Posted: March 18th, 2023
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 image plane where there occurs destructive interference and reduction of intensity giving rise to relatively dark areas. 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 lenses lead to what is known as aberrations or lens errors in optical microscopy. There are two major causes of aberration that have been identified: geometrical or spherical aberrations, and chromatic aberrations. The first cause relates to the spherical nature of the lens and approximation used to obtain the Gaussian lens equation, while the second one arises from the variation in the refractive indices of the wide range of frequencies existing in visible light (Davidson and Abramowitz, 2009). Generally, optical aberrations bring about faults in the features of an image that is being observed under a microscope.
Spherical aberration: These artifacts are experienced when light waves passing through the periphery and those passing closer to the center are not brought into identical focus. The waves passing closer to the center undergo a slight refraction while those in the periphery are greatly refracted giving rise to different focal points along the optical axis (Davidson and Abramowitz, 2009). Since this artifact makes the image of the specimen to spread out instead of being focused, it is considered as one of the most serious resolutions artifacts. In order to reduce these lens defects, the outer edged of the lens can be limited from exposure to light using diaphragms and also by using aspherical lens surfaces within the system.
Chromatic aberration: The fact that lights is composed of numerous wavelengths is the cause of this optical defect. These different wavelengths of light are refracted differently when they pass through a convex lens depending on their frequency. Blue light experiences the greatest refraction then green and red lights, this is referred to as dispersion. Since the lens is not capable of bringing all colors into a common focus, the resultant is a slightly different image size and focal point for each predominant wavelength group. The outcome will be an image surrounded by color fringes (Davidson and Abramowitz, 2009). When the lens thickness, curvature, refractive index and dispersion are properly combined then the doublet reduces chromatic aberration by bringing two of the wavelength groups into a common focal plane. Introducing fluorspar into the glass formulation used to fabricate the lens will bring the three colors red, green, and blue into a single focal point resulting in a negligible amount of chromatic aberration.
Eyepieces or oculars are used together with microscope objectives to increase the magnification of the intermediate image so that specimen details can be observed. In order to get best results in microscopy, objectives must be used together with the appropriate eyepieces. There are two types of eyepieces which are categorized using the lens and diaphragm arrangement, these are the negative and positive eyepieces. The negative eyepiece have the internal diaphragm located between the lenses while that of the positive eyepiece is located below the lenses. The negative eyepieces have two lenses: the upper lens closest to the observer’s eyes (eye-lens) and the lower lens (field lens). The positive eyepiece has an eye lens, but the field lens is mounted with the curved surface facing towards the eye lens (Davidson and Abramowitz, 2009).
The work of the substage condenser is to gather light from the microscope light source and concentrate it into a cone of light. This cone of light will then illuminate the specimen through parallel beams having uniform intensity from all azimuths in the whole viewfield. Adjusting the condenser light cone properly is critical in order to achieve the optimal intensity and angle of light entering the objective front lens (Davidson and Abramowitz, 2009). Whenever a change is made on the objective, a corresponding adjustment on the substage condenser aperture iris diaphragm must follow, this provides the appropriate light cone for the numerical aperture of the new objective.
Numerical aperture and Resolution
This value is very significant in microscope objectives as it gives the indication of light acceptance angle, which then determines the light gathering power, the resolving power, and depth of field of the objective. Some objectives which are specifically designed for transmitted light fluorescence and darkfield imaging are equipped with internal iris diaphragm thus allowing for adjustment of the effective numerical aperture.
When dealing with resolution in optical microscopy, emphasis is mostly placed on point-to-point resolution in the plane perpendicular to the optical axis. Axial resolution power of an objective is also an important aspect to resolution, this is measured parallel to the optical axis and is usually referred to as depth of field. Axial resolution is determined by the numerical aperture of the objective only, the work of the eyepiece is just magnification of details resolved and then this is projected in the intermediate image plane.
The following formula can be used to calculate resolution, this is a formula that was introduced by Ernst Abbe.
Resolution = ?/2[?.sin (?)],
Where; is the wavelength of light, ? is the refractive index of the imaging medium, and the combined term ?.sin (?) represents the objective numerical aperture (Webb, 1996).
