Scanning Electron Microscopy (SEM): Its application and Working

 

Introduction

The electron microscopy technique was first time laid by Max Knoll in 1935. However, various scientists worked for many years to enhance its functionality. In recent times, cost of this microscope can be anywhere between $70,000 to $2000,000. In the field of Microbiology, Scanning Electron Microscopy (SEM) describes topography or surface morphology of the specimen, but not the internal structures for which Transmission Electron Microscopy (TEM) is required. Thus, SEM emerges as a crucial tool for exploring the complexity of microbial structures. Its capability helps to provide fundamental understanding of the ultra-structures like bacterial cell walls, flagella, pili, and various cellular structures.

Beyond mere visualization, SEM serves in exploration of various hidden structures such as “biofilms” made by complex microbial communities which allow them to attach onto diverse surfaces, including host cells. This information helps to understand the secrets of pathogenesis caused by harmful microbes, and analyze the dynamics of host-microbe relationships. The versatile application of SEM also extends to pharmaceutical research, where it inspects the effectiveness of antimicrobial agents and methods of drug delivery systems at the microscopic level.

SEM utilizes a focused beam of electrons, light microscopy relies on visible light

 

Difference between resolution of SEM Versus Conventional Light Microscopy

Why to use Electrons?

·       It’s all about resolution. In microscopy, resolution is often described as the ability to recognize the smallest gap between two points in an image, allowing these points to be seen as separate and distinct. For this to occur, the wavelength (λ) should be smaller than the distance between those two points in an image.

     A smaller wavelength, resolved finer details of a specimen, leading to higher magnification. Wavelength(λ) of electrons in SEM remains 5000 times less than λ of visible light (380-700nm).  That is why, an electron beam of λ 0.02 to 0.008 nm is used in SEM.

Comparing resolution: Large λ in light microscopy gives low resolution as compared to SEM using electron beam of smaller λ giving high resolution 

·       This much low wavelength allowing it to be effectively resolve structure of all microorganisms including viruses (averaging 50nm of size) in high resolution. In some conditions, SEM can resolve structure as small as 1nm (size of DNA). So, based upon its resolution, the useful magnification of SEM can be up to 2 million times.

·    The electron beam scans the surface of the specimen in raster pattern, providing detailed information about the topography, including its microstructures present on the surface of specimen.

Raster scanning pattern

·      The scanning process must be conducted in a vacuum chamber because negatively charged electrons can scatter upon interacting with air molecules, resulting in diffraction from the path and energy loss.

 Working of SEM

Preparation of specimen for SEM

•  Dip the specimen in primary fixative i.e., Aldehydes (glutaraldehyde and formaldehyde) for at least 2 hours to crosslink the protein and stabilize the structure of the specimen.
• Use a Secondary fixative (osmium tetraoxide) to fix the lipid membranes as during dehydration process use of solvents may remove the lipid membrane.
• Dehydrate the specimen by dipping the specimen in increasing concentration of alcohol or acetone (70%, 80, 90 and 100%). This stepwise dehydration protects the sample from shrinkage to some extent. Dehydration is a crucial step because, in a vacuum, the water in the specimen would begin to boil even at room temperature. This can compromise the structure of the specimen.
• Dry the specimen to remove acetone/alcohol. These solvents can evaporate by itself but this process makes cracks on the surface of specimen, therefore drying was carried out using liquid carbon dioxide.
• Take a copper stub to mount the dried specimen. Stick a double-sided conductive carbon tape onto the surface of the stub, and then stick the dried specimen on the surface of carbon tape to fix it in a place.
  
  
  
  
  
• As the specimen is non-metallic so obviously it is not conductive. Therefore, there is a need to coat the specimen with a thin (in nanometers) layer of heavy metal (gold, chromium, platinum, iridium or silver) using a sputter coater (using argon gas) under vacuum to make it conductive.

Scanning of specimen

    SEM has various parts working together to scans the specimen such as:

a)     An Electron Gun

Scanning is done by electrons, released by electron gun. The gun may have different sources to release electrons such as:

• Tungsten wire filaments used in first generation of electron microscopes to fire electrons. The tungsten used is a relatively cheap source of electron.
• Solid-state hexaboride crystals made up of cerium hexaboride (CeB₆) or lanthanum hexaboride (LaB₆). These crystals are known for their stability, long life and high electron emission, releasing 10 times more electrons than tungsten.
• Field emission guns (FEG) uses fine tip (single crystal of tungsten) of 100 nm with a strong electrostatic field around the tip which provides a very small diameter electron beam. This ensures release of bright and stable beam helps to produce a very high-resolution image.
  

Simplified SEM diagram

b)     Condenser lenses:

SEM uses electromagnets rather than conventional lenses. These lenses use magnetic fields to converge the electrons into a focused beam. A well-focused beam ensures the electrons to follow a vertical path and interact with the sample surface more effectively.

c)     Scanning Coil

These coils generate magnetic fields that move across the focused electron beam enabling electron beam to scans the sample back and forth, in a raster pattern. This raster scan collects signals from different points on the sample surface, contributing to the generation of an high resolution image.

d)     The objective lens

The objective lens is used to magnify the focused electron beam. The electron beam when hit the surface of the specimen, it emits:

• X-rays
• Primary backscattered electrons (trajectory of primary electron deviates due to positive charge of the nuclei present at the surface of the specimen).
• Secondary electrons (primary electrons transfers its energy to the specimen which knock off the electron from the surface of the specimen).

The objective lens is also designed to collect the backscattered electrons emitted from the sample surface.

e)      Detectors

Different detectors are used to collect signals from the specimen in the form of X-rays, backscattered electrons, and secondary electrons. Such detectors are:

• Backscattered Electron (BSE) Detector
• Secondary Electron (SE) Detector
• Energy-Dispersive X-ray Spectroscopy (EDS) Detector
• Wavelength-Dispersive X-ray Spectroscopy (WDS) Detector

 These detectors work together to capture different signals, that gives information about regions of the specimen having different atomic numbers, sample's surface characteristics and its chemical composition.

 

High Resolution SEM image

All the above information collected by different detectors of SEM creates a detailed and realistic image of the specimen's surface, highlighting features such as texture, morphology, and composition. This comprehensive imaging capability makes SEM invaluable in various scientific fields, including materials science, microbiology, geology, and nanotechnology, allowing researchers to study the fine details of specimens at the micro- and nanoscale.