Microscopy Home PageThe microscope has a long and distinguished career in the service of mankind. Simple magnifying glasses were in use in the early and middle 1600s and development has continued ever since.
People with inquiring minds have always wanted to see beyond the limits of smallness and distance so the need for magnification with adequate resolution has always been (and remains) a goal for both microscopists and astronomers.
Description of form and behaviour are prerequisites for understanding, magnification with resolution is necessary if small or distant objects are to be described.
The simple magnifying glass was developed by the early spectacle makers. It was refined to a peak of performance by the Dutchman Leeuwenhoek, who was able to use very tiny spherical lenses to view bacteria only a few micrometres in diameter.
Holland remained a centre for microscope development and the compound microscope was developed there around the beginning of the 17th century. Due to problems of chromatic and spherical aberration it was probably less effective than Leeuwenhoek's simple lens, but it was easier to use and became the most popular type of instrument by the early 19th century.
For a much fuller early history, try Thomas Jones' History of the Light Microscope. It's also well worth looking at Naples University's Museum where you can see images of the microscopes in the collection, and there's an on-line index on the history of scientific instruments for more general coverage.
Chromatic aberration remained a serious problem until the middle 1800s. Once dispersion of light was understood, and a suitable variety of glasses became available, it was possible for both microscope and refracting telescope designers to improve performance. Multi-element objective lenses were perfected.
Spherical aberration was dealt with as lens designers and makers developed the mathematics and manufacturing skills to handle non-spherical surfaces with precision.
Good illumination is required for full performance of higher power objectives, and the substage mirror and condenser arrangement became the norm. Kohler in Germany formalised this, but even today poor illumination (through operator ignorance of the principles) is a common problem.
At the start of the 20th century microscopes had reached the theoretical limits of resolution imposed by the nature of light. The microscope had by now taken on pretty much its modern form. By this time, too, advances in techniqes for preparing specimens meant that microscopists could use thin, flat samples obtained by embedding and sectioning or by grinding and polishing.
Although the thin specimens helped to improve resolution, they had an unfortunate effect on contrast. They reduced it, in many cases to almost nothing!
Clearly, something had to be done. Various staining techniques were tried and some of these are still in regular use today. Other methods of contrast enhancement employed optical methods instead, relying on properties of the specimen other than colour and optical density.
Oblique illumination, darkfield illumination and its colourful Rheinberg offshoot all depend on scattering. Polarising filters provide information about an object's ability to rotate polarised light.
The best-known of these innovations must be the phase-contrast design brought onto the market by Zeiss between the wars, this effectively converts subtle phase effects into visible amplitude differences. Later developments include Nomarski interference contrast (also from Zeiss originally).
Fluorescence microscopy, first tried early in the 20th century, is another method of introducing visible contrast into an object. Specialised microscopes for this purpose were manufactured from around 1960 onwards, today's instruments often use epi-fluorescence instead of the standard sub-stage condenser.
By about 1970 the optical microscope had again reached a state which seemed incapable of further improvement. But the confocal technique invented by Marvin Minsky in the US about 1960 was already in use (though little known), and today's laser confocal scanning instruments derived from it have made further advances possible by the removal of out-of-focus blur and better penetration of thick samples.
Further innovation since 1985 has once more taken light microscopy way beyond the limits of what was once thought possible. The near field microscope uses a scanned probe with an aperture much smaller than a wavelength of visible light. Useful resolutions of around a fortieth of a wavelength are possible with equipment of this type, the scanned aperture being used either to illuminate the object or to feed the detector.
To see objects or details smaller than about half a wavelength of visible light, microscopists soon realised that they must use smaller waves. The first steps in this direction were taken early this century with ultra-violet light sources and photographic emulsions to capture the images. (The UV light could not be seen by eye and photomicrography was already in use with visible light.)
Focussing was difficult, and the gains in resolution were modest, so the technique was never very popular. Electron microscopy has its origins in the years before the Second World War, early experiments were made in Germany, Britain and the United States, and by 1950 transmission EM was becoming a very useful tool as methods of specimen preparation improved. Serious aberrations in electron lenses were more than compensated for by the dramatic improvement in wavelengths over those of the light microscope. Suddenly, for the right specimens, resolution increased 50 to 100 times or more.
Improved electron lenses and more stable power supplies, better vacuum systems and better beam sources have gradually enhanced the instrument until it is now capable of imaging atomic lattices in crystalline structures.
The scanning electron microscope (SEM) made its appearance a little later, being developed in Britain in the mid 50s following much earlier beginnings. The SEM improved resolution and depth of field as dramatically for solid surfaces as the TEM had done for thin sections.
Once again, specimen preparation lagged behind instrument capabilities, particularly for biological microscopy. Today resin embedding, thin sectioning with glass or diamond knives, cryo-preservation by rapid freezing, etching, ion beam thinning, sputtering and coating techniques are available to enable the microscopes to give good quality, meaningful results.
Analytical microscopy was developed early. Samples for the light microscope can be stained in ways that colour specific substances. One of the earliest methods must be the use of iodine solutions to localise starch in plant tissues.
Other substances involved more complex chemistry, performed in situ on the specimen before sectioning, or more commonly on the tissue sections immediately before examination. From the late 19th century until the middle of the 20th ever more elegant methods were developed. During the 1970s immunochemical methods came into favour and were rapidly developed in the 1980s. These highly specific and very sensitive procedures are now widely used in all kinds of plant and animal research, hospital pathology laboratories and in forensic science.
Similar methods are now available for electron microscopy, gold-labelling is used to locate the binding sites in the tissues. Much of this development took place in the 1980s.
Also in the 80s detectors were developed for electron microscopes which enabled the X-rays emitted by the specimen to be collected and analysed. The energies of the X-ray photons depend on the energy of the electron beam and on the elements present in the sample. Element maps can therefore be constructed by processing the data in a computer.
Light and electron beams are not the only possible sources of microscopic images. Many alternatives have been tried, mainly since about 1960. Some of the methods used are X-ray microscopy by shadow projection using a very small source, acoustic microscopy, and scattering of accelerated protons or neutrons.
The newest type of instruments are those which use a fine probe close to the surface of the specimen. The probe is kept close but not touching, controlled by sensitive feedback loops measuring (for example) atomic forces or tunneling currents. These microscopes are capable of resolving individual atoms. Brief details on the history and development of scanned probe microscopy are available from TopoMetrix
Over the years microscopy has developed from the use of a crude hand lens to sensitive and very high resolution methods demanding the use of modern computing facilities and exotic beam sources and detectors.
As the technology developed and commercially manufactured instruments reached the market, microscopy became commonplace and even essential in many aspects of modern life. From medicine to the microchip the microscope benefits us all in so many ways. And it won't stop here. New and improved instruments and specimen preparation methods are appearing faster today than ever before, so we can look forward confidently to more exciting advances over the coming decades.