Chapter 2- 2.3- More microscopy Flashcards
In light microscopes what is the limiting factors?
The resolution.
Why can we see more detail with electron microscopes?
In electron microscopy a beam of electrons with a wavelength of less than 1nm is used to illuminate the specimen. More details of cell ultrastructure can be seen because electrons have a much smaller wavelength than light waves.
They can produce images with magnifications of up to x500000 and still have clear resolution.
What are the disadvantages of using electron microscopes?
Electron microscopes have changed the way we understand cells but there are some disadvantages to this technique. They are very controlled environments in a dedicated space. Specimens can also be damaged by the electron beam and because the preparation process is very complex, there is a problem with artefacts (structures that are produced due to the preparation process). However, as techniques improved a lot of these artefacts can be eliminated.
There are two types of electron microscope?
Transmission electron microscope (TEM) a beam of electrons is transmitted through a specimen and focused to produce an image.
This is similar to light microscopy. This has the best resolution with a resolving power of 0.5 nm.
Scanning electron microscope (SEM) a beam of electrons is sent across the surface of a specimen and the reflected electrons are collected. The resolving power is from 3-10 nm, so the resolution is not as good as with transmission electron microscopy but stunning 3D images of surfaces are produced, giving us valuable information about the appearance of different organisms.
How is a sample prepared for electron microscopes?
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.
Specimens preparation involves fixation using chemicals of freezing, staining with heavy metals and dehydration with solvents.
Samples for a TEM will then be set in resin and may be stained again.
Samples for a SEM may be fractured to expose the inside and will then need to be coated with heavy metals.
What are the comparison points for light microscopes and electron microscopes?
Light microscope-Inexpensive to buy and operate whereas electron microscopes are expensive to buy and operate.
Light microscopes are small and portable whereas electron microscopes are large and needs to be installed
Light microscopes are simple sample preparation whereas electron microscopes are complex sample preparation.
Light microscopes sample preparation does not usually lead to distortion whereas electron microscopes sample preparation often distorts material.
Light microscope vacuum is not required whereas in electron microscopes a vacuum is required.
Light microscope- the natural colour of sample is seen (or stains are used up) whereas in electron microscopes black and white images produced (but can be coloured digitally).
Light microscopes- up to x2000 magnification and electron microscope is over x500000 magnification
Light microscope resolving power is 200nm whereas electron microscopes have a resolving power of transmission electron microscope is 0.5 nm and a scanning electron microscope is 3-10nm.
Light microscope- specimens can be living or dead whereas electron microscope- specimens are dead.
What is an artefact?
An artefact is a visible structural detail 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 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 continuity in membranes, distortion of organelles and empty spaces in the cytoplasm of cells.
Experience enables scientists to distinguish between an artefact and a true structure.
What is laser confocal microscopy?
Light microscopy has also continued to develop. Some of the latest technology produces images that are very different from electron micrographs but are just as useful.
Conventional optical microscopes use visible light to illuminate specimens and a lens to produce a magnified image. In fluorescent microscopes a higher light intensity is used to illuminate a specimen that has been treated with a fluorescent chemical (a fluorescent dye).
Fluorescence is the absorption and re-radiation of light. Light of a longer wavelength and lower energy is emitted and used to produce a magnified image.
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Explain what a laser scanning confocal microscope does?
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 a pinhole aperture. Only light radiated from very close to the focal plane (the distance that gives the sharpest focus) is detected.
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 intensities, which improves the illumination.
As very tin sections of specimen are examined and light from elsewhere is removed, very high resolution images can be obtained.
The spot illuminating the specimen is moved across the specimen and a 2D image is produced. A 3D image can be produced by creating images at different focal planes.
Is laser scanning confocal microscopy invasive?
Laser scanning confocal microscopy is non-invasive and is currently used in the diagnosis of disease of the eye and is also being developed for use in endoscopic procedures. The fact that it can be used to see the distribution of molecules within cells means it is also used in the development of new drugs.
The future uses for advanced optical microscopy include virtual biopsies, particularly in cases of suspected skin cancer.
The beamsplitter is a dichroic mirror, which only reflects one wavelength (from the laser) but allows 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.
Explain how fluorescent tags work.
By using antibodies with fluorescent “tags” specific features can be targeted and therefore studied by confocal microscopy with much more precision then when using staining and light microscopy.
Green fluorescent protein (GFP) is produced by the jellyfish Aequorea Victoria.
The protein emits bright green light when illuminated by ultraviolet light. GFP molecules have been engineered to fluoresce 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 proteins under investigation. The fluorescence indicates that a protein I being made and is used to see where it goes within the cell organism.
Bacterial, fungal, plant, and human cells have all been modified to express this gene and fluoresce. The use of these fluorescing proteins provides a non-invasive technique to study the production and distribution in cells and organisms.
