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Of all the techniques used in biology microscopy is probably the most important. The vast majority of living organisms are too small to be seen in any detail with the human eye, and cells and their organelles can only be seen with the aid of a microscope. Cells were first seen in 1665 by Robert Hooke (who named them after monks' cells in a monastery), and were studied in more detail by Leeuwehoek using a primitive microscope.

Units of measurement

= 1 m
= 10-3 m
= 10-6 m
= 10-9 m
= 10-12 m
= 10-10 m (obsolete)

Magnification and Resolution �623�**[back to top]**�1055�

By using more lenses microscopes can magnify by a larger amount, but this doesn't always mean that more detail can be seen. The amount of detail depends on the resolving power of a microscope, which is the smallest separation at which two separate objects can be distinguished (or resolved).
The resolving power of a microscope is ultimately limited by the wavelength of light (400-600nm for visible light). To improve the resolving power a shorter wavelength of light is needed, and sometimes microscopes have blue filters for this purpose (because blue has the shortest wavelength of visible light).
Magnification is how much bigger a sample appears to be under the microscope than it is in real life.
Overall magnification = Objective lens x Eyepiece lens
Resolution is the ability to distinguish between two points on an image i.e. the amount of detail
  • The resolution of an image is limited by the wavelength of radiation used to view the sample.
  • This is because when objects in the specimen are much smaller than the wavelength of the radiation being used, they do not interrupt the waves, and so are not detected.
  • The wavelength of light is much larger than the wavelength of electrons, so the resolution of the light microscope is a lot lower.
  • Using a microscope with a more powerful magnification will not increase this resolution any further. It will increase the size of the image, but objects closer than 200nm will still only be seen as one point.

Different kinds of Microscopes: �670�**[back to top]**�1102�

Light Microscopy: This is the oldest, simplest and most widely-used form of microscopy. Specimens are illuminated with light, which is focussed using glass lenses and viewed using the eye or photographic film. Specimens can be living or dead, but often need to be stained with a coloured dye to make them visible. Many different stains are available that stain specific parts of the cell such as DNA, lipids, cytoskeleton, etc. All light microscopes today are compound microscopes, which means they use several lenses to obtain high magnification. Light microscopy has a resolution of about 200 nm, which is good enough to see cells, but not the details of cell organelles. There has been a recent resurgence in the use of light microscopy, partly due to technical improvements, which have dramatically improved the resolution far beyond the theoretical limit. For example fluorescence microscopy has a resolution of about 10 nm, while interference microscopy has a resolution of about 1 nm.

Preparation of Slide Samples

  • Fixation: Chemicals preserve material in a life like condition. Does not distort the specimen.
  • Dehydration: Water removed from the specimen using ethanol. Particularly important for electron microscopy because water molecules deflect the electron beam which blurs the image.
  • Embedding: Supports the tissue in wax or resin so that it can be cut into thin sections.
    Sectioning Produces very thin slices for mounting. Sections are cut with a microtome or an ulramicrotome to make them either a few micrometres (light microscopy) or nanometres
    (electron microscopy) thick.
  • Staining: Most biological material is transparent and needs staining to increase the contrast between different structures. Different stains are used for different types of tissues. Methylene blue is often used for animal cells, while iodine in KI solution is used for plant tissues.
  • Mounting: Mounting on a slide protects the material so that it is suitable for viewing over a long period.
Electron Microscopy. This uses a beam of electrons, rather than electromagnetic radiation, to "illuminate" the specimen. This may seem strange, but electrons behave like waves and can easily be produced (using a hot wire), focused (using electromagnets) and detected (using a phosphor screen or photographic film). A beam of electrons has an effective wavelength of less than 1 nm, so can be used to resolve small sub-cellular ultrastructure. The development of the electron microscope in the 1930s revolutionised biology, allowing organelles such as mitochondria, ER and membranes to be seen in detail for the first time.
The main problem with the electron microscope is that specimens must be fixed in plastic and viewed in a vacuum, and must therefore be dead. Other problems are that the specimens can be damaged by the electron beam and they must be stained with an electron-dense chemical (usually heavy metals like osmium, lead or gold). Initially there was a problem of artefacts (i.e. observed structures that were due to the preparation process and were not real), but improvements in technique have eliminated most of these.
There are two kinds of electron microscope. The transmission electron microscope (TEM) works much like a light microscope, transmitting a beam of electrons through a thin specimen and then focusing the electrons to form an image on a screen or on film. This is the most common form of electron microscope and has the best resolution. The scanning electron microscope (SEM) scans a fine beam of electron onto a specimen and collects the electrons scattered by the surface. This has poorer resolution, but gives excellent 3-dimentional images of surfaces.
Transmission Electron Microscope (TEM)
Scanning Electron Microscope (SEM)
  • Pass a beam of electrons through the specimen. The electrons that pass through the specimen are detected on a fluorescent screen on which the image is displayed.
  • Thin sections of specimen are needed for transmission electron microscopy as the electrons have to pass through the specimen for the image to be produced.
  • This is the most common form of electron microscope and has the best resolution
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Bacterium (TEM)
e provided by:

