Scanning Electron Transmission Phase Contrast
Feature Fluorescent Microscope
Microscope (SEM) Electron Microscope
Microscope
Uses electron beams Uses electron Enhances contrast Uses fluorescent dyes to
Principle to scan the surface beams transmitted by converting emit light when excited
of a specimen. through the phase shifts in by specific wavelengths.
Resolution ~1–10 nm specimen.
~0.1–0.5 nm light.
~200 nm ~10–30 nm
Magnificat Up to ~100,000x Up to ~2,000,000x Up to ~1,500x Up to ~2,000x
ion
Image 2D internal High-contrast live Bright fluorescent
3D surface images
Type structure images cell images images
Specimen Dry, coated with Ultra-thin, stained Live, unstained Fluorescently stained
Type metal (gold/ with heavy metals cells samples
Sample platinum)
Complex (fixation, Very complex Simple (no Requires fluorescent
Preparatio dehydration, (fixation, ultrathin staining required) dyes or antibodies
n
Live coating) sectioning, Limited (mostly fixed
No No Yes
Sample samples)
Observatio
Depth of High Low Moderate Low
Field
Contrast Electron Phase shift of
Electron scattering Fluorescent emission
Mechanis transmission & light
m
Usage/ scattering
Surface morphology Ultrastructure of Live cell imaging, Immunofluorescence,
Applicatio of cells, bacteria, cells, organelles, motility studies protein localization, cell
ns
Cost viruses,
High viruses,
Very Highproteins Moderate tracking
High
Radiation Electron Beam Electron Beam Visible Light UV or visible light
Used
Vacuum
Yes Yes No No
Requireme
nt
Artifact Possible due to Possible due to Possible due to
Minimal
Formation coating staining and photobleaching
Staining Yes (coating with sectioning
Yes (heavy metal
No Yes (fluorescent dyes)
Required? metal) stains)
Working Principles of Different Microscopes
1. Scanning Electron Microscope (SEM)::
• Uses a focused beam of electrons to scan the surface of the specimen.
• Electrons interact with the surface, generating secondary electrons and backscattered
electrons.
• A detector captures these emitted electrons and converts them into a digital image.
• Provides a detailed 3D surface morphology of the specimen.
• Requires the specimen to be coated with a thin layer of conductive material (gold/platinum)
to prevent charging.
2. Transmission Electron Microscope (TEM)::
• Uses a high-energy electron beam that passes through an ultrathin specimen.
• Some electrons are scattered, while others pass through to form an image on a fluorescent
screen or detector.
Feature Fluorescent Microscope
Microscope (SEM) Electron Microscope
Microscope
Uses electron beams Uses electron Enhances contrast Uses fluorescent dyes to
Principle to scan the surface beams transmitted by converting emit light when excited
of a specimen. through the phase shifts in by specific wavelengths.
Resolution ~1–10 nm specimen.
~0.1–0.5 nm light.
~200 nm ~10–30 nm
Magnificat Up to ~100,000x Up to ~2,000,000x Up to ~1,500x Up to ~2,000x
ion
Image 2D internal High-contrast live Bright fluorescent
3D surface images
Type structure images cell images images
Specimen Dry, coated with Ultra-thin, stained Live, unstained Fluorescently stained
Type metal (gold/ with heavy metals cells samples
Sample platinum)
Complex (fixation, Very complex Simple (no Requires fluorescent
Preparatio dehydration, (fixation, ultrathin staining required) dyes or antibodies
n
Live coating) sectioning, Limited (mostly fixed
No No Yes
Sample samples)
Observatio
Depth of High Low Moderate Low
Field
Contrast Electron Phase shift of
Electron scattering Fluorescent emission
Mechanis transmission & light
m
Usage/ scattering
Surface morphology Ultrastructure of Live cell imaging, Immunofluorescence,
Applicatio of cells, bacteria, cells, organelles, motility studies protein localization, cell
ns
Cost viruses,
High viruses,
Very Highproteins Moderate tracking
High
Radiation Electron Beam Electron Beam Visible Light UV or visible light
Used
Vacuum
Yes Yes No No
Requireme
nt
Artifact Possible due to Possible due to Possible due to
Minimal
Formation coating staining and photobleaching
Staining Yes (coating with sectioning
Yes (heavy metal
No Yes (fluorescent dyes)
Required? metal) stains)
Working Principles of Different Microscopes
1. Scanning Electron Microscope (SEM)::
• Uses a focused beam of electrons to scan the surface of the specimen.
• Electrons interact with the surface, generating secondary electrons and backscattered
electrons.
• A detector captures these emitted electrons and converts them into a digital image.
• Provides a detailed 3D surface morphology of the specimen.
• Requires the specimen to be coated with a thin layer of conductive material (gold/platinum)
to prevent charging.
2. Transmission Electron Microscope (TEM)::
• Uses a high-energy electron beam that passes through an ultrathin specimen.
• Some electrons are scattered, while others pass through to form an image on a fluorescent
screen or detector.
