The scientific method requires evidence for the determination of facts. If our senses are limited, we need tools to increase the power and accuracy of our observations and, thus, our knowledge of the world. For biologists, light and electron microscopes have become indispensible as "eyes" for discovery about the detailed structure of cells. How do microscopes extend the limits of human vision?

1) They magnify an image, enlarging it so that it can be seen by the human eye, and

2) they increase resolution, the detail provided by magnification.

Thus, resolution is the ability of a viewing instrument (the eye, a camera, a microscope) to distinguish adjacent objects as separate rather than as one larger object. For example, how close can two lines get before you will no longer be able to see both of them? How thin can a single line become and still be visible?

 

UNRESOLVED	PARTIAL RESOLUTION	RESOLVED

I'd like to review dimensions before I proceed.

The lens of the human eye has a resolving power of approximately 100 µm. This means that the eye will see two objects as distinct if the space between them is at least 100 µm. If the space is 90 µm, they will appear as a single object. The best light microscopes have resolving powers of about 1 nm. They will "see" objects or spaces between objects as small as 1 nm. If you look at the period at the end of this sentence you will see a dot. Imagine that it is a cell composed of many parts. If you magnified the dot 100 fold without increasing resolution (as in photographs) you would have a larger image but not greater detail. (This is called empty magnification.) Magnifying the dot 100 fold with a microscope, however, resolves ultra-fine structures of the image. Thus, the useful magnification of a viewing system depends on its resolving power.

The two most important factors that determine resolving power are the quality of the lens system and the wavelength of the source of illumination. Resolution varies inversely with wavelength; a shorter wavelength allows for greater resolution.

Light Microscopes : Standard laboratory microscopes are compound microscopes, constructed of two lenses in series that focus rays of light to illuminate the object. The total magnification is obtained by multiplying the magnification of both lenses. High quality lenses give light microscopes resolving powers of about 200 nanometers, a 500-fold increase over that of the eye. This allows the observer to visualize the outlines of bacterial cells and only glimpse at the larger internal structures in cells of plants and animals.

Brightfield Microscope

The most common light microscope is a brightfield microscope - one that uses visible light to illuminate the specimen. Click on the parts of the microscope to learn their function.To return to the image, click on the higlighted term. You will be required to identify all of these parts and know their functions during the next laboratory period.

Ocular lens (eyepiece) - magnifies the image (usually 10X)

Arm - supports the head and stage

Revolving nosepiece - rotates objective lenses into viewing position

Objective lenses - magnify the image (scanning-4X; low power - 10X; high dry - 40X; oil immersion - 100X)

Mechanical stage controls- moves slide holder (specimen) to view different regions of the specimen

Stage - holds slide and contains opening to allow light to pass to slide

Coarse adjustment knob - brings the specimen into focus with the 4X and 10X objectives

Fine adjustment knob- brings the specimen into focus with the HiDri and oil immersion objectives

Condenser - focuses the light on the specimen; can be raised and lowered but RP greatest when fully raised

Light - Illumination of specimen

Base - support for the microscope; contains control for light intensity

Care and Use of the Microscope

1. Always carry the microscope upright and with two hands - one hand should grip the arm and the other hand should be placed firmly underneath the base. Be careful not to bump or drop the instrument. Make sure that your lab bench is clear before you bring the microscope.

2. Use only optically safe lens paper to clean the objective, ocular lenses and the condenser before and after each lab period. If you should find a microscope that has not been cleaned, please notify your instructor. Do not use any solvents or soap without the permission of your instructor.

3. Clean all immersion oil from the objective. Make sure that the oil is not used on the dry objectives.

4. Before returning the microscope to its designated space storage cabinet

a. remove any slide,

b. clean the optical system,

c. rotate the lowest power objective into position,

d. center the mechanical stage,

e. wrap the light cord around the base, and

f. cover.

Oil Immersion

As previously stated, resolving power (RP) varies with wavelength of light and quality (light gathering capacity) of a lens. The light gathering property is termed numerical aperture (NA).