  • Pass a beam of electrons over the surface of the specimen in the form of a ‘scanning’ beam.
  • Electrons are reflected off the surface of the specimen as it has been previously coated in heavy metals.
  • It is these reflected electron beams that are focussed of the fluorescent screen in order to make up the image.
  • Larger, thicker structures can thus be seen under the SEM as the electrons do not have to pass through the sample in order to form the image. This gives excellent 3-dimensional images of surfaces
  • However the resolution of the SEM is lower than that of the TEM.

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A head and the right eye of a fly
(Image provided by: Goran Drazic)to focus at the plane of the fixed diaphragm in the eyepiece. The distance from the back focal plane of the objective (not necessarily its back lens) to the plane of the fixed diaphragm of the eyepiece is known as the **optical tube length** of the objective.
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In the last case, the object is situated at the front focal plane of the convex lens. In this case, the rays of light emerge from the lens in parallel. The image is located on the **same** side of the lens as the object, and it appears upright (see Figure 1). The image is a virtual image and appears as if it were 10 inches from the eye, similar to the functioning of a simple magnifying glass; the magnification factor depends on the curvature of the lens.

The last case listed above describes the functioning of the observation eyepiece of the microscope. The "object" examined by the eyepiece is the magnified, inverted, real image projected by the objective. When the human eye is placed above the eyepiece, the lens and cornea of the eye "look" at this secondarily magnified virtual image and see this virtual image as if it were 10 inches from the eye, near the base of the microscope.
This case also describes the functioning of the now widely used infinity-corrected objectives. For such objectives, the object or specimen is positioned at exactly the front focal plane of the objective. Light from such a lens emerges in parallel rays from every azimuth. In order to bring such rays to focus, the microscope body or the binocular observation head must incorporate a **tube lens** in the light path, between the objective and the eyepiece, designed to bring the image formed by the objective to focus at the plane of the fixed diaphragm of the eyepiece. The magnification of an infinity-corrected objective equals the focal length of the tube lens (for Olympus equipment this is 180mm, Nikon uses a focal length of 200mm; other manufacturers use other focal lengths) divided by the focal length of the objective lens in use. For example, a 10X infinity-corrected objective, in the Olympus series, would have a focal length of 18mm (180mm/10).

