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Basic Concepts in Optical Microscopy

Section Overview:

Microscopes are instruments designed to produce magnified visual or photographic images of small objects. 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 designs with objectives and condensers, but also very simple single lens devices that are often hand-held, such as a magnifying glass.

Review Articles

  • Introduction

    The microscope is an instrument designed to make fine details visible. This section discusses the evolution of the microscope from its beginning in the 1600s to modern-day sophisticated microscopes.

  • 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 & Geometrical Optics

    The concepts explored in this discussion 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 components contained within microscopes are mounted on a stable, ergonomically designed base that allows precision centering and careful alignment between those assemblies that are optically interdependent.

  • Microscope Illumination

    Discussions about microscope illumination cover the theory of Köhler illumination, and the practical aspects of adjusting a microscope for illumination in both transmitted and reflected light.

  • Light Sources

    Microscopes usually have an integral light source that can be controlled to a relatively high degree. Such sources are an incandescent tungsten-halogen bulb, arc-discharge lamps, light emitting diodes (LEDs), and lasers.

  • Köhler Illumination

    Köhler Illumination provides bright, even illumination throughout the viewfield and is the method of choice in all modern microscopy and photomicrography for transmitted as well as reflected light techniques.

  • Köhler Illumination with Transmitted Light

    Adjustment of a transmitted light microscope for Köhler illumination is a relatively easy process as each time a microscope is turned on, it should be carefully inspected to ensure proper alignment of all optical components.

  • Köhler Illumination with Reflected Light

    Köhler illumination in reflected light microscopy is similar to that in transmitted light. This section discusses the fundamentals and importance of aligning a reflected light microscope for Köhler illumination.

  • Köhler Illumination Interactive Java Tutorials

    These tutorials explore various aspects in preparing a microscope for Köhler illumination, and allow students to practice alignment of the microscope without the burden of requiring the presence of a physical instrument.

  • 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.

  • 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.

  • Microscope Objectives:
    Specifications and Identification

    Learn about the objective designs that manufacturers offer to meet the needs of specialized imaging methods in order to compensate for cover glass thickness variations, and to increase the working distance of the objective.

  • Microscope Objectives:
    Objectives for Specialized Applications

    Objectives designed for phase contrast, Hoffman modulation contrast, and differential interference contrast require the assistance of optical detectors to modify events occurring at the objective rear focal plane.

  • Microscope Objectives:
    Numerical Aperture and Resolution

    Numerical aperture is a measure of the ability to gather light and resolve specimen detail at a fixed object distance. Resolution is the smallest distance between two points on a specimen that can still be labeled as separate entities.

  • Microscope Objectives:
    Image Formation

    In optical microscopes, image formation occurs at the intermediate image plane through interference between direct light that has passed through specimen unaltered and light diffracted by minute features in the specimen.

  • Microscope Objectives:
    Optical Aberrations

    Departures from the conditions of Gaussian optics are known as optical aberrations. Microscope optical trains typically suffer from five common aberrations: spherical, chromatic, curvature of field, comatic, and astigmatic.

  • Microscope Objectives:
    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.

  • Microscope Objectives:
    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.

  • Microscope Objectives:
    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.

  • Microscope Objectives:
    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 as shown in this section.

  • Microscope Objectives:
    Selected Literature References

    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. In many instances, eyepieces are designed to eliminate chromatic aberration.

  • Substage Condensers

    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

    Microscopes are designed to include a stage where the specimen is placed for observation. Stages are equipped with a device that holds the specimen slide in place and translates the slide back and forth and/or side to side.

  • 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.

  • Basic Microscope Ergonomics

    Microscope operators 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, and the hand reaching high up for a focusing control.

  • Microscope Anatomy Interactive Java Tutorials

    We have constructed a variety of interactive tutorials to help explain some of the more difficult concepts in optical microscopy.

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.

Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.

Brian O. Flynn, John C. Long, Matthew 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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