2.3 More microscopy Flashcards
What is the limiting factor in light microscopy?
Resolution
Define electron microscopy.
Microscopy using a microscopy that employs a beam of electrons to illuminate the specimen.
Since electrons have a much smaller wavelength than light, they produce images with higher resolutions than light microscopes.
Electron microscopy - what is used to illuminate the specimen?
A beam of electrons with a wavelength of less than 1 nm.
Electron microscopy - how can more detail of cell ultra-structure be seen?
Electrons have a much smaller wavelength than light waves - they can magnify x500 000 with clear resolution
What are the disadvantages of electron microscopy?
- Expensive
- Only usable in controlled environment
- Specimens can be damaged by the electron beam
- Artefacts often made due to complex preparation process
What are the two types of electron microscope?
- Transmission electron microscope (TEM)
- Scanning electron microscope (SEM)
How does a transmission electron microscope (TEM) work? What is its resolving power?
A beam of electrons is transmitted through a specimen and focused to produce an image
Produces the best resolution with resolving power of 0.5nm
How does a scanning electron microscope (SEM) work? What is its resolving power?
What’s the good thing about SEM?
A beam of electrons is transmitted across the surface of a specimen and reflected electrons are collected
- Resolving power is 3-10 nm
- 3D images can be produced, providing us info about the appearance of organisms
Compare light microscopy with electron microscopy
LIGHT vs ELECTRON
- Cheap - Expensive
- Small/portable - Large, requires installation
- Simple sample prep - Complex sample prep
- Sample prep doesn’t - Sample prep often causes
cause distortion distortion
- Vacuum not required - Vacuum required
- Natural colour of sample - Black and white images
- x2000 magnification - x500 000 magnification
- 200nm resolving power - RP of: TEM = 0.5nm
SEM - 3-10nm
-Specimens can be live/dead -Specimens are dead
In terms of microscopy, what is an artefact?
A visual structural detail caused by processing the specimen - not a feature of the specimen
(appears in both electron and light microscopy)
E.g. bubbles trapped under cover slip during prep for electron microscopy are artefacts
How does a laser scanning confocal (LSC) microscope work?
-Moves a single spot of focused light across a specimen, causing fluorescence from components labelled with a dye
(Fluorescence is the absorption and re-radiation of light)
-Light emitted from the specimen is filtered through the pinhole aperture
-Only light radiated from close to the focal plane is detected
Why are high resolution images obtained using a laser scanning confocal (LSC) microscope?
- Thin sections of specimen are examined
- Light from elsewhere is removed
Define resolution
Shortest distance between 2 objects that are still seen as separate objects.
Define fluorescence
Absorption and re-radiation of light
How do fluorescent microscopes work?
Higher light intensity used to illuminate a specimen treated with a fluorescent dye
What is a laser scanning confocal microscope?
Microscope that uses a laser beam and pin-hole aperture to produce an image with very high resolution
Why is blurring reduced in laser scanning confocal microscopy?
Light emitted from other parts of the specimen doesn’t pass through the pinhole aperture.
So high resolution images are obtained.
What is the benefit of using a laser instead of light in laser scanning confocal microscopy?
To get higher illumination
How are 2D images produced with LSCM?
Spot illuminating the specimen is moved across the specimen
How are 3D images produced with LSCM?
By creating images at different focal planes
What is the beamsplitter in LSCM?
A dichroic mirror which only reflects one wavelength, from the laser, but allows other wavelengths, from the sample, to pass through
What does the position of the 2 pinholes in LSCM mean?
Light waves from the laser illuminating the sample follow the same path as light waves radiated when the sample fluoresces.
This means they have the same focal plane, hence the name confocal