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Interactive Java Tutorial
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Objective Focal LengthDiscover how changes in magnification and tube length affect objective focal length.
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An easy way to understand the microscope is by means of a comparison with a slide projector, a device familiar to most of us. Visualize a slide projector turned on its end with the lamp housing resting on a table. The light from the bulb passes through a condensing lens, and then through the transparency, and then through the projection lens onto a screen placed at right angles to the beam of light at a given distance from the projection lens. The real image on this screen emerges inverted (upside down and reversed) and magnified. If we were to take away the screen and instead use a magnifying glass to examine the real image in space, we could further enlarge the image, thus producing another or second-stage magnification.
Now we will describe how a microscope works in somewhat more detail. The first lens of a microscope is the one closest to the object being examined and, for this reason, is called the objective. Light from either an external or internal (within the microscope body) source is first passed through the substage condenser, which forms a well-defined light cone that is concentrated onto the object (specimen). Light passes through the specimen and into the objective (similar to the projection lens of the projector described above), which then projects a real, inverted, and magnified image of the specimen to a fixed plane within the microscope that is termed the intermediate image plane (illustrated in Figure 6). The objective has several major functions:
//* The objective must gather the light coming from each of the various parts or points of the specimen.
  • The objective must have the capacity to reconstitute the light coming from the various points of the specimen into the various corresponding points in the image (Sometimes called anti-points).
  • The objective must be constructed so that it will be focused close enough to the specimen so that it will project a magnified, real image up into the body tube.
The intermediate image plane is usually located about 10 millimeters below the top of the microscope body tube at a specific location within the fixed internal diaphragm of the eyepiece. The distance between the back focal plane of the objective and the intermediate image is termed the optical tube length. Note that this value is different from the mechanical tube length of a microscope, which is the distance between the nosepiece (where the objective is mounted) to the top edge of the observation tubes where the eyepieces (oculars) are inserted.
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The eyepiece or ocular, which fits into the body tube at the upper end, is the farthest optical component from the specimen. In modern microscopes, the eyepiece is held into place by a shoulder on the top of the microscope observation tube, which keeps it from falling into the tube. The placement of the eyepiece is such that its eye (upper) lens further magnifies the real image projected by the objective. The eye of the observer sees this secondarily magnified image as if it were at a distance of 10 inches (25 centimeters) from the eye; hence this virtual image appears as if it were near the base of the microscope. The distance from the top of the microscope observation tube to the shoulder of the objective (where it fits into the nosepiece) is usually 160 mm in a finite tube length system. This is known as the mechanical tube length as discussed above. The eyepiece has several major functions:
* The eyepiece serves to further magnify the real image projected by the objective.
  • In visual observation, the eyepiece produces a secondarily enlarged virtual image.
  • In photomicrography, it produces a secondarily enlarged real image projected by the objective. This augmented real image can be projected on the photographic film in a camera or upon a screen held above the eyepiece.
  • The eyepiece can be fitted with scales, markers or crosshairs (often referred to as graticules, reticules or reticles) in such a way that the image of these inserts can be superimposed on the image of the specimen.
The factor that determines the amount of image magnification is the objective magnifying power, which is predetermined during construction of the objective optical elements. Objectives typically have magnifying powers that range from 1:1 (1X) to 100:1 (100X), with the most common powers being 4X (or 5X), 10X, 20X, 40X (or 50X), and 100X. An important feature of microscope objectives is their very short focal lengths that allow increased magnification at a given distance when compared to an ordinary hand lens (illustrated in Figure 1). The primary reason that microscopes are so efficient at magnification is the two-stage enlargement that is achieved over such a short optical path, due to the short focal lengths of the optical components.
Eyepieces, like objectives, are classified in terms of their ability to magnify the intermediate image. Their magnification factors vary between 5X and 30X with the most commonly used eyepieces having a value of 10X-15X. Total visual magnification of the microscope is derived by multiplying the magnification values of the objective and the eyepiece. For instance, using a 5X objective with a 10X eyepiece yields a total visual magnification of 50X and likewise, at the top end of the scale, using a 100X objective with a 30X eyepiece gives a visual magnification of 3000X.
