Lab Number 2 for Biology

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  • Topic: Microscopy, Microscope slide, Lens
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  • Published : January 29, 2013
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Lab No. 2: The Microscope

Light microscopy

The bright field microscope is best known to students and is most likely to be found in a classroom. Visible light is focused through a specimen by a condenser lens, then is passed through two more lenses placed at both ends of a light-tight tube. The latter two lenses each magnify the image. Limitations to what can be seen in bright field microscopy are not so much related to magnification as they are to resolution, illumination, and contrast. Resolution can be improved using oil immersion lenses.

Other than the compound microscope, a simpler instrument for low magnification use may also be found in the laboratory. This is the stereo microscope, or dissecting microscope. Stereo microscopes usually have a binocular eyepiece tube, a long working distance, and a range of magnifications, typically from 4x to 40x. Some instruments supply lenses for higher magnifications, but there is no improvement in resolution. Such "false magnification" is rarely worth the expense.

Magnification is the increasing in size of an object.
Resolution is ability to distinguish between two separate objects.

We will be using a compound light microscope throughout this course to view various cells and tissues. It is very important that you learn to use the microscope correctly, and can efficiently get images into the proper focus for study.

1. Bright Field Microscopy

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. Some microscopes have a built-in illuminator, while others use a mirror to reflect light from an external source. 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 times the ocular magnification.

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 & contrast is low.

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Using a bright field microscope

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.

Mount the specimen on the stage

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.

Optimize the lighting

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...
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