Visualizing the Universe: Light Microscopes

By: Jerry Flattum, Performer/Songwriter & Writer/Editor

The first light microscopes were developed in the late 1500s by Robert Hooke, Antoni van Leeuwenhoek, and others. Since then, the light microscope has evolved to include a variety of special techniques and optics used in biomedical research, medical diagnostics and materials science.

visualizing-the-universe-light-microscope

Classic Microscope

Microscopes are instruments designed to produce magnified visual or photographic images of objects too small to be seen with the naked eye. A microscope magnifies an image (whatever is being used as a specimen) and makes details visible to the eye or a camera. How lens work is based on the principles of refraction and reflection.

Light microscopes can magnify objects up to 1,000 times. Electron microscopes go up to 10,000 times with some transmission and scanning electron microscopes ranging in the millions.

Microscopes range from ancient sixteenth-century single-lens Dutch models to modern microprocessor-powered research microscopes. Very simply, microscope plus optics equals optical microscopy. Some microscopes are multiple-lens (compound microscopes) with objectives and condensers. Others are simple single lens instruments (includes the magnifying glass). Many microscopes now use charge-coupled devices (CCDs) and digital cameras to capture images.

Modern compound microscopes feature a two-stage magnifying design built around separate lens systems, the objective and the eyepiece (called 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).

The intermediate image is further magnified by the eyepiece. The viewer is able to see an enlarged virtual image through the eyepieces. Total magnification is the combination of the objective and eyepiece. By combining a number of lenses a microscope can produce extreme magnification, and microscopic levels can reach the atomic and sub-atomic levels.

Microscopes are designed with a great deal of precision. They must be mounted solidly, with precise centering and adjustment capability. Specimens are placed on a glass slide with a cover, and like the various lenses, can be subject to aberration. Illumination needs to be bright, no glare, and evenly dispersed. Apertures with numbers are used to make adjustments like brightness and magnification level. There is a wide range of accessories for an equally wide range of microscopes to help fine tune or provide different perspectives.

Precision and variety in design is critical since microscopes are used to view a wide range of specimens in different contexts, such as living cells immersed in water, or semiconductors, ceramics, metals, and polymers under varying conditions.

The first reported measurements performed with an optical microscope took place 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. Now, various micrometry techniques are used to make more precise measurements.

In photomicrography, the primary medium was film until the past decade when improvements in electronic cameras and computer technology made digital imaging cheaper and easier to use than conventional photography. In digital imaging, digitizing a video or electronic image captured through an optical microscope allows a significant increase in the ability to enhance features, extract information, or modify the image.

A light microscope is similar to a refracting telescope. A telescope gathers large amounts of light from a dim, distant object, and uses a large objective lens to gather as much light as possible and bring it to a bright focus. Because the objective lens is large, it brings the image of the object to a focus at some distance away, which is why telescopes are much longer than microscopes. The eyepiece of the telescope then magnifies that image as it brings it to your eye.

A microscope gathers light from a tiny area of a thin, well-illuminated specimen that is close-by. So the microscope does not need a large objective lens. Instead, the objective lens of a microscope is small and spherical, which means that it has a much shorter focal length on either side. It brings the image of the object into focus at a short distance within the microscope’s tube. The image is then magnified by a second lens, called an ocular lens or eyepiece.

A microscope has a light source and a condenser. The condenser is a lens system that focuses the light from the source onto a tiny, bright spot of the specimen, which is the same area that the objective lens examines. A telescope has a fixed objective lens and interchangeable eyepieces.

Microscopes have interchangeable objective lenses and fixed eyepieces. By changing the objective lenses (going from relatively flat, low-magnification objectives to rounder, high-magnification objectives), a microscope can bring increasingly smaller areas into view. Light gathering is not the primary task of a microscope’s objective lens, like it is with a telescope.

A simple way to demonstrate how a microscope works is to use two magnifying glasses and printed words on paper. One magnifying glass makes the print look larger. When a second magnifying glass is held between the eye and the first magnifying glass, moving the first magnifier brings the print into focus and makes the print even larger than just one magnifier.

Image Quality is based on brightness, focus, resolution and contrast. Brightness is how light and dark an image is. Brightness is related to the illumination system and can be changed by changing the voltage to the lamp (rheostat) and adjusting the condenser and diaphragm/pinhole apertures. Brightness is also related to the numerical aperture of the objective lens (the larger the numerical aperture, the brighter the image).

Focus is how blurred or detailed an image is. Focus is related to focal length and can be controlled with the focus knobs. The thickness of the cover glass on the specimen slide can also affect focus. Resolution is how close two points can be in the image before they are no longer seen as two separate points.