Total magnification is also dependent upon the tube length of the microscope. Most standard fixed tube length microscopes have a tube length of 160, 170, 200, or 210 millimeters with 160 millimeters being the most common for transmitted light biomedical microscopes. Many industrial microscopes, designed for use in the semiconductor industry, have a tube length of 210 millimeters. The objectives and eyepieces of these microscopes have optical properties designed for a specific tube length, and using an objective or eyepiece in a microscope of different tube length will lead to changes in the magnification factor (and may also lead to an increase in optical aberration lens errors). Infinity-corrected microscopes also have eyepieces and objectives that are optically-tuned to the design of the microscope, and these should not be interchanged between microscopes with different infinity tube lengths.
Modern research microscopes are very complex and often have both episcopic and diascopic illuminators built into the microscope housing. Design constrictions in these microscopes preclude limiting the tube length to the physical dimension of 160 millimeters resulting the need to compensate for the added physical size of the microscope body and mechanical tube. This is done by the addition of a set of parallelizing lenses to shorten the apparent mechanical tube length of the microscope. These additional lenses will sometimes introduce an additional magnification factor (usually around 1.25-1.5X) that must be taken into account when calculating both the visual and photomicrographic magnification. This additional magnification factor is referred to as a tube factor in the user manuals provided by most microscope manufacturers. Thus, if a 5X objective is being used with a 15X set of eyepieces, then the total visual magnification becomes 93.75X (using a 1.25X tube factor) or 112.5X (using a 1.5X tube factor).
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In addition to the parallelizing lenses used in some microscopes, manufacturers may also provide additional lenses (sometimes called magnification changers) that can be rotated into the optical pathway to increase the magnification factor. This is often done to provide ease in specimen framing for photomicrography. These lenses usually have very small magnification factors ranging from 1.25X up to 2.5X, but use of these lenses may lead to empty magnification, a situation where the image is enlarged, but no additional detail is resolved. This type of error is illustrated in Figure 7 with photomicrographs of liquid crystalline DNA. The photomicrograph in Figure 7(a) was taken with a 20X plan achromat objective under polarized light with a numerical aperture of 0.40 and photographically enlarged by a factor of 10X. Detail is crisp and focus is sharp in this photomicrograph that reveals many structural details about this hexagonally-packed liquid crystalline polymer. Conversely, the photomicrograph on the right (Figure 7(b)) was taken with a 4X plan achromat objective, having a numerical aperture of 0.10 and photographically enlarged by a factor of 50X. This photomicrograph lacks the detail and clarity present in Figure 7(a) and demonstrates a significant lack of resolution caused by the empty magnification factor introduced by the enormous degree of enlargement.
Care should be taken in choosing eyepiece/objective combinations to ensure the optimal magnification of specimen detail without adding unnecessary artifacts. For instance, to achieve a magnification of 250X, the microscopist could choose a 25X eyepiece coupled to a 10X objective. An alternative choice for the same magnification would be a 10X eyepiece with a 25X objective. Because the 25X objective has a higher numerical aperture (approximately 0.65) than does the 10X objective (approximately 0.25), and considering that numerical aperture values define an objective's resolution, it is clear that the latter choice would be the best. If photomicrographs of the same viewfield were made with each objective/eyepiece combination described above, it would be obvious that the 10x eyepiece/25x objective duo would produce photomicrographs that excelled in specimen detail and clarity when compared to the alternative combination.
The range of useful total magnification for an objective/eyepiece combination is defined by the numerical aperture of the system. There is a minimum magnification necessary for the detail present in an image to be resolved, and this value is usually rather arbitrarily set as 500 times the numerical aperture (500 x NA). At the other end of the spectrum, the maximum useful magnification of an image is usually set at 1000 times the numerical aperture (1000 x NA). Magnifications higher than this value will yield no further useful information or finer resolution of image detail, and will usually lead to image degradation, as discussed above. Exceeding the limit of useful magnification causes the image to suffer from the phenomenon of empty magnification (see Figures 7 (a) and (b)), where increasing magnification through the eyepiece or intermediate tube lens only causes the image to become more magnified with no corresponding increase in detail resolution. Table 1 lists the common objective/eyepiece combinations that lie in the range of useful magnification.

