2.3 More microscopy Flashcards
Light microscopy advantages
Magnification between x1500 - x2000.
A range of specimens can be viewed (dead and life).
Euglena, daphnia
Smear preparations of blood cells.
Disadvantages of light microscope
maximum resolution power is only 200 nm (0.2μm) - any object that is closer than 200nm will seem as if it is one object.
Resolution power is small because of the magnitude of the wavelength of light - objects can only be distinguished if light can pass through them.
Electron microscopy
A beam of electrons with a wavelength of less than 1 nm is used to illuminate the specimen.
More detail can be seen because electrons have a much smaller wavelength than light waves.
They can produce images with magnifications up to x500 000 times.
Clear resolution.
TEM
Transmission electron microscope
Electrons passees through a very thin specimen to provide an image.
Electrons pass through the dense part of the sample less easily, giving more contrast.
Image is two dimensional.
Magnification is x500 000.
Resolving power of 0.5nm.
SEM
Scanning electron microscope
Electron beam does not pass through the sample.
Beams reflect or are bounced off the sample.
Only travels on the surface and are collected.
The final image is a 3D view.
Magnification is x100 000.
Resolving power of 3-10nm.
Sample preparation for electron microscope
The inside of an electron microscope is a vacuum to ensure the electron beams travel in straight lines.
Because of this, samples need to be processed in a specific way.
Specimen preparation involved fixation using chemicals or freezing, staining with heavy metals and dehydration with solvents.
Samples for a TEM will then be set in resin and may be stained again.
Samoks for a SEM may be fractured to expose the inside and will then need to be coated with heavy metals.
Suggest reasons for the following steps in the preparation of samples for electron microscopy
- Fixation
- dehydration
- embedding in resin
- staining with heavy metals
Fixation
fixation stabilise sample / prevents decomposition (1) /
Dehydration
dehydration prevent vaporisation of water in vacuum (1) vaporisation would damage sample (1) /
Embedding in resin
embedding allows thin slices to be obtained (1)
Staining with heavy metals
staining with heavy metals creates contrast (1) in electron beams (1)
Light microscope vs electron microscope
Inexpensive to buy and operate
Expensive to buy and operate
Small and portable
Large and needs to be installed
Simple sample preparation
Complex sample preparation
Does not lead to distortion
Sample preparation often distorts material
Vacuum is not required
Vacuum is required
Natural colour of sample is seen
Black and white images produced
Up to x2000 magnification
Over x500 000 magnification
Resolving power is 200nm
TEM: 0.5nm
SEM: 3-10nm
Specimens can be living or dead
Specimens are dead
Creation of artefacts
Structures that are produced due to the preparation process.
Specimens can be damaged by the electron beam.
An artefact is a visible structural detailed caused by processing the specimen and not a feature of the specimen.
Artefacts appear in both light and electron microscopy.
The bubbles that get trapped under the cover slip as you prepare a slide for light microscopy are artefacts.
When preparing specimens for electron microscopy, changes in the ultrastructure of cells are inevitable during the processing that the samples must undergo.
They are seen as the loss of cintiuity in membranes, distortion of organelles and empty spaces in the cytoplasm of cells.
Laser Scanning Confocal Microscopy
In fluorescent microscopes a higher light intensity is used to illuminate a specimen that has been treated with a fluorescent chemical (‘dye’).
Fluorescence id the absorption and re-radiation of light.
Light of a longer wavelength and lower energy is emitted and used to produce a magnified image.
A laser scanning confocal microscope
Moves a single spot of focused light across a specimen (point illumination)
This causes fluorescence from the components labelled with a ‘dye’.
The emitted light from the specimen is filtered through pinhole aperture.
Only light radiated from very close to the focal plane is detected
(the distance that gives the sharpest focus).
Light emitted from other parts of the specimen would reduce the resolution and cause blurring.
This unwanted radiation does not pass through the pinhole and is not detected.
A laser is used instead of light to get higher intensitues, which improves the illumination.
As very thin section of specimen are examined and light from eleswehere is removed, very high resolution images can be obtained.
The spot illuminating the specimen is moved across the specimen adn a 2D image is produced.
A 3D image can be produced by creating images at different focal planes.
LSCM is non invasive
Currently used in the diagnosis of diseases of the eye.
The future used for advanced for advanced optical microscopy include virtual biopsies, particularly in cases of suspected skin cancer
The beamspliiter us a dichroic mirror
Which only reflects one wavelength (from the laser)
But allows other wavelengths (produced by the sample) to pass through.
The position of the two pinholes means the light waves from the laser follow the same path as the light waves radiated when the sample fluoresces.
This means they will both have the same focal plane
Hence the term confocal.
Fluorescent tags
By using antibodies with fluorescent tages, specific features can be targeted and therefore studied by confocal microscopy
With much more precision than staining and LM.
Green fluorescent protein (GFP) is produced by a jellyfish,
The protein emits brigh green light when illuminated by ultraviolet light.
GFP molecules have been engineered to fluorescence different colours, meaning different components of a specimen can be studied at the same time.
The gene for this protein has been isolated and can be attached by genetic engineering to genes coding for protein under investigation.
The fluorescent indicates that a protein is being made and is used to see where it goes within the cell.
Provide a non-invasive technique to study the production and distribution of proteins.
Suggest whether fluorescent microscopy has a higher resolution than normal light microscopy
resolution the same (1) /
resolution limited by wavelength of light (1) /
fluorescence is light emitted (1) /
super resolved fluorescent microscopy has higher resolution (1)
Give one way that an image produced by a laser scanning confocal microscope differs from that produced by an electron microscope.
- lower resolution than an electron microscope.
- can have a fluorescent tag.
- can see movement (as can be used on living cells).
- can see, different layers / at different depths.
Explain why you would see more detail with an EM than an EM
Electron microscopes use electrons instead of light and electrons have a shorter wavelength than light (1)
which produces images with a higher resolution (1).
Define the term artefact
An artefact is a visible object (1) or distorted cell structure (1) present in an electron micrograph (or other micrograph) due to the sample preparation process (1).
