Basic Concepts in Optical Microscopy
Modern compound microscopes feature a two-stage magnifying design built around separate lens systems, the objective and the eyepiece (commonly termed an ocular), mounted at opposite ends of a tube, known as the body tube. The objective is composed of several lens elements that together form a magnified real image (the intermediate image) of the specimen being examined. The intermediate image is further magnified by the eyepiece. The microscopist is able to observe a greatly enlarged virtual image of the specimen by peering through the eyepieces. The total magnification of a microscope is determined by multiplying the individual magnifications of the objective and eyepiece. This section discusses the basic concepts associated with optical microscopy, including objectives, eyepieces, condensers, stages, magnification, numerical aperture, optical aberrations, and a variety of related topics.
Introduction to Microscopy - Microscopes are instruments designed to produce magnified visual or photographic images of objects too small to be seen with the naked eye. The microscope must accomplish three tasks: produce a magnified image of the specimen, separate the details in the image, and render the details visible to the human eye or camera. This group of instruments includes not only multiple-lens (compound microscopes) designs with objectives and condensers, but also very simple single lens instruments that are often hand-held, such as a loupe or magnifying glass.
The Concept of Magnification - The image of an object can be magnified when viewed through a simple lens. By combining a number of lenses in the correct manner, a microscope can be produced that will yield very high magnification values.
Introduction to Lenses and Geometrical Optics - The action of a simple lens, similar to many of those used in the microscope, is governed by the principles of refraction and reflection and can be understood with the aid of a few simple rules about the geometry involved in tracing light rays through the lens. The basic concepts explored in this discussion, which are derived from the science of Geometrical Optics, will lead to an understanding of the magnification process, the properties of real and virtual images, and lens aberrations or defects.
Microscope Optical Components - The optical components contained within modern microscopes are mounted on a stable, ergonomically designed base that allows rapid exchange, precision centering, and careful alignment between those assemblies that are optically interdependent. Together, the optical and mechanical components of the microscope, including the mounted specimen on a glass micro slide and coverslip, form an optical train with a central axis that traverses the microscope base and stand.
Microscope Illumination - One of the most critical aspects in optical microscopy is to ensure the specimen is illuminated with light that is bright, glare-free, and evenly dispersed in the field of view. Discussions about microscope illumination cover the theory of Köhler illumination (accompanied by interactive tutorials), and the practical aspects of adjusting a microscope for proper illumination in both transmitted and reflected light.
Light Sources for Optical Microscopy - The performance of the various illumination sources available for optical microscopy depends on the emission characteristics and geometry of the source, as well as the focal length, magnification and numerical aperture of the collector lens system. In gauging the suitability of a particular light source, the important parameters are structure (the spatial distribution of light, source geometry, coherence, and alignment), the wavelength distribution, spatial and temporal stability, brightness, and to what degree these various parameters can be controlled.
Image Brightness - Regardless of the imaging mode utilized in optical microscopy, image brightness is governed by the light-gathering power of the objective, which is a function of numerical aperture. Just as brightness of the microscope source illumination is determined by the square of the condenser working numerical aperture, brightness of the specimen image is proportional to the square of the objective numerical aperture.
Microscope Objectives - Microscope objectives are the most important components of an optical microscope because they determine the quality of images that the microscope is capable of producing. There is a wide range of objective designs available that feature excellent optical performance and provide for the elimination of most optical aberrations.
Eyepieces (Oculars) - Eyepieces work in combination with microscope objectives to further magnify the intermediate image so that specimen details can be clearly observed. There are two major types of eyepieces that are grouped according to lens and aperture diaphragm arrangement: the negative eyepieces with an internal diaphragm and positive eyepieces that have a diaphragm below the lenses of the eyepiece. In many instances, eyepieces are designed to work together with objectives to eliminate chromatic aberration.
Linear Measurements (Micrometry) - The first reported measurements performed with an optical microscope were undertaken in the late 1600s by the Dutch scientist Antonie van Leeuwenhoek, who used fine grains of sand as a gauge to determine the size of human erythrocytes. Since then, countless approaches have been employed for measuring linear, area, and volume specimen dimensions with the microscope (a practice known as micrometry or morphometrics), and a wide variety of useful techniques have emerged over the past few hundred years.
Substage Condensers - The substage condenser gathers light from the microscope light source and concentrates it into a cone of light that illuminates the specimen with uniform intensity over the entire viewfield. It is critical that the condenser light cone be properly adjusted to optimize the intensity and angle of light entering the objective front lens. Perhaps the most poorly understood component in the optical train, the condenser is nevertheless one of the most important factors in obtaining high quality images in the microscope.
Specimen Stages - All microscopes are designed to include a stage where the specimen (usually mounted onto a glass slide) is placed for observation. Stages are often equipped with a mechanical device that holds the specimen slide in place and can smoothly translate the slide back and forth as well as from side to side. Other stages are designed to allow rotation of the specimen through 360 degrees or to provide anchors for auxiliary light sources, specimen manipulation tools, and other accessories.
Reflected Light Microscopy - Microscopy using oblique or epi-illumination is utilized for the study of specimens that are opaque, including semiconductors, ceramics, metals, polymers, and many others.
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.
Basic Microscope Ergonomics - In order to view specimens and record data, microscope operators must assume an unusual but exacting position, with little possibility to move the head or the body. They are often forced to assume an awkward work posture such as the head bent over the eye tubes, the upper part of the body bent forward, the hand reaching high up for a focusing control, or with the wrists bent in an unnatural position.
Cleaning, Care, and Maintenance of Microscopes - Microscopes often represent a significant investment of funds and are sophisticated optical instruments that require periodic maintenance and cleaning to guarantee production of high-contrast images equal to the quality of the optical, electronic, and mechanical components. When neglected by exposure to dust, lint, pollen, and dirt, failure to remove immersion oil in a timely manner, or when expensive objectives are abused, optical performance can experience a serious decline that increases over time.
Microscope Anatomy Interactive Java Tutorials - We have constructed a variety of interactive Java-driven microscopy tutorials to help explain some of the more difficult concepts in optical microscopy. Students can view and utilize these tutorials using a web browser without the addition of plug-in software.
Digital Image Galleries
Brightfield Microscopy Digital Image Gallery - Brightfield illumination has been one of the most widely used observation modes in optical microscopy for the past 300 years. The technique is best suited for utilization with fixed, stained specimens or other kinds of samples that naturally absorb significant amounts of visible light. Images produced with brightfield illumination appear dark and/or highly colored against a bright, often light gray or white, background. This digital image gallery explores a variety of stained specimens captured with an Olympus BX51 microscope coupled to a 12-bit QImaging Retiga camera system and a three-color liquid crystal tunable filter.
Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.
H. Ernst Keller - Carl Zeiss Inc., One Zeiss Dr., Thornwood, NY, 10594.
Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.
Brian O. Flynn, John C. Long, Matthew J. Parry-Hill, Kirill I. Tchourioukanov, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.
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