Range of Useful Magnification
(500-1000 x NA of Objective)

x = good combination

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Table 1

These basic principles underlie the operation and construction of the compound microscope which, unlike a magnifying glass or simple microscope, employs a group of lenses aligned in series. The elaboration of these principles has led to the development, over the past several hundred years, of today's sophisticated instruments. Modern microscopes are often modular with interchangeable parts for different purposes; such microscopes are capable of producing images from low to high magnification with remarkable clarity and contrast.

Contributing Authors
Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.
Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

====Specialized Microscopy Techniques====
Modern microscopists and optical engineers have developed a wide spectrum of useful techniques designed to aid in contrast enhancement, provide better observation, and assist in the collection of photomicrographs and digital images of a wide variety of specimens. This section of the Molecular Expressions Microscopy Primer describes many of these techniques in detail.
Contrast Enhancing Techniques
Contrast in Optical Microscopy - With the assistance of Dr. Robert Hoffman, we review the problems of contrast enhancement with both amplitude and phase specimens and review techniques that have been developed to assist with specimen contrast.
Darkfield Microscopy - Oblique illumination can be used to increase the visibility of specimens lacking in sufficient contrast that are difficult to observe with standard brightfield microscopy. This section discusses various aspects of the theory and practice of condenser design and other important concepts in both transmitted and reflected light darkfield microscopy.
Differential Interference Contrast - An excellent mechanism for rendering contrast in transparent specimens, differential interference contrast (DIC) microscopy is a beam-shearing interference system in which the reference beam is sheared by a minuscule amount, generally somewhat less than the diameter of an Airy disk. The technique produces a monochromatic shadow-cast image that effectively displays the gradient of optical paths for both high and low spatial frequencies present in the specimen. Those regions of the specimen where the optical paths increase along a reference direction appear brighter (or darker), while regions where the path differences decrease appear in reverse contrast. As the gradient of optical path difference grows steeper, image contrast is dramatically increased.
Hoffman Modulation Contrast - Hoffman modulation contrast is an oblique illumination technique that enhances contrast in both stained and unstained specimens by detection of optical phase gradients. This section includes discussions of transmitted and reflected light applications using Hoffman modulation contrast and links to interactive Java tutorials designed to aid in understanding the technique. Also included are virtual microscopes and an image gallery of photomicrographs made using modulation contrast either alone or in combination with other illumination mechanisms.
Oblique or Anaxial Illumination - Achieving conditions necessary for oblique illumination, which has been employed to enhance specimen visibility since the dawn of microscopy, can be accomplished by a variety of techniques with a simple transmitted optical microscope. Perhaps the easiest methods are to offset a partially closed condenser iris diaphragm or the image of the light source. In former years, some microscopes were equipped with a condenser having a decenterable aperture iris diaphragm. The device was engineered to allow the entire iris to move off-center in a horizontal plane so that closing the circular diaphragm opening would result in moving the zeroth order to the periphery of the objective rear focal plane. In advanced models, the entire diaphragm was rotatable around the axis of the microscope so that oblique light could be directed toward the specimen from any azimuth to achieve the best desired effect for a given specimen.
Phase Contrast - A large spectrum of living biological specimens are virtually transparent when observed in the optical microscope under brightfield illumination. To improve visibility and contrast in such specimens, microscopists often reduce the opening size of the substage condenser iris diaphragm, but this maneuver is accompanied by a serious loss of resolution and the introduction of diffraction artifacts. Phase contrast was introduced in the 1930's for testing of telescope mirrors, and was adapted by Zeiss laboratories into a commercial microscope several years later. This technique provides an excellent method of improving contrast in unstained biological specimens without significant loss in resolution, and is widely utilized to examine dynamic events in living cells.
