| Microscopy is any technique for producing visible images of structures or details too small to otherwise be seen by the
human eye.
In classical light microscopy, this involves passing light transmitted through or
reflected from the subject through a series of lenses, to be detected
directly by the eye, imaged on a photographic plate or captured digitally.
As resolution depends on the wavelength of the light, electron microscopy has been developed since the 1930's that use
electron beams instead of light. Because of the much lower wavelenth of the electron beam, resolution is far higher.
Microscopy usually involves the diffraction, reflection, or refraction of radiation incident upon the
subject of study.
There are also forms of microscopy, which work based on a very small probe, and recognizing perturbations of the end of the
probe, due to electrical effects. Examples are force
microscopy, electron tunnel microscopy and
derivatives.
The development of microscopy revolutionized biology and remains an essential tool
in that science.
See also: Microscope.
Types of Transmitted Light Microscopy
There are many different types of microscopes.
Light Microscopy: the contrast issue
Light microscopy can distinguish objects separated by down to 0.2 micrometers. Several optical configurations exist, depending
on the amount of contrast a specimen under study needs.
- Bright field
- Dark field
- Phase contrast
- Differential interference contrast (DIC)
Live cells in general lack sufficient contrast to be studied successfully. The problem here is that the internal structures of
the cell are colourless and transparent ie without enough contrast to see detail. In a normal (brightfield) light microscope,
contrast can generally be enhanced by closing the condenser aperture; however this will inherently reduce resolution to the point
that the image becomes useless.
The most obvious way is to stain the different structures with selective dyes, but this generally involves killing and
fixing the material followed by staining. Every part can and generally will induce artefacts. With the lifesciences nowadays
focussing on living cells, there was a need to develop optical methods to enhance the contrast. In general, these techniques make
use of differences in refractive index of e.g. the different cell organelles. It is comparable to looking through a glass window:
you don't see the glass but merely the dirt on the glass. There is however a difference as glass is a more dense material, and
this creates a difference in phase of the light passing through. The human eye is not sensitive to this difference in phase but
clever optical solutions have been thought out to change this difference in phase into a difference in amplitude (ie light
intensity).
Very old is the use of sideways (oblique) illumination, by covering part of the light entrance to the condenser. This
method will give the specimen a sense of relief. A more recent technique based on this method is Hoffmann’s modulation
contrast. This system is most often found on inverted microscopes for use in cell culture. Dark field illumination is
another well known technique where a cone of light is being produced by the condenser that will not reach the objective. Minute
particles will show up brightly on a dark background much like the dust that shows up in a beam of sunlight in an otherwise
darkened room (Tyndal effect).
More sophisticated techniques will show differences in optical density in proportion. Phase contrast is a widely used
technique that shows differences in refractive index as difference in contrast. It was developed by the Dutch physisist Frits
Zernike in the 1930's (for which he was awarded the Nobel Prize in 1953). The nucleus in a cell for example will show up darkly
against the surrounding cytoplasm. Contrast is excellent; however it is not for use with thick objects. Frequently, a halo is
formed even around small objects, which obscure detail. The system consists of a circular annulus in the condenser which produces
a cone of light. This cone is superimposed on a similar sized ring within the phase-objective. Every objective has a different
size ring, so for every objective another condenser setting has to be chosen. The ring in the objective has special optical
properties: it first of all reduces the direct light in intensity, but more importantly, it creates an artificial phase
difference of about a quarter wavelength. As the physical properties of this direct light have changed, interference with the
diffracted light occurs, resulting in the phase contrast image.
Superior and much more expensive is the use of interference contrast. Differences in optical density will show up as
differences in relief. A nucleus within a cell will actually show up as a globule in the most often used differential
interference contrast system according to Nomarski. However, it has to be kept in mind that this is an optical
effect, and the relief does not necessarily resemble the true shape! Contrast is very good and the condenser aperture can be
used fully open, thereby reducing the depth of field and maximising resolution. The system consists of a special prism in the
condenser that splits light in a ‘normal’ and a ‘reference’ beam. The spatial difference between the two
beams is minimal (less than the maximum resolution of the objective). After passage through the specimen, the beams are reunited
by a similar prism in the objective. In a homogenous specimen, there is no difference between the two beams, and no contrast is
being generated. However, near a refractive boundary (say a nucleus within the cytoplasm), the difference between the normal and
the reference beam will generate a relief in the image. Differential interference contrast uses polarised light to work properly. Two polarising filters have to be fitted in the light path, one
below the condenser (the polarizer), and the other above the objective (the analyser).
Fluorescence Microscopy
Fluorescence is the effect that certain compounds will send out light when illuminated with more energetic light. Often
specimen show their own characteristic autofluorescence image, based on their chemical makeup.
This method took a high flight in the modern lifesciences, as it can be extremely sensitive, with even detection possible of
single molecules. Many different fluorescent dyes can be used to stain different structures
or chemical compounds. Very powerful is the combination of antibodies coupled to a
fluorochrome as in immunostaining. Examples of commonly used
fluorochromes are fluorescein or rhodamine. The antibodies can be made very specific towards a chemical compound. For example, one strategy often
in use nowadays is by producing proteins artificially, based on the genetic code (DNA). These proteins can then be used to
immunize rabbits. The antibodies developed against those proteins are then coupled chemically to a fluorochrome and then used to
trace back the proteins in the cells under study.
Since recently, highly efficient fluorescent proteins such as the Green Fluorescent Protein GFP can be specifically fused on DNA level to the protein of interest. This combined fluorescent protein is not toxic
and hardly ever impedes the original task of the protein under study. Genetically modified cells or organisms directly express the fluorescently tagged proteins, which enables the study of the
function of the original protein in vivo.
Since fluorescence emission differs in wavelength (color) from the excitation light, a fluorescent image ideally only shows the structure of interest
that was labelled with the fluorescent dye. This high specificity lead to the widespread use of fluorescence light microscopy in
biomedical reseach. Different fluorescent dyes can be used to stain different biological structures, which can then be detected
simultaneously still being specific due to the individual color of the dye.
To block the excitation light from reaching the observed or the detector, filtersets of high quality are needed. These typically consist of an excitation filter selecting the range of excitation wavelengths, a dichroic mirror, and an emission filter blocking the excitation light. Most fluorescence microscopes are operated in the Epi-illumination mode (illumination and detection from one side of the sample)
to further decrease the amount of excitation light entering the detector.
See also total internal reflection fluorescence microscope.
Confocal scanning
- main article see Confocal
laser scanning microscopy
Generates the image by a completely different way then the normal visual bright field microscope. It gives slightly higher
resolution, but most importantly it provides optical sectioning without disturbing out-of-focus light degrading the image.
Therefore it provides sharper images of 3D objects. This is often used in conjunction with fluorescence microscopy.
Removing unwanted out-of-focus light is also possible by computer based methods (deconvolution). By supplying a stack of
images from a 3D object at different focal levels, it is possible to calculate which part of the image is out of focus and can
then be removed from the image.
Electron microscopy
For Light Microscopy the wavelength of the light limits the resolution to around 0.2 micrometer. In order to gain higher
resolution, the use of an electron beam with far smaller wavelength is used in Electron Microscopes.
- Transmission electron
microscopy (TEM) is principally quite similar to the compound light microscope, by sending an electron beam through a very
thin slice of the specimen. Resolution limit nowadays (2005) is around 0.05 nanometer.
- Scanning electron microscopy (SEM)
visualizes details on the surfaces of cells and particles and gives a very nice 3D view. It gives results much like the stereo
light microscope and akin to that its most useful magnification is in the lower range then that of the transmission electron
microscope.
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