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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.

  • Specifications and Identification - Microscope manufacturers offer a wide range of objective designs to meet the performance needs of specialized imaging methods, to compensate for cover glass thickness variations, and to increase the effective working distance of the objective. Often, the function of a particular objective is not obvious simply by looking at the construction of the objective, but many specifications are permanently engraved on the objective barrel.

  • Objectives for Specialized Applications - Standard brightfield objectives, corrected for varying degrees of optical aberration, are the most common and are useful for examining specimens with traditional illumination techniques. Other, more complex, methods require specific objective configurations, which often include placement of a detector on or near the rear focal plane. To complicate the issue, the objective rear focal plane can reside in the center of an internal glass lens element, an area that is not easily accessible to the microscopist.

  • Water Immersion Objectives - Microscopic investigations of thinly cut fixed tissue sections and living cells adhered to glass substrates routinely produce superb high-resolution images when employing plan apochromat or fluorite objectives having a high numerical aperture. However, a significant amount of current biological research involves the investigation of cellular dynamics inside living tissue where important events can occur deep within the specimen, far away from the cover glass. Attempts to image cellular details and activities at a micrometer distances from the specimen cover glass with conventional oil immersion techniques often suffer from artifacts, including severe optical (spherical) aberration. The use of water in place of oil, as the immersion medium, is an effective approach to overcoming the aberration problems, and highly corrected water immersion objectives have been introduced by several manufacturers for applications involving living cells and tissues.

  • Adjustment of Objective Correction Collars - Most microscope objectives are designed to be used with a cover glass that has a standard thickness of 0.17 millimeters and a refractive index of 1.515, which is satisfactory when the objective numerical aperture is 0.4 or less. However, when using high numerical aperture dry objectives (numerical aperture of 0.8 or greater), cover glass thickness variations of only a few micrometers result in dramatic image degradation due to aberration, which grows worse with increasing cover glass thickness. To compensate for this error, the more highly corrected objectives are equipped with a correction collar to allow adjustment of the central lens group position to coincide with fluctuations in cover glass thickness. This interactive tutorial explores how a correction collar is adjusted to achieve maximum image quality.

  • Numerical Aperture & Resolution - Numerical aperture as applied to microscope objectives is a measure of the ability to gather light and resolve fine specimen detail at a fixed object distance. The resolution of a microscope objective is defined as the smallest distance between two points on a specimen that can still be distinguished as two separate entities. Resolution is a somewhat subjective value in microscopy because at high magnification, an image may appear unsharp but still be resolved to the maximum ability of the objective. Numerical aperture determines the resolving power of an objective, but the total resolution of a microscope system is also dependent upon the numerical aperture of the substage condenser. The higher the numerical aperture of the total system, the better the resolution.

  • Image Formation - In the optical microscope, image formation occurs at the intermediate image plane through interference between direct light that has passed through the specimen unaltered and light diffracted by minute features present in the specimen. The image produced by an objective lens is conjugate with the specimen, meaning that each image point at the intermediate plane is geometrically related to a corresponding point in the specimen.

  • Optical Aberrations - Departures from the idealized conditions of Gaussian optics are known as optical aberrations. Microscope optical trains typically suffered from as many as five common aberrations: spherical, chromatic, curvature of field, comatic, and astigmatic. Geometrical distortion is another artifact often encountered in the zoom lens systems found in stereoscopic microscopes.

  • Immersion Media - Most low-power objectives are designed to be used "dry" with air as the imaging medium. Higher magnification objectives commonly use liquid immersion media to help correct aberrations and increase numerical aperture.

  • Mechanical Tube Length - The mechanical tube length of an optical microscope is defined as the distance from the nosepiece opening, where the objective is mounted, to the top edge of the observation tubes where the eyepieces (oculars) are inserted. Until the 1980s, most microscopes had a fixed tube length ranging from 160 to 210 millimeters, depending upon the manufacturer and application. Modern microscopes are equipped with infinity-corrected objectives that utilize a tube lens in the microscope body to form a parallel region of light waves into which optical accessories can be inserted without seriously affecting image quality.

  • Modulation Transfer Function - The modulation transfer function of a lens, microscope objective, or other optical system is a measurement of its ability to transfer contrast at a particular resolution level from the object (or specimen) to the image. Computation of the modulation transfer function is a mechanism that is often utilized by optical manufacturers to incorporate resolution and contrast data into a single specification.

  • Infinity Optical Systems - In modern research-grade microscopes equipped with infinity-corrected optical systems, the objective no longer projects the intermediate image directly into the intermediate image plane. Instead, the objectives are designed so that light emerging from the rear aperture is focused to infinity, and a second lens, known as the tube lens, form the image at its focal plane.

  • Selected Literature References - The subject of microscope objective lenses has been reviewed on numerous occasions by a number of distinguished scientists. Many references listed in this section are comprehensive and cover a majority of topics concerning the structure and function of objectives, while others concentrate on various aspects and specialized applications of these lenses.

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.

Contributing Authors

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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