Resolution is related to the numerical aperture of the objective lens (the higher the numerical aperture, the better the resolution) and the wavelength of light passing through the lens (the shorter the wavelength, the better the resolution). Contrast is the difference in lighting between adjacent areas of the specimen. Contrast is related to the illumination system and can be adjusted by changing the intensity of the light and the diaphragm/pinhole aperture. Chemical stains are applied to a specimen to enhance contrast.

Most light microscopes feature the same components although can vary from manufacturer to manufacturer, with some microscopes designed for specific purposes. Light microscopes can reveal the structures of living cells and non-living specimens such as rocks and semiconductors.

Microscopes come in two basic configurations: upright and inverted. An upright microscope has the illumination system below the stage and the lens system above the stage. An inverted microscope has the illumination system above the stage and the lens system below the stage. Inverted microscopes are better for looking through thick specimens, such as dishes of cultured cells, because the lenses can get closer to the bottom of the dish, where the cells grow.

The stage is a platform where the specimen rests. Clips hold the specimen still on the stage. Various lenses form the image, while objective lenses gather light from the specimen. The eyepiece transmits and magnifies the image from the objective lens to the eye. The nosepiece rotating mount can hold several objective lenses.

The tube holds the eyepiece at the proper distance from the objective lens and blocks out stray light. The tube is also connected to the arm of the microscope with a rack and pinion gear. It allows refocusing when changing lens, observers or specimens. The arm is a curved piece that that aligns and holds all of the optical parts at a fixed distance. Focus is achieved when the objective lens is positioned at a distance from the specimen that produces the clearest image.

Microscopes are sensitive and must be sturdy since even the smallest movement of a specimen can throw an image out of focus. The base supports the microscope. Further adjustments are made with course and fine tuning knobs. A micromanipulator is a device that allows moving the specimen in controlled, small increments along the x and y axes, such as in for scanning a slide.

A simple illumination system is a mirror reflecting room light up through the specimen. Lamps are usually tungsten-filament light bulbs. For specialized applications, mercury or xenon lamps may be used to produce ultraviolet light while other microscopes use lasers to scan a specimen.

The rheostat alters the current applied to the lamp to control light intensity and the condenser aligns and focuses the light from the lamp onto the specimen. Diaphragms or pinhole apertures are placed in the light path to alter the amount of light that reaches the condenser (for enhancing contrast in the image.

The depth of field is the vertical distance from above to below the focal plane that yields an acceptable image. The field of view is the area of the specimen that can be seen through the microscope with an objective lens. The focal length is the distance required for a lens to bring the light to a focus (measured in microns). The focal point is where the light from a lens comes together to form an image.

Magnification is generated by the magnifying powers of the objective and eyepiece lenses. A numerical aperture measures the light-collecting ability of the lens. Resolution is the closest two objects can be before they’re no longer detected as separate objects (usually measured in nanometers.

When looking at a specimen with transmitted light, the light must pass through the specimen in order to form an image. The thicker the specimen, the less light passes through. The less light that passes through, the darker the image. Consequently, specimens must be thin, in the 0.1 to 0.5 mm range. Many living specimens must be cut into thin sections before observation. Specimens of rock or semiconductors are too thick to be sectioned and observed by transmitted light, so they are observed by the light reflected from their surfaces.

A major problem in observing specimens under a microscope is that their images do not have much contrast. This is especially true of living things (such as cells), although natural pigments, such as the green in leaves, can provide good contrast. One way to improve contrast is to treat the specimen with colored pigments or dyes that bind to specific structures within the specimen.

Different types of microscopy have been developed to improve the contrast in specimens. The specializations are mainly in the illumination systems and the types of light passed through the specimen. For example, a darkfield microscope uses a special condenser to block out most of the bright light and illuminate the specimen with oblique light, much like the moon blocks the light from the sun in a solar eclipse. This optical set-up provides a totally dark background and enhances the contrast of the image to bring out fine details (bright areas at boundaries within the specimen).

The basic idea involves splitting the light beam into two pathways that illuminate the specimen. Light waves that pass through dense structures within the specimen slow down compared to those that pass through less-dense structures. As all of the light waves are collected and transmitted to the eyepiece, they are recombined, causing interference. The interference patterns provide contrast. Some patterns will show dark areas (more dense) on a light background (less dense), or create a type of false three-dimensional (3-D) image.

Brightfield is a basic microscope and technique that has very little contrast. Contrast is usually achieved by staining the specimens. In turn, the Darkfield technique enhances contrast. Rheinberg illumination is similar to darkfield, but uses a series of filters to produce an “optical staining” of the specimen. Phase contrast is best for looking at living specimens, such as cultured cells.

