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BRIGHT FIELD MICROSCOPY

LIGHT MICROSCOPY

Optical or light microscopy involves passing visible light transmitted through or reflected from the sample through a single or multiple lenses to allow a magnified view of the sample. The resulting image can be detected directly by the eye, imaged on a photographic plate or captured digitally. The single lens with its attachments, or the system of lenses and imaging equipment, along with the appropriate lighting equipment, sample stage and support, makes up the basic light microscope.

Limitations

Limitations of standard optical microscopy (bright field microscopy) lie in three areas;

The technique can only image dark or strongly refracting objects effectively.

Diffraction limits resolution to approximately 0.2 micrometres.

Out of focus light from points outside the focal plane reduces image clarity.

Live cells in particular generally lack sufficient contrast to be studied successfully, internal structures of the cell are colourless and transparent. The most common way to increase contrast is to stain the different structures with selective dyes, but this involves killing and fixing the sample. Staining may also introduce artifacts, apparent structural details that are caused by the processing of the specimen and are thus not a legitimate feature of the specimen.

These limitations have all been overcome to some extent by specific microscopy techniques that can non-invasively increase the contrast of the image. In general, these techniques make use of differences in the refractive index of cell structures. It is comparable to looking through a glass window: you (bright field microscopy) don't see the glass but merely the dirt on the glass. There is however a difference as glass is a denser material, and this creates a difference in phase of the light passing through. The human eye is not sensitive to this difference in phase but clever optical solutions have been thought out to change this difference in phase into a difference in amplitude (light intensity).

Types of light microscopy:

- bright field

- dark field

- phase contrast

- differential interference contrast

- Nomarski contrast

- Hoffman modulation contrast

- fluorescence

- confocal

Four examples of transilumination techniques used to generate contrast in a sample of tissue paper. 1.559 μm/pixel.

Bright field Dark field Cross-polarized Phase contrast

 

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. 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.



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.

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 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.


Date: 2015-12-11; view: 1167


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