Visiblelight

The lowest power lens is usually 3.5 or 4x, and is used primarily for initially finding specimens. We sometimes call it the scanning lens for that reason. The most frequently used objective lens is the 10x lens, which gives a final magnification of 100x with a 10x ocular lens. For very small protists and for details in prepared slides such as cell organelles or mitotic figures, you will need a higher magnification. Typical high magnification lenses are 40x and 97x or 100x. The latter two magnifications are used exclusively with oil in order to improve resolution.

X-ray: A dentist uses X-rays to image your teeth, and airport security uses them to see through your bag. Hot gases in the Universe also emit X-rays.

Are radio waves completely different physical objects than gamma-rays? They are produced in different processes and are detected in different ways, but they are not fundamentally different. Radio waves, gamma-rays, visible light, and all the other parts of the electromagnetic spectrum are electromagnetic radiation.

A disadvantage of having to rely solely on an aperture diaphragm for contrast is that beyond an optimum point the more contrast you produce the more you distort the image. With a small, unstained, unpigmented specimen, you are usually past optimum contrast when you begin to see the image.

Infraredwavelength range in nm

The electromagnetic spectrum from lowest energy/longest wavelength (at the top) to highest energy/shortest wavelength (at the bottom). (Credit: NASA's Imagine the Universe)

Infraredfrequency

The cover slip must be up if there is one. High magnification objective lenses can't focus through a thick glass slide; they must be brought close to the specimen, which is why coverslips are so thin. The stage may be equipped with simple clips (less expensive microscopes), or with some type of slide holder. The slide may require manual positioning, or there may be a mechanical stage (preferred) that allows precise positioning without touching the slide.

Move up in magnification by steps. Each time you go to a higher power objective, re-focus and re-center the specimen. Higher magnification lenses must be physically closer to the specimen itself, which poses the risk of jamming the objective into the specimen. Be very cautious when focusing. By the way, good quality sets of lenses are parfocal, that is, when you switch magnifications the specimen remains in focus or close to focused.

The light microscope, so called because it employs visible light to detect small objects, is probably the most well-known and well-used research tool in biology. Yet, many students and teachers are unaware of the full range of features that are available in light microscopes. Since the cost of an instrument increases with its quality and versatility, the best instruments are, unfortunately, unavailable to most academic programs. However, even the most inexpensive "student" microscopes can provide spectacular views of nature and can enable students to perform some reasonably sophisticated experiments.

Bigger is not always better. All specimens have three dimensions, and unless a specimen is extremely thin you will be unable to focus with a high magnification objective. The higher the magnification, the harder it is to "chase" a moving specimen.

With a conventional bright field microscope, light from an incandescent source is aimed toward a lens beneath the stage called the condenser, through the specimen, through an objective lens, and to the eye through a second magnifying lens, the ocular or eyepiece. We see objects in the light path because natural pigmentation or stains absorb light differentially, or because they are thick enough to absorb a significant amount of light despite being colorless. A Paramecium should show up fairly well in a bright field microscope, although it will not be easy to see cilia or most organelles. Living bacteria won't show up at all unless the viewer hits the focal plane by luck and distorts the image by using maximum contrast.

Start with the lowest magnification objective lens, to home in on the specimen and/or the part of the specimen you wish to examine. It is rather easy to find and focus on sections of tissues, especially if they are fixed and stained, as with most prepared slides. However it can be very difficult to locate living, minute specimens such as bacteria or unpigmented protists. A suspension of yeast cells makes a good practice specimen for finding difficult objects.

Bright field microscopy is best suited to viewing stained or naturally pigmented specimens such as stained prepared slides of tissue sections or living photosynthetic organisms. It is useless for living specimens of bacteria, and inferior for non-photosynthetic protists or metazoans, or unstained cell suspensions or tissue sections. Here is a not-so-complete list of specimens that might be observed using bright-field microscopy, and appropriate magnifications (preferred final magnifications are emphasized).

One or both of the eyepieces may be a telescoping eyepiece, that is, you can focus it. Since very few people have eyes that are perfectly matched, most of us need to focus one eyepiece to match the other image. Look with the appropriate eye into the fixed eyepiece and focus with the microscope focus knob. Next, look into the adjustable eyepiece (with the other eye of course), and adjust the eyepiece, not the microscope.

Electromagnetic radiation can be described in terms of a stream of mass-less particles, called photons, each traveling in a wave-like pattern at the speed of light. Each photon contains a certain amount of energy. The different types of radiation are defined by the the amount of energy found in the photons. Radio waves have photons with low energies, microwave photons have a little more energy than radio waves, infrared photons have still more, then visible, ultraviolet, X-rays, and, the most energetic of all, gamma-rays.