Comparison of Phase Contrast and DIC Microscopy - Phase contrast and differential interference contrast (DIC) microscopy are complementary techniques capable of producing high contrast images of transparent biological phases that do not ordinarily affect the amplitude of visible light waves passing though the specimen. The most fundamental distinction between differential interference contrast and phase contrast microscopy is the optical basis upon which images are formed. Phase contrast yields image intensity values as a function of specimen optical path length magnitude, with very dense regions (those having large path lengths) appearing darker than the background. The situation is quite distinct for differential interference contrast, where optical path length gradients (in effect, the rate of change in the direction of wavefront shear) are primarily responsible for introducing contrast into specimen images.
Polarized Light Microscopy - The polarized light microscope is designed to observe and photograph specimens that are visible primarily due to their optically anisotropic character. In order to accomplish this task, the microscope must be equipped with both a polarizer, positioned in the light path somewhere before the specimen, and an analyzer (a second polarizer), placed in the optical pathway between the objective rear aperture and the observation tubes or camera port. Image contrast arises from the interaction of plane-polarized light with a birefringent (or doubly-refracting) specimen to produce two individual wave components that are each polarized in mutually perpendicular planes. The velocities of these components are different and vary with the propagation direction through the specimen. After exiting the specimen, the light components become out of phase, but are recombined with constructive and destructive interference when they pass through the analyzer. Polarized light is a contrast-enhancing technique that improves the quality of the image obtained with birefringent materials when compared to other techniques such as darkfield and brightfield illumination, differential interference contrast, phase contrast, Hoffman modulation contrast, and fluorescence.
Rheinberg Illumination - Rheinberg illumination, a form of optical staining, was initially demonstrated by the British microscopist Julius Rheinberg to the Royal Microscopical Society and the Quekett Club (England) over a hundred years ago. This technique is a striking variation of low to medium power darkfield illumination using colored gelatin or glass filters to provide rich color to both the specimen and background. In Rheinberg illumination, the oblique light rays traversing a brightfield condenser pass through an annular filter of one or more colors, while the central rays of light pass through another spot-shaped filter fitted into the circular opening of the annular-shaped filter. The objective is used at full aperture.
Fundamentals of Stereomicroscopy - Considering the wide range of accessories currently available for stereomicroscope systems, this class of microscopes is extremely useful in a multitude of applications. Stands and illuminating bases for a variety of contrast enhancement techniques are available from all of the manufacturers, and can be adapted to virtually any working situation. There are a wide choice of objectives and eyepieces, enhanced with attachment lenses and coaxial illuminators that are fitted to the microscope as an intermediate tube. Working distances can range from 3-5 centimeters to as much as 20 centimeters in some models, allowing for a considerable amount of working room between the objective and specimen.
Deconvolution in Optical Microscopy - Deconvolution is a computationally intensive image processing technique that is being increasingly utilized for improving the contrast and resolution of digital images captured in the microscope. The foundations are based upon a suite of methods that are designed to remove or reverse the blurring present in microscope images induced by the limited aperture of the objective. Practically any image acquired on a digital fluorescence microscope can be deconvolved, and several new applications are being developed that apply deconvolution techniques to transmitted light images collected under a variety of contrast enhancing strategies. One of the most suitable subjects for improvement by deconvolution are three-dimensional montages constructed from a series of optical sections.