The brightfield microscope often uses visible light with a wavelength of 550 nm. The NA of most low power objectives is 0.25. Thus the smallest object that can be seen through the lens is 1100 nm or _______ µm 1.1 .

To increase resolution, either the wavelength of light transmitted through the specimen must be decreased or the NA of the objective must be increased. Many brightfield microscopes use a blue filter over the light source to reduce the wavelength of transmitted light to 475nm. What is the RP now? 950 nm

NA depends on the configuration of the lens and the material between the lens and the light source. Some light is lost when it passes from the specimen on a glass slide through air to the objective lens. This occurs because the refractive index (ability to bend light) of glass and air differ. The air space can be replaced by a drop of immersion oil which has a higher refractive index, closer to that of glass. When the objective lens is lowered into the oil, more light is transmitted to the lens. Check your microscope to determine the NA of your oil immersion lens. It is usually imprinted on the objective. What is the resolving power of the oil immersion lens?

Because of the increased resolving power, most bacteriological microscopy uses the oil immersion lens. It is important to be aware that the working distance, the distance between the specimen and the objective lens decreases with higher magnification. The 97X lens has an average working distance of approximately 0.1 mm. Care must be taken not to break the slide or the coverslip when viewing at this magnification.

Become familiar with the brightfield microscope. Use the low and high power dry lenses and the oil immersion on each slide. Quality brightfield instruments are parfocal and parcentric. This means that the specimen remains in focus and at the center of the viewing field when objective lenses are rotated. You should therefore not have to use the coarse adjustment at higher magnifications. Compare what you can see at each magnification. (Do not be concerned if you do not see anything clearly at low power.) Describe your observations in your lab notebook.

Fluorescence Microscope

This variation of the brightfield microscope uses a mercury arc vapor lamp as a source ultraviolet to illuminate the specimen. Specimens that are fluorescent will absorb the illuminating light and emit light at longer wavelengths in the visible range. A filter is placed between the objective and ocular lenses to prevent all but the longer wavelength light passing to the eyepiece. A special condenser is used so that the background field is dark, thereby resulting in a high contrast fluorescent image.

One of the most important applications of fluorescence microscopy is immunofluorescence used to identify microbial pathogens. An antibody to a specific pathogen is linked to a fluorescent dye and is reacted with a clinical specimen. If the pathogen is present in the specimen, the antibody (and the fluorescent dye) will bind to the specimen. Later in the semester you will perform this procedure.

Phase Contrast Microscope

Living cells can best be viewed with a phase-contrast microscope. The optics of the microscope translate differences between the refractive indices of cellular components and the surrounding medium into differences in light intensity.

Darkfield Microscope

In this microscope, the condenser is designed to prevent light from passing to the objective unless it strikes the specimen. Thus the background is dark. The high contrast between background and the specimen makes it possible to view living unstained specimens.

Electron Microscopes

Two types of electron microscopes have been developed over the past 50 years: the transmission electron microscope (TEM) and the scanning electron microscope (SEM). Both instruments contain magnetic lenses that focus a beam of electrons on the specimen. Electrons used in this fashion generate a wavelength that may be 100,000 times shorter than that of visible light. As a result, electron microscopes have resolving powers as much as 400 times that of light microscopes and 200,000 times that of the human eye.

The TEM bombards a thin specimen with electrons. Depending on their composition, the components of the specimen either transmit, absorb or deflect the electrons. The image produced on a photographic plate is a visual translation of this interaction of electrons with the specimen. The transmission electron microscope exposes many secrets of subcellular structure. It gave scientists their first look at the world of viruses, invisible by light microscopy, and today permits us to see molecules and atoms.

The SEM is quite different from the TEM. It is designed to generate dramatic three-dimensional pictures of surface detail. In this microscope an electron beam is moved back and forth over the surface of a metal-coated specimen causing the emission of secondary electrons from the specimen. The secondary electrons produce the stunning images characteristic of scanning electron microscopy.

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