A compound light microscope has two lenses - the objective lens, which is placed near to the specimen, and an eyepiece lens, through which the specimen is viewed.
The objective lens produces a magnified image, which is magnified again by the eyepiece lens.
This objective/eyepiece lens configuration allows for much higher magnification and reduced chromatic aberration than that in a simple light microscope.
Illumination is usually provided by a light underneath the sample.
Opaque specimens can be illuminated from above with some microscopes.
Maximum magnification of 1000x
Maximum magnification of 200nm
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Labelled Scanning Electron Microscope
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Labelled Transmission Electron Microscope
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Electron microscopes
In light microscopy, increased magnification can be achieved easily using the appropriate lenses, but if the image is blurred no more detail will be seen.
Resolution is the limiting factor.
In electron microscopy, a beam of electrons with a wavelength of less than one nm is used to illuminate the specimen.
More detail of cell ultrastructure can be seen because electrons have a much smaller wavelength that light waves.
They can produce images with magnifications of up to x500,000 and still have clear resolution.
Electron microscopes have changed the way we understand cells but there are some disadvantages to this technique.
They are very expensive pieces of equipment and can only be used inside a carefully controlled environment 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 improve a lot of these artefacts can be eliminated.
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Electron microscopes (continued)
There are two types of electron microscope:
In a 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 (0.5 nm).
In a 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 3-10nm, so the resolution is not as good as with transmission electron microscopy but stunning three-dimensional images of surfaces are produced, giving us valuable information about the appearance of different organisms.
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 flourescent microscopes a higher light intensity is used to illuminate a specimen that has been treated with a flourescent chemical .
Flourescence 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.
A laser scanning confocal microscope moves a single spot of focused light across a specimen (point illumination).
This causes flourescence 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 is detected.
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Laser scanning confocal microscopy (continued)
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 it not detected.
A laser is used instead of light to get higher intensities, which improves the illumination.
As very thin 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 two dimensional image is produced.
A three dimensional image can be produced by creating images at different focal planes.
Laser scanning confocal microscopy is non-invasive and is currently used in the diagnosis of diseases 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.
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Laser scanning confocal microscopy (continued 2)
The future uses for advanced optical microscopy include virtual biopsies, particularly in cases of suspected skin cacer.
The beam splitter is a dichroic mirror, which only reflects one wavelength (from the laser) but allows other wavelengths (produced by the sample) to pass through.
The positions of the two pinholes means the light waves from the laser follow the same path as the light waves radiated when the sample flouresces.
This means they will both have the same focal plane, hence the term confocal.
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