Light microscopy
The light microscope is the main tool that has been used to look at biological specimens for many years and is still very much in use today. Very small subjects, such as bacteria, can be looked at whole under a microscope; larger tissues must first be chemically preserved, embedded in a supporting material such as wax and sliced very thinly. They are then mounted on glass slides and often stained before viewing to pick out particular features. The microscope works by light being focused by a concave mirror and/or a condenser before it passes through the specimen and into an 'objective', which magnifies the subject before it is viewed through the eyepiece. Certain components of the tissue, such as nerves or particular proteins, can be viewed by staining them with specific dyes. Some of these are weakly fluorescent and show up only when filters are used to block out all light except that of the same wavelength as the fluorescence.

One kind of light microscopy, seen in this display, uses differential interference contrast optics. This relies on polarized light being slightly bent as it passes through tissues. The differences in the composition of different cells cause slight changes in the bending of the light. After the light has passed through the tissue it passes through another polarizer. Tissues that have bent the light to differing degrees appear darker or lighter, thus enhancing contrast and giving an almost three-dimensional appearance. An example of this technique is the chick neural tube image.

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Transmission electron microscopy
This type of microscopy uses an electron beam instead of light to visualize biological tissues. The beam is focused with electromagnetic lenses and goes through a thin section of the specimen. It is then expanded thousands of times by another series of lenses until it reaches a photographic plate or a light-emitting screen to form an image. The entire process takes place within a high vacuum chamber. Typically, magnifications of 1000 to 200 000 times actual size are obtained, and structures which are less than one nanometre (one- thousandth of a millimetre) apart can be resolved. The electron beam - generated by electric current through a filament - is accelerated by a high- voltage potential and thus can penetrate through the section with limited distortion or absorption; for this the specimen section has to be very thin (typically one ten-thousnadth of a millimetre) and firmly supported. To make thin sections, specimens are chemically hardened and then embedded, usually in an epoxy resin that is prepared as a fluid and then polymerized as extremely hard blocks. The sections are soaked with a solution of heavy atoms (such as lead, tungsten and uranium) to increase differentially the density, or 'contrast', of its components. Different stains can also be used to highlight different features in the sample. This can be seen in the images of collagen fibrils where the cationic dye, cuprolinic blue, shows proteoglycans projecting out from the fibrils.

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Scanning electron microscopy
This technique uses an electron microscope to visualize surface features of a subject from low to very high magnifications. Biological specimens must be chemically preserved and impregnated with a chemical called osmium, then dehydrated with increasing concentrations of alcohol. They are then dried, mounted on an aluminium stud and viewed under the electron microscope. The incident electron beam causes electrons to be emitted from the surface of the subject and it is the pattern of this electron emission that forms the image. Sometimes specimens are first sprayed with a very thin layer of gold to improve the emission of electrons from the surface. The image can either be photographed directly from a high-resolution screen, or processed by a computer to generate a digital image. The images produced by both transmission and scanning electron microscopy are always black and white. They can however be colour-enhanced on the computer to help distinguish particular features.

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Confocal microscopy
Traditionally, biologists have physically sliced through specimens in order to look at internal structures with a conventional light or electron microscope. The laser scanning confocal microscope, however, makes optical sections through a whole intact subject. It uses a computer-controlled laser beam to scan a pin point of light at a fixed depth within the specimen and rejects the out-of-focus information from other planes, thus providing a crisp, high-resolution image at that depth. One or more specific components of the specimen, such as a protein, is 'labelled' with a fluorescent stain. The laser stimulates this fluorescent stain to emit coloured light, which is detected by a photomultiplier tube and digitally stored by the computer. Up to three different-coloured fluorescent markers, each indicating a different component, can be used at once. By progressively changing the plane of focus, one can section the entire specimen optically, producing a sharp image of the fluorescently marked components for many different depths. A confocal reconstruction is produced when all these layers are put together to provide a sharp two-dimensional representation of the three-dimensional information.

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Scanning probe microscopy and atomic force microscopy
Scanning probe microscopy (SPM) is the generic name for a family of local probe techniques that operates at the nanoscale (a nanometre is one-millionth of a millimetre). Atomic force microscopy (AFM) is one member of this family and provides true three-dimensional images of a surface at nanometre resolution. It does this by measuring the nanonewton-sized forces (a newton is a unit of force) between the surface and the ultra-sharp tip of a force-sensing cantilever (this may be thought of as a soft spring). The cantilever is scanned in a grid pattern over the surface by piezoelectric transducers, which are capable of moving sub nanometre distances in response to applied voltages. This nanomechanical tool then responds to the surface topography in much the same way as a record player stylus responds to the varying topography of the groove in a record. The deflection of the cantilever is detected by a simple and elegant optical lever Ð laser light shone onto the back of the cantilever is reflected onto a photodiode and the voltage measured is proportional to deflection. Measured at each point of a two-dimensional array, this information builds up to yield a three-dimensional image. Colours may be generated by a computer linked to the microscope. Colour is often used to pick out the contours of the surface relief, making it easier to interpret the image.

AFM is ideally suited to biological samples as it is a low-energy technique, the material does not require any specialized pre-treatment, and the microscope may be used in liquid. So living cells can be imaged, as can a variety of dynamic processes such as crystal growth, protein film formation, and degradation of starch by enzymes. Recent advances in this rapidly developing field are permitting even the softest materials to be imaged through intermittent contact (tapping) and non-contact oscillatory modes of tip motion. For further information, please visit the web site of the Bristol Physics SPM group: spm.phy.bris.ac.uk

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