Live-Cell Imaging - An increasing number of investigations are using live-cell imaging techniques to provide critical insight into the fundamental nature of cellular and tissue function, especially due to the rapid advances that are currently being witnessed in fluorescent protein and synthetic fluorophore technology. As such, live-cell imaging has become a requisite analytical tool in most cell biology laboratories, as well as a routine methodology that is practiced in the wide ranging fields of neurobiology, developmental biology, pharmacology, and many of the other related biomedical research disciplines. Among the most significant technical challenges for performing successful live-cell imaging experiments is to maintain the cells in a healthy state and functioning normally on the microscope stage while being illuminated in the presence of synthetic fluorophores and/or fluorescent proteins.
Introduction to Confocal Microscopy - Confocal microscopy offers several advantages over conventional optical microscopy, including controllable depth of field, the elimination of image degrading out-of-focus information, and the ability to collect serial optical sections from thick specimens. The key to the confocal approach is the use of spatial filtering to eliminate out-of-focus light or flare in specimens that are thicker than the plane of focus. There has been a tremendous explosion in the popularity of confocal microscopy in recent years, due in part to the relative ease with which extremely high-quality images can be obtained from specimens prepared for conventional optical microscopy, and in its great number of applications in many areas of current research interest.
Principles and Applications of Interferometry - The foundation for interferometry (often referred to as microinterferometry) dates back to the nineteenth century with the introduction of the first interference microscope, which was based on the principles of the Jamin interferometer. Since that period, a number of commercial interference microscopes, both with transmitted and reflected light capabilities, have been produced by a number of manufacturers. Primarily designed to yield quantitative data from interference images, these microscopes utilize various technologies to determine parameters such as refractive index, birefringence, and thickness for a wide spectrum of materials.
* Two Beam Interferometry - A two-beam interferometer functions by dividing originally coherent light into two beams of equal intensity, directing one beam onto the reference mirror and the other onto the specimen, and measuring the optical path difference (the difference in optical distances) between the resulting two reflected light waves.
  • Multiple-Beam Interferometry - The technique of multiple-beam interferometry is based upon situating two surfaces of high reflectivity in close proximity and using a lens to converge beams which have undergone multiple-reflection between the surfaces.
Near-Field Scanning Optical Microscopy - For ultra-high optical resolution, near-field scanning optical microscopy (NSOM) is currently the photonic instrument of choice. Near-field imaging occurs when a sub-micron optical probe is positioned a very short distance from the sample and light is transmitted through a small aperture at the tip of this probe. The near-field is defined as the region above a surface with dimensions less than a single wavelength of the light incident on the surface. Within the near-field region evanescent light is not diffraction limited and nanometer spatial resolution is possible. This phenomenon enables non-diffraction limited imaging and spectroscopy of a sample that is simply not possible with conventional optical imaging techniques.
Fluorescence Microscopy Techniques
Fluorescence Microscopy - Used primarily with episcopic illumination, fluorescence microscopy is rapidly becoming a standard tool in the fields of genetics, embryology, and cell biology.
Fluorescence and Differential Interference Contrast Combination Microscopy - Fluorescence microscopy can be combined with contrast enhancing techniques such as differential interference contrast (DIC) illumination to minimize the effects of photobleaching by locating a specific area of interest in a specimen using DIC then, without relocating the specimen, switching the microscope to fluorescence mode.
Fluorescence and Phase Contrast Combination Microscopy - To minimize the effects of photobleaching, fluorescence microscopy can be combined with phase contrast illumination. The idea is to locate the specific area of interest in a specimen using the non-destructive contrast enhancing technique (phase) then, without relocating the specimen, switch the microscope to fluorescence mode.
Olympus FluoView Laser Scanning Confocal Microscopy - The new Olympus FluoViewTM FV1000 is the latest in point-scanning, point-detection, confocal laser scanning microscopes designed for today's intensive and demanding biological research investigations. Excellent resolution, bright and crisp optics, and high efficiency of excitation, coupled to an intuitive user interface and affordability are key characteristics of this state-of-the-art optical microscopy system.
Interactive Java Tutorials - A gallery of interactive Java applets designed to aid students in understanding difficult concepts in specialized microscopy techniques.