In a phase-contrast microscope, the annular rings in the objective lens and the condenser separate the light. The light that passes through the central part of the light path is recombined with the light that travels around the periphery of the specimen. The interference produced by these two paths produces images in which the dense structures appear darker than the background.

Differential interference contrast (DIC) uses polarizing filters and prisms to separate and recombine the light paths, giving a 3-D appearance to the specimen (DIC is also called Nomarski after the man who invented it). Hoffman modulation contrast is similar to DIC except that it uses plates with small slits in both the axis and the off-axis of the light path to produce two sets of light waves passing through the specimen, producing a 3-D image.

A polarized-light microscope uses two polarizers, one on either side of the specimen, positioned perpendicular to each other so that only light that passes through the specimen reaches the eyepiece. Light is polarized in one plane as it passes through the first filter and reaches the specimen. Regularly-spaced, patterned or crystalline portions of the specimen rotate the light that passes through. Some of this rotated light passes through the second polarizing filter, so these regularly spaced areas show up bright against a black background.

A fluorescence microscope uses high-energy, short-wavelength light (usually ultraviolet) to excite electrons within certain molecules inside a specimen, causing those electrons to shift to higher orbits. When they fall back to their original energy levels, they emit lower-energy, longer-wavelength light (usually in the visible spectrum), which forms the image.

Lens

The term lens is the common name given to a component of glass or transparent plastic material, usually circular in design, with two primary surfaces ground and polished in a specific manner designed to produce either a convergence or divergence of light. Lenses operate according to the principles of refraction and reflection.

A microscope forms an image of a specimen placed on the stage (specimen mounting area) by passing light from the illuminator through a series of glass lenses and focusing this light either into the eyepieces, on the film plane in a traditional camera system, or onto the surface of a digital image sensor. Errors in the lens are called aberrations, and are found throughout all microscopes and other optical devices.

A simple thin lens has two focal planes that are defined by the geometry of the lens and the relationship between the lens and the focused image. Light rays passing through the lens will intersect and are physically combined at the focal plane. Extensions of the rays passing through the lens will intersect with the rays emerging from the lens at the principal plane.

The focal length of a lens is defined as the distance between the principal plane and the focal plane, and every lens has a set of these planes on each side (front and rear).

A magnifying glass consists of a single thin bi-convex lens that produces a modest magnification useful for reading or viewing things enlarged to a magnification level similar to making words bigger. Single lenses like the bi-convex lens are useful for simple magnification commonly found in magnifying glasses, eyeglasses, single-lens cameras, loupes, viewfinders, and contact lenses.

Positive, or converging, thin lenses unite incident light rays that are parallel to the optical axis and focus them at the focal plane to form a real image. Negative lenses diverge parallel incident light rays and form a virtual image by extending traces of the light rays passing through the lens to a focal point behind the lens. In general, these lenses have at least one concave surface and are thinner in the center than at the edges.

Mirrors

In addition to being used in microscope illumination systems, mirrors are found everywhere, from fun houses to bathrooms to portable make-up kits. They vary widely in design, construction and reflectivity. Some mirrors magnify, like make-up kits. Others are highly polished, coated with metals that reflect both visible and infrared wavelengths.

Reflection of light is an inherent and important fundamental property of mirrors, and is quantitatively gauged by the ratio between the amount of light reflected from the surface and that incident upon the surface, a term known as reflectivity.

The images formed by a mirror are either real or virtual, depending upon the proximity of the object to the mirror, and can be accurately predicted with respect to size and location from calculations based on the geometry of any particular mirror. Real images are formed when the incident and reflected rays intersect in front of the mirror, whereas virtual images occur at points where extensions from incident and reflected rays converge behind the mirror. Planar (flat) mirrors produce virtual images because the focal point, at which extensions from all incident light rays intersect, is positioned behind the reflective surface.

In order to reflect light waves with high efficiency, the surface of a mirror must be perfectly smooth over a long range, with imperfections that are much smaller than the wavelength of light being reflected. This requirement applies regardless of the shape of the mirror, which can be irregular or curved, in addition to the planar mirror surfaces commonly seen in households.

Curved mirrors are roughly divided into two categories, concave and convex, terms that are also used to describe the geometry of simple thin lenses. With mirrors, the curved surface is referred to as either concave or convex depending upon whether the center of curvature occurs on the side of the reflecting surface or the opposite side.

Concave mirrors have a curved surface with a center of curvature equidistant from every point on the mirror’s surface. An object beyond the center of curvature forms a real and inverted image between the focal point and the center of curvature. Moving the object farther away from the center of curvature affects the size of the real image formed by the mirror.