Confocal microscopy and Multiphoton microscopy
This is a technique that increases the contrast of microscope images, particularly in specimens that are thick. This technique keeps the overlying or nearby scatters from contributing to the detected signal through the restriction of the observed volume. The condition therefore is that only one point must be observed by the machine at a time (laser version) or the machine can observe a group of separated points with very little light (disc version) (Webb, 1996). The alternative to confocal microscopy is multiphoton microscopy. This technique provides a three-dimensional imaging which is an obvious advantage. Multiphoton microscopy particularly does well where living cells imaging is done, more so when there are intact tissues such as embryos, brain slices, and even whole organs. Whenever thick specimens are used, the effective sensitivity of fluorescence microscopy is reduced by out-of-focus flare. The confocal microscope reduces this limitation by using confocal pinhole which rejects out-of-focus background fluorescence giving rise to thin and clear optical sections.
Confocal microscopy makes use of a pinhole in excluding out-of-focus background fluorescence from detection thereby allowing three-dimensional sectioning into thicker tissues. However, fluorescence is generated by the excitation light thereby producing photobleaching and phototoxicity in the whole specimen, even though the collection of the signal just happens within the plane of focus. In addition to this, there is limited penetration depth in confocal microscopy due to absorption of excitation energy throughout the beam path, and by scattering of specimen of both the excitation and emission photons (Webb, 1996). Multiphoton microscopy on the other hand provides the three dimensional optical sectioning without absorption above and below the plane of focus. Consequently, there is increased depth penetration as compared to confocal microscopy, and can have less toxicity. However, since these two excitation methods are governed by different photophysics, negative effects are occasionally experienced with multi-photon excitation of certain fluorophores which then limit the application of multiphoton microscopy for optical sectioning in thin specimens.
Application of optical microscope
The common application is in microelectronics, nanophysics, biotechnology, pharmaceutic research, mineralogy and microbiology. It is also applied in medical diagnosis especially where tissues or smear tests on free cells are involved. In the industrial sector it is commonly presented as the binocular microscopes.
In the pharmaceutical research, optical microscopy has been recommended for particle characterization and is preferred for characterizing and identifying particulate matter in medical products (Herman and Lemasters, 1993). This is very important in quality control where it helps in examination of liquid dosage forms for undissolved particles of the active pharmaceutical ingredient. Another application in quality control is the comparison of particle characteristics of incoming material, for lot-to-lot variation. Optical microscopy is also important in the characterization of extraneous matter or particulate matter, through optical microscopy extraneous matter can be identified whenever good reference materials are available (Herman and Lemasters, 1993). This is made possible because microscopic images have greater clarity, the details revealed are far much beyond the human eye resolution and there are additional optical data which cannot be realized through unaided eye. The combination of clarity and fine details gives an easy understanding of optical data by the end users.
Optical microscopy has been very instrumental in laboratory science and other fields in the industrial sector. It has advanced over time since the early 1880s. Even though microscopists faced various challenges initially, there were several developments that eliminated these challenges and led to better results for the microscopists. The use of optical and confocal microscopes also became wider as their efficiency increased and they have more popular in the current world.
Bradbury, S, 1967.The Evolution of the Microscope. New York: Pergamon Press.
Davidson, M.W., & Abramowitz, M. 2009. Optical Microscopy. Olympus America, Inc., 2
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Herman, B. And Lemasters J.J. (eds.), 1993. Optical Microscopy: Emerging Methods and Applications. Academic Press, New York, 1993.
Hoffman, R, 1977, “Journal of Microscopy,” 110: 205-222.
Nikon MicroscopyU, Fluorescence Microscopy, Multiphoton Excitation. 2015. [ONLINE]
Available at: http://www.microscopyu.com/articles/fluorescence/multiphoton/multiphotonintro.html. [Accessed 30 March 2015].
Webb, H. Robert. 1996. Confocal optical microscopy. Rep. Prog. Phys. 59, 427 — 471
Zeiss Group Microscopes Business Unit, 1996. Anticipating the Future. Jena, Germany.
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