How Light Microscopes Work

Ever since their invention in the late 1500s, light microscopes have enhanced our knowledge in basic biology, biomedical research, medical diagnostics and materials science. Light microscopes can magnify objects up to 1,000 times, revealing microscopic details. Light-microscopy technology has evolved far beyond the first microscopes of Robert Hooke and Antoni van Leeuwenhoek. Special techniques and optics have been developed to reveal the structures and biochemistry of living cells­. Microscopes have even entered the digital age, using charge-coupled devices (CCDs) and digital cameras to capture images. Yet the basic principles of these advanced microscopes are a lot like those of the student microscope you may have used in your first biology class.

We will enter the tiny world of light microscopes and examine the various technologies that let them expose what is otherwise undetectable to the human eye.



The Basics

A light microscope works very much like a refracting telescope, but with some minor differences. Let's briefly review how a telescope works.

A telescope must gather large amounts of light from a dim, distant object; therefore, it needs 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.

In contrast to a telescope, a microscope must gather 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, as it is brought to your eye.

The other major difference between a telescope and a microscope is that 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.

Also unlike a telescope, which has a fixed objective lens and interchangeable eyepieces, microscopes typically 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, as it is a telescope's.

We'll take a detailed look at the parts of a microscope later in the article.

Image Quality



Brightness - How light or dark is the image? 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 the image blurry or well-defined? 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 your ability to focus the image -- it can be too thick for the objective lens. The correct cover-glass thickness is written on the side of the objective lens.



Resolution - How close can two points in the image be 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 - What 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. Also, chemical stains applied to the specimen can enhance contrast

The Parts of a Light Microscope

A light microscope, whether a simple student microscope or a complex research microscope, has the following basic systems:

Specimen control - hold and manipulate the specimen stage - where the specimen rests clips - used to hold the specimen still on the stage (Because you are looking at a magnified image, even the smallest movements of the specimen can move parts of the image out of your field of view.)
micromanipulator - device that allows you to move the specimen in controlled, small increments along the x and y axes (useful for scanning a slide)
Illumination - shed light on the specimen (The simplest illumination system is a mirror that reflects room light up through the specimen.)
lamp - produces the light (Typically, lamps are tungsten-filament light bulbs. For specialized applications, mercury or xenon lamps may be used to produce ultraviolet light. Some microscopes even use lasers to scan the specimen.) rheostat - alters the current applied to the lamp to control the intensity of the light produced condenser - lens system that aligns and focuses the light from the lamp onto the specimen diaphragms or pinhole apertures - placed in the light path to alter the amount of light that reaches the condenser (for enhancing contrast in the image) Diagram of a typical student light microscope, showing the parts and the light path
Lenses - form the image objective lens - gathers light from the specimen eyepiece - transmits and magnifies the image from the objective lens to your eye nosepiece - rotating mount that holds many objective lenses tube - holds the eyepiece at the proper distance from the objective lens and blocks out stray light


Focus - position the objective lens at the proper distance from the specimen coarse-focus knob - used to bring the object into the focal plane of the objective lens fine-focus knob - used to make fine adjustments to focus the image


Support and alignment arm - curved portion that holds all of the optical parts at a fixed distance and aligns them base - supports the weight of all of the microscope parts The tube is connected to the arm of the microscope by way of a rack and pinion gear. This system allows you to focus the image when changing lenses or observers and to move the lenses away from the stage when changing specimens.

Some of the parts mentioned above are not shown in the diagram and vary between microscopes. Microscopes come in two basic configurations: upright and inverted. 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.

Light microscopes can reveal the structures of living cells and tissues, as well as of non-living samples such as rocks and semiconductors. Microscopes can be simple or complex in design, and some can do more than one type of microscopy, each of which reveals slightly different information. The light microscope has greatly advanced our biomedical knowledge and continues to be a powerful tool for scientists.