Regardless of the position of the object reflected by a convex mirror, the image formed is always virtual, upright, and reduced in size.

Beamsplitters and Prisms

Beamsplitters and prisms are not only found in a wide variety of common optical instruments, such as cameras, binoculars, microscopes, telescopes, periscopes, range finders, and surveying equipment, but also in many sophisticated scientific instruments including interferometers, spectrophotometers, and fluorimeters. Both of these important optical tools are critical for laser applications that require tight control of beam direction to precise tolerances with a minimum of light loss due to scatter or unwanted reflections.

Binocular microscopes use prisms and beamsplitters. In order to divert light collected by the objective into both eyepieces, it is first divided by a beamsplitter and then channeled through reflecting prisms into parallel cylindrical optical light pipes. Thus, the binocular observation tube utilizes both prism and beamsplitter technology to direct beams of light having equal intensity into the eyepieces.

Prisms and beamsplitters are essential components that bend, split, reflect, and fold light through the pathways of both simple and sophisticated optical systems. Prisms are polished blocks of glass or other transparent materials cut and ground to specific tolerances and exact angles. They are used to deflect a light beam, rotate or invert an image, separate polarization states, or disperse light into its component wavelengths.

A beamsplitter is a common optical component that partially transmits and partially reflects an incident light beam, usually in unequal proportions. In addition to the task of dividing light, beamsplitters can be employed to recombine two separate light beams or images into a single path.

There are many different kinds of prisms and beamsplitters, such as reflecting prisms, right-angle prisms, equilateral prisms, dielectric plate beamsplitters, circular prisms, wedge prisms, birefringent polarizing prisms and others.

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.

Light emitting diodes (LEDs) (miniature semiconductor devices) could conceivably replace the light bulb. This is revolutionary, considering the light bulb might single-handedly be responsible for modern society. Light emitting diodes (LEDs) are a general source of continuous light with high luminescence efficiency, and are based on the general properties of a simple twin-element semiconductor diode encased in a clear epoxy dome that acts as a lens.

In order to generate enough excitation light intensity to furnish secondary fluorescence emission capable of detection, powerful light sources are needed. These are usually either mercury or xenon arc (burner) lamps, which produce high-intensity illumination powerful enough to image faintly visible fluorescence specimens. Mercury and xenon arc lamps are now widely in fluorescence microscopy.

Nearly every source of light depends, at the fundamental level, on the release of energy from atoms that have been excited in some manner. Standard incandescent lamps, derived directly from the early models of the 1800s, now commonly utilize a tungsten filament in an inert gas atmosphere, and produce light through the resistive effect that occurs when the filament temperature increases as electrical current is passed through (see Color Temperature).

Fluorescence Microscopy

In the mid-19th century, British scientist Sir George G. Stokes made the observation that the mineral fluorspar exhibits fluorescence when illuminated with ultraviolet light. Hence, fluorescence.

Fluorescence microscopy is an excellent method of studying material that can be made to fluoresce, either in its natural form (termed primary or auto fluorescence) or when treated with chemicals capable of fluorescing (known as secondary fluorescence). The fluorescence microscope came into being during the early 20th century through the work of August Kohler, Carl Reichert, Heinrich Lehmann, and others. Fluorescence microscopy is now used extensively in cellular biology.

Epifluorescence is an optical set-up for a fluorescence microscope in which the objective lens is used both to focus ultraviolet light on the specimen and collect fluorescent light from the specimen. Epifluorescence is more efficient than transmitted fluorescence, in which a separate lens or condenser is used to focus ultraviolet light on the specimen. Epifluorescence also allows fluorescence microscopy to be combined with another type on the same microscope.

A fluorescence microscope uses a mercury or xenon lamp to produce ultraviolet light. The light comes into the microscope and hits a dichroic mirror, which is a mirror that reflects one range of wavelengths and allows another range to pass through. The dichroic mirror reflects the ultraviolet light up to the specimen. The ultraviolet light excites fluorescence within molecules in the specimen. The objective lens collects the fluorescent-wavelength light produced. This fluorescent light passes through the dichroic mirror and a barrier filter (that eliminates wavelengths other than fluorescent), making it to the eyepiece to form the image.

Fluorescence-microscopy techniques are useful for seeing structures and measuring physiological and biochemical events in living cells. Various fluorescent indicators are available to study many physiologically important chemicals such as DNA, calcium, magnesium, sodium, pH and enzymes. In addition, antibodies that are specific to various biological molecules can be chemically bound to fluorescent molecules and used to stain specific structures within cells.