It is a lot harder to find something when you have no expectations as to its apprearance. How big is it? Will it be moving? Is it pigmented or stained, and if so what is its color? Where do you expect to find it on a slide? For example, students typically have a lot of trouble finding stained bacteria because with the unaided eye and at low magnifications the stuff looks like dirt. It helps to know that as smears dry down they usually leave rings so that the edge of a smear usually has the densest concentration of cells.

Infrared light definitionand examples

The Earth's atmosphere stops most types of electromagnetic radiation from space from reaching Earth's surface. This illustration shows how far into the atmosphere different parts of the EM spectrum can go before being absorbed. Only portions of radio and visible light reach the surface. (Credit: STScI/JHU/NASA)

First, think about what you want to do with the microscope. What is the maximum magnification you will need? Are you looking at a stained specimen? How much contrast/resolution do you require? Next, start setting up for viewing.

To adjust and align the microscope, start by reading the manual. If no manual is available, try using these guidelines. If the condenser is focusable, position it with the lens as close to the opening in the stage as you can get it. If the condenser has selectable options, set it to bright field. Start with the aperture diaphragm stopped down (high contrast). You should see the light that comes up through the specimen change brightness as you move the aperture diaphragm lever.

The Earth's atmosphere stops most types of electromagnetic radiation from space from reaching Earth's surface. This illustration shows how far into the atmosphere different parts of the EM spectrum can go before being absorbed. Only portions of radio and visible light reach the surface. (Credit: STScI/JHU/NASA)

Infraredradiation examples

A light source should have a wide dynamic range, to provide high intensity illumination at high magnifications, and lower intensities so that the user can view comfortably at low magnifications. Better microscopes have a built-in illuminator, and the best microscopes have controls over light intensity and shape of the light beam. If your microscope requires an external light source, make sure that the light is aimed toward the middle of the condenser. Adjust illumination so that the field is bright without hurting the eyes.

The smallest objects that are considered to be living are the bacteria. The smallest bacteria can be observed and cell shape recognized at a mere 100x magnification. They are invisible in bright field microscopes, though. These pages will describe types of optics that are used to obtain contrast, suggestions for finding specimens and focusing on them, and advice on using measurement devices with a light microscope.

The electromagnetic (EM) spectrum is the range of all types of EM radiation. Radiation is energy that travels and spreads out as it goes – the visible light that comes from a lamp in your house and the radio waves that come from a radio station are two types of electromagnetic radiation. The other types of EM radiation that make up the electromagnetic spectrum are microwaves, infrared light, ultraviolet light, X-rays and gamma-rays.

Electromagnetic radiation can be expressed in terms of energy, wavelength, or frequency. Frequency is measured in cycles per second, or Hertz. Wavelength is measured in meters. Energy is measured in electron volts. Each of these three quantities for describing EM radiation are related to each other in a precise mathematical way. But why have three ways of describing things, each with a different set of physical units?

Infrareduses

The short answer is that scientists don't like to use numbers any bigger or smaller than they have to. It is much easier to say or write "two kilometers" than "two thousand meters." Generally, scientists use whatever units are easiest for the type of EM radiation they work with.

Infrared and optical astronomers generally use wavelength. Infrared astronomers use microns (millionths of a meter) for wavelengths, so their part of the EM spectrum falls in the range of 1 to 100 microns. Optical astronomers use both angstroms (0.00000001 cm, or 10-8 cm) and nanometers (0.0000001 cm, or 10-7 cm). Using nanometers, violet, blue, green, yellow, orange, and red light have wavelengths between 400 and 700 nanometers. (This range is just a tiny part of the entire EM spectrum, so the light our eyes can see is just a little fraction of all the EM radiation around us.)

Radio: Your radio captures radio waves emitted by radio stations, bringing your favorite tunes. Radio waves are also emitted by stars and gases in space.

Ultraviolet: Ultraviolet radiation is emitted by the Sun and are the reason skin tans and burns. "Hot" objects in space emit UV radiation as well.

The apparent field of an eyepiece is constant regardless of magnification used. So it follows that when you raise magnification the area of illuminated specimen you see is smaller. Since you are looking at a smaller area, less light reaches the eye, and the image darkens. With a low power objective you may have to cut down on illumination intensity. With a high power you need all the light you can get, especially with less expensive microscopes.

What isinfrared

Gamma ray: Doctors use gamma-ray imaging to see inside your body. The biggest gamma-ray generator of all is the Universe.

