MicroscopyMicroscopy is a technique for producing visible images of structures or details too small to otherwise be seen by the human eye. With the exception of techniques such as Force microscopy and electron tunnel microscopy, microscopy usually involves the diffraction, reflection, or refraction of radiation incident upon the subject of study. 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 or imaged on a photographic plate. There is also a form of microscopy, which works based on a very small probe, and recognizing perturbations of the end of the probe, due to electrical effects. The development of microscopy revolutionized biology by enabling the discovery of microbes. Microscopes remain an essential tool in that science.
TypesThere are many types of microscopy. White Light: The Contrast IssueLight microscopy can distinguish objects separated by down to .2 micrometers.
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. It is like 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 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 heve been thought out to change this difference in phase into a difference in amplitude (ie light intensity). In the microscope, contrast can generally be enhanced by closing the condenser aperture; however this will reduce resolution to the point that the image will become 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 lifesciences nowadays focussing on living cells, the need was there 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. Very old is the use of sideway (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 in a dark background. 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. 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 accorsding to Nomarski. However, it has to be kept in mind that this is an optical effect, and it 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 resolution of the microscope). 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). FluorescenceFluorescence 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 fuorochromes are fluorescein or rhodamine. The antibodies can be made very specific towards a chemical compound. Very often nowadays, proteins are being made artificially, based on the genetic code (DNA). These proteins can then be used to immunize rabbits, and the antibodies developed against those proteins are 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 fluorescent protein is not toxic and hardly ever impedes the original task of the protein under study. Genetically modified cells or organisms then directly express the fluorescently tagged proteins, which enables the study of the 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. Confocal scanningGenerates 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 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
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