Evolution, Principle and Application of the Optical Microscope
The application of optical microscopy has grown tremendously over the last few decades, this has been so in various disciplines where micron and submicron level investigations are applicable. The spreading out of fluorescence microscopy in research and laboratory applications has been fast-tracked by the instantaneous development of new fluorescent labels. Microscopists have also been able to get quantitative measurements faster and efficiently due to the developments in digital imaging and analysis. It is also possible to obtain very thin optical sections when optical microscopy is enhanced using digital video, the application of confocal optical systems is as well becoming common in a number of major research institutions. Before the nineteenth century, microscopists faced various shortcomings including, optical aberration, blurred images, and poor lens design (Davidson and Abramowitz, 2009). However, in the mid-nineteenth century there was partial correction to aberration through the use of Lister and Amici achromatic objectives. This led to a reduction of the chromatic aberration and raised numerical apertures to around 0.65 for dry objectives and up to 1.25 for homogenous immersion objectives (Bradbury, 1967). Ernst Abbe's and Carl Zeiss also worked together in 1886 to produce apochromatic objectives which were based on sound optical principles and lens design, this was a first one of its kind (Zeiss Group Microscopes Business Unit, 1996). With these advanced objectives, it was possible to obtain images having reduced spherical aberration without color distortions but at high numerical apertures.
Evolution of the optical microscope
Towards the end of the nineteenth century, Professor August Kohler developed a method of illumination which intended to optimize photomicrography thereby giving microscopists the opportunity of fully utilizing the resolving power of Abbe's objectives. It is within the last decade of the nineteenth century that various innovations in optical microscopy were made, such as metallographic microscopes, anastigmatic photo lenses, binocular microscopes with image-erecting prisms, and the first stereomicroscope (Zeiss Group Microscopes Business Unit, 1996). Further advancements were made in the early twentieth century such as par focalization of objectives by manufacturers which gave the microscopists the advantage of retaining the image in focus while exchanging objectives on the rotating nosepiece. In 1824, a LeChatelier-style metallograph with infinity-corrected optics was introduced by Zeiss, but this method took time to be widely applied. Zeiss later on, just before the beginning of Second World War came up with a number of prototype phase contrast microscopes based on Frits Zernike's optical principles, these microscopes were later modified leading to the development of the first time-lapse cinematography of cell division photographed with phase contrast optics (Davidson and Abramowitz, 2009). This technique which enhanced contrast was not immediately recognized until 1950s when it received a universal acceptance and many biologists still prefer it to-date. The Wollaston prism design was improved by physicist Georges Normarski giving rise to another strong contrast-generating microscopy theory in 1955. This new technique, commonly known as Nomarski interference or differential interference contrast (DIC) microscopy coupled with phase contrast has given scientists a chance of exploring various arenas in biology using living cells or unstained tissues. Another method of increasing contrast was introduced by Robert Hoffman (Hoffman, 1977), this utilized the advantage of phase gradients near cell membranes, a technique now referred to as Hoffman Modulation Contrast. Until the late 1980s, most microscopes had fixed mechanical tube lengths (between 160 to 210 millimeters), after which the infinity-corrected optics was largely adopted.
Fundamentals of Image Formation
In considering the optical microscope, when light produced by the microscope lamp is directed to go through the condenser and then through the specimen, a portion of the light will go around and through the specimen without experiencing any disturbance in its path, thus referred to as direct light or undeviated light. The light that passes around the specimen to form the background light is also undeviated light. A portion of the light that passes through the specimen encountering parts of the specimen is deviated. This deviated light compared to the undeviated light has half wavelength or is 180 degrees out of step. This leads to destructive interference with the direct light at the intermediate image plane found at the fixed diaphragm of the eyepiece. This image is further magnified by the eye lens of the eye piece and finally projected onto the retina, the film plane of a camera, or the surface of a light sensitive chip. Basically, the objective projects the direct or undeviated light spreading it evenly across the whole image plane at the diaphragm of the eyepiece. This diffracted light is then focused at various localized points on the same...
It is these light and dark patterns that are recognized as image of the specimen (Davidson and Abramowitz). Since human eyes are sensitive to variations in brightness, the image is seen as a relatively realistic reconstitution of the original specimen. Image formation is thus based on the principle combining or manipulating direct and diffracted light. The rear focal plane of the objective and the front focal plane of the substage condenser then become significant locations for such manipulation. Various contrast improvement methods in optical microscopy are based on this core principle, this is particularly important when it comes to high magnification of small details whose size are close to the wavelength of light.
The figure below shows a diffraction spectra generated at the rear focal plane of the objective by undeviated and diffracted light (Davidson and Abramowitz, 2009).
: (a) Spectra visible through a focusing telescope at the rear focal plane of an objective. (b) Schematic diagram of light both diffracted and undeviated by a line grating on the microscope
In microscopy and critical photomicrography it is very important that specimen is properly illuminated for purposes of achieving high-quality images. August Kohler first introduced an elaborate procedure for microscope illumination in 1893, this was to give optimum specimen illumination. With this technique, users of the microscope were able to achieve a uniformly bright and glare free specimen thus utilizing the microscope adequately. In most modern microscopes, the collector lens and other optical parts built into the base are such that they will project an enlarged and focused image of the lamp filament onto the plane of the aperture diaphragm of a properly positioned substage condenser. The angle of the light rays emerging from the condenser is controlled by closing and opening the condenser diaphragm thus reaching the specimen from all azimuths. Since the focusing of the light source is not done at the specimen level, a grainless and extended illumination at specimen level is achieved, this is free from deterioration caused by dust and imperfections on the glass surfaces of the condenser (Davidson and Abramowitz, 2009). The resulting numerical aperture of the microscope system is determined by the opening size of the condenser aperture diaphragm and the aperture of the objective. On opening the condenser diaphragm, the numerical aperture of the microscope is increased giving rise to greater light transmittance and resolving power. The parallel light rays that pass through and illuminate the specimen are focused at the rear focal plane of the objective where there is simultaneous observation of the image of the variable condenser aperture diaphragm and the light source.
: Light paths in Kohler Illumination The figure below shows light paths in Kohler illumination. The left side illustrates illuminating ray paths while the right side are image-forming ray paths. When the lamp emits light it goes through a collector lens and subsequently field diaphragm. The size and shape of the illumination cone on the specimen plane is determined by the aperture diaphragm in the condenser. Before the light is focused at the back focal plane of the objective, it passes through the specimen and eventually is magnified by the ocular and finally into the eye (Davidson and Abramowitz, 2009).
Microscope Objectives, Eyepieces, Condensers, and Optical Aberrations
The design of the finite microscope objectives is such that they should project a diffraction-limited image at a fixed plane that is determined by the microscope tube length and located at a pre-specified distance from the rear focal plane of the objective. The imaging of the specimens happens at a very short distance beyond the front focal plane of the objective through a medium of defined refractive index, normally air, water, glycerin, or specialized immersion oils (Davidson and Abramowitz, 2009). In order to meet the performance needs of specialized imaging methods, microscope manufacturers provide a wide range of objective designs. These designs also compensate for thickness of cover glass and increase the effective working distance of the objective. The most commonly used design now is the infinity-corrected objectives which project imaging rays in parallel bundles from every azimuth to infinity. In order to focus the image at the intermediate image plane, a tube lens is necessary in the light path.
Artifacts arising from the interaction of light with glass…
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