Infrareddangers

Astronomers who study radio waves tend to use wavelengths or frequencies. Most of the radio part of the EM spectrum falls in the range from about 1 cm to 1 km, which is 30 gigahertz (GHz) to 300 kilohertz (kHz) in frequencies. The radio is a very broad part of the EM spectrum.

You know more about the electromagnetic spectrum than you may think. The image below shows where you might encounter each portion of the EM spectrum in your day-to-day life.

Students are usually aware of the use of the coarse and fine focus knobs, used to sharpen the image of the specimen. They are frequently unaware of adjustments to the condenser that can affect resolution and contrast. Some condensers are fixed in position, others are focusable, so that the quality of light can be adjusted. Usually the best position for a focusable condenser is as close to the stage as possible. The bright field condenser usually contains an aperture diaphragm, a device that controls the diameter of the light beam coming up through the condenser, so that when the diaphragm is stopped down (nearly closed) the light comes straight up through the center of the condenser lens and contrast is high. When the diaphragm is wide open the image is brighter and contrast is low.

A service of the High Energy Astrophysics Science Archive Research Center (HEASARC), Dr. Andy Ptak (Director), within the Astrophysics Science Division (ASD) at NASA/GSFC

A good quality microscope has a built-in illuminator, adjustable condenser with aperture diaphragm (contrast) control, mechanical stage, and binocular eyepiece tube. The condenser is used to focus light on the specimen through an opening in the stage. After passing through the specimen, the light is displayed to the eye with an apparent field that is much larger than the area illuminated. The magnification of the image is simply the objective lens magnification (usually stamped on the lens body) times the ocular magnification.

Most electromagnetic radiation from space is unable to reach the surface of the Earth. Radio frequencies, visible light and some ultraviolet light makes it to sea level. Astronomers can observe some infrared wavelengths by putting telescopes on mountain tops. Balloon experiments can reach 35 km above the surface and can operate for months. Rocket flights can take instruments all the way above the Earth's atmosphere, but only for a few minutes before they fall back to Earth.

The wavelengths of ultraviolet, X-ray, and gamma-ray regions of the EM spectrum are very small. Instead of using wavelengths, astronomers that study these portions of the EM spectrum usually refer to these photons by their energies, measured in electron volts (eV). Ultraviolet radiation falls in the range from a few electron volts to about 100 eV. X-ray photons have energies in the range 100 eV to 100,000 eV (or 100 keV). Gamma-rays then are all the photons with energies greater than 100 keV.

With a single ocular, there is nothing to do with the eyepiece except to keep it clean. With a binocular microscope (preferred) you need to adjust the eyepiece separation just like you do a pair of binoculars. Binocular vision is much more sensitive to light and detail than monocular vision, so if you have a binocular microscope, take advantage of it.

Why do we put telescopes in orbit? The Earth's atmosphere stops most types of electromagnetic radiation from space from reaching Earth's surface. This illustration shows how far into the atmosphere different parts of the EM spectrum can go before being absorbed. Only portions of radio and visible light reach the surface. (Credit: STScI/JHU/NASA) Most electromagnetic radiation from space is unable to reach the surface of the Earth. Radio frequencies, visible light and some ultraviolet light makes it to sea level. Astronomers can observe some infrared wavelengths by putting telescopes on mountain tops. Balloon experiments can reach 35 km above the surface and can operate for months. Rocket flights can take instruments all the way above the Earth's atmosphere, but only for a few minutes before they fall back to Earth. For long-term observations, however, it is best to have your detector on an orbiting satellite and get above it all! Updated: March 2013

Other than the compound microscope, a simpler instrument for low magnification use may also be found in the laboratory. The stereo microscope, or dissecting microscope usually has a binocular eyepiece tube, a long working distance, and a range of magnifications typically from 5x to 35 or 40x. Some instruments supply lenses for higher magnifications, but there is no improvement in resolution. Such "false magnification" is rarely worth the expense.

Infrared: Night vision goggles pick up the infrared light emitted by our skin and objects with heat. In space, infrared light helps us map the dust between stars.

A beginner tends to think that the challenge of viewing small objects lies in getting enough magnification. In fact, when it comes to looking at living things the biggest challenges are, in order,

Microwave: Microwave radiation will cook your popcorn in just a few minutes, but is also used by astronomers to learn about the structure of nearby galaxies.

The bright field microscope is best known to students and is most likely to be found in a classroom. Better equipped classrooms and labs may have dark field and/or phase contrast optics. Differential interference contrast, Nomarski, Hoffman modulation contrast and variations produce considerable depth of resolution and a three dimensional effect. Fluorescence and confocal microscopes are specialized instruments, used for research, clinical, and industrial applications.