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 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, and the practical aspects of adjusting a microscope for proper illumination in both transmitted and reflected light.

• Light Sources - Modern microscopes usually have an integral light source that can be controlled to a relatively high degree. The most common source for today's microscopes is an incandescent tungsten-halogen bulb positioned in a reflective housing that projects light through the collector lens and into the substage condenser. Other sources include arc-discharge lamps, light emitting diodes (LEDs), and lasers.

• Köhler Illumination - This procedure 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.

• Illumination with Transmitted Light - The important aspects of establishing the conditions of Köhler illumination in transmitted light microscopes are described in this section.

• Illumination with Reflected Light - Köhler illumination in reflected light microscopy is similar to that in transmitted light. This section discusses the fundamentals of aligning a reflected light microscope for Köhler illumination.

• Interactive Java Tutorials on Microscope Illumination - 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. 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.

• 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.

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.

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.

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.

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.