to less than a nanometer.

What is the role of the electron gun in an SEM?

The electron gun in SEM serves as the source of electrons. It generates the electron beam that is then focused and directed toward the sample using electromagnetic lenses.

Why is the wavelength of the electron beam in SEM smaller than that of light in optical microscopes?

The wavelength of the electron beam is smaller due to the higher energy and smaller size of electrons. The wavelength decreases as the accelerating voltage increases, allowing SEM to achieve finer resolution.

What are the three types of electrons detected in SEM, and what information do they provide?

The three types of electrons detected in SEM are secondary electrons, backscattered electrons, and X-rays. Secondary electrons provide surface topology details, backscattered electrons give compositional information, and X-rays are used for elemental analysis.

How does the accelerating voltage affect the SEM imaging?

The accelerating voltage determines the penetration depth and surface resolution. Higher voltage increases the penetration depth but reduces surface resolution, while lower voltage enhances surface detail but limits penetration.

Why must biological samples be coated before imaging with SEM?

Biological samples must be coated because they are typically non-conductive. Coating them with materials like gold or platinum prevents damage to the sample and improves imaging quality by allowing electrons to flow properly.

What are some advantages and disadvantages of using SEM?

Advantages of SEM include its high resolution, ability to analyze a variety of materials, and usefulness in surface topology analysis. Disadvantages include its inability to image live cells, the need for extensive sample preparation, and its focus on surface details rather than internal structures.

What is the main limitation of SEM when studying biological samples?

The main limitation is that SEM cannot image live biological samples. Only fixed, processed, and non-viable cells can be imaged, which restricts its use in studying living organisms.

What types of materials can be analyzed using SEM?

SEM can analyze a wide range of materials, including biological samples, metals, polymers, and nanomaterials, making it versatile for applications in various scientific fields like biology, physics, and materials science.