Contributing Authors
Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.
Robert Hoffman - Modulation Optics, Inc., 100 Forest Drive, Greenvale, New York 11548.
Tatsuro Otaki - Optical Design Department, Instruments Company, Nikon Corporation, 1-6-3 Nishi-Ohi, Shinagawa-ku, Tokyo, 140-8601, Japan.
Philip C. Robinson - Department of Ceramic Technology, Staffordshire Polytechnic, College Road, Stroke-on-Trent, ST4 2DE, United Kingdom.
Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.
Kirill I. Tchourioukanov and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

Laboratory Investigation:(Images will be given in print out)
The Micrometer Eyepiece

A microscope can be used not only to see very small things but also to measure them. Things seen in microscopes are so small that centimeters or even millimeters are too big. As a result, micrometers (or microns) are used. A micrometer, also written µm, is one thousandth of a millimeter - it's 10-6m.
For this, a micrometer eyepiece is used in place of the standard eyepiece of the microscope. This has a series of numbered lines inside of it which make it look like a ruler (see image to the right, click on it to see a bigger version).
The images below show what the eyepiece looks like (with its protective box) and where to put it on the microscope.

Method - How to use it:
1. After placing the special eyepiece, it is necessary to calibrate the microscope. To do this, a calibration slide must be used. This is a glass slide with one one-hundredth of a millimeter, 0.01mm, engraved on to its top surface (see photo to the right). Use care when handling this little piece of glass - it costs a lot of money to replace! Since a hundredth of a millimeter is very small and difficult to see, a circle is drawn around it. This slide allows us to find out how big things are as we look at them through the microscope at different powers of magnification. Put the slide on the stage as shown in the photo. Be sure that the top of the slide (the surface with the microscopic lines engraved on it) is pointing up.

2. Set the microscope to low power and focus on the lines engraved on the surface of the calibration slide. You should see the following:

3. The number of lines must be counted. As shown in the figure above, the eyepiece lines have numbers on them whereas the calibration slide's lines do not. The total number of eyepiece lines (which will be called X) are from line 21 to 59. That's a total of 38 lines. The number of calibration slide lines (which will be called Y) show a total of 10 lines (note that the little lines mark off half spaces and that the first line is not counted because is shows the zero mark).
4. Calculate how much each line of the eyepiece measures. In other words find out what distance is shown between each line of the eyepiece. To do so, use this equation:

If we plug in the numbers from the example in step 3, we get 10/38 x 10µm = 2.63 µm. That means that as you are looking through the microscope at low power, the space between each line on the micrometer eyepiece measures 2.63µm. Try it on the cell to the right. The cell goes from line 22 to line 29 on the eyepiece so it measures about 7 lines across.
If a cell is 7 lines wide when observed on low power, that means that in reality, it measures 7 x 2.63µm = 18.4µm. Since we know that cells are supposed to be between 10 and 30µm, it makes sense that the human cheek cell in the photo above measures just over 18µm.
5. After calibrating for low power observations, medium power should be calibrated. Switch to the medium power objective lens and focus once again on the lines etched into the top of the calibration slide. Use the same technique as steps 3 and 4 counting the lines and calculating the measurement between two lines on the eyepiece. Try with this example (click to see what is viewed in the microscope):

• What is the value for Y? (click on the ??? to see the answer)
• What is the value for X?
• Using the calculation, what is the value for the measurment between any two lines on the eyepiece?
• Now look at the cell below and measure its size in µm. It is a bit difficult to see where its cell membrane ends because it is stuck together with other cells. What do you get? Size = Is this close to what we got for the size of a cell in low power? It should be!

6. Do the same for high power. Here is what the calibration slide looks like:

If you follow the same steps, you should get a value of 0.26µm for the space between the lines on the eyepiece. Question: How many lines would an 18µm-wide cell take up on the eyepiece as you look at it through high power? If you work it out, you should get about 70 lines.
7. Now it is possible to use the microscope to observe and measure things. Take the calibration slide out and carefully put it back in its protective box so that it does not get damaged. Now place the slide of what you would like to look at on the stage.
Note that if you change microscopes, the calibration process must be done again for each of the objective lenses that you are using. Why? Because the magnification is different on different microscopes. To save time, only calibrate the eyepiece for the objective lenses that you will be using.

(Optional) Using a calculation to get other powers:
(If you don't want to do this part, go down to the bottom of the page where there are more examples.)
Another time-saving method is to calibrate one power (low power, for example) and then calculate the proportions rather than measuring the other two. Note that you do not need to know this for an exam.
Look at our example above: for 40x (low power), the calibration was 2.63µm. For 160 power (medium power), the calibration was 1.05µm and for 400 power, it was 0.26µm. A proportion can be set up:

Notice that each time we zoomed in with ojective lens, it increased the value of the proportion by 10 fold. So, if you have the number for low power (2.63µm for 40x), find out what the magnification power is for the other objective lenses and use the formula below to calculate the calibration for medium power:

In our examle this means:

or for medium power to high power...


More examples:
An onion skin at medium power. How big are the cells?
A tomato cell's nucleus seen at high power. How big is the nucleus?
An onion skin at high power. How big is a typical nucleus?
A banana cell at high power. How big are the starch grains?// ||