Saturday, 26 October 2019

Fluorescence Microscope

Fluorescence Microscope

Learning Objectives

Each student should be able to
1. Understand the principles behind the fluorescence microscope
2. Correctly use the fluorescence microscope by observing prepared slides of known bacteria
stained with a fluorescent dye

Materials per Group of Students

fluorescence microscope lens paper and lens cleaner low-fluorescing immersion oil protective glasses that filter UV light prepared slides of known bacteria (M. tuberculosis) stained with fluorescent dye

Why Is the Following Bacterium Used in This Exercise?

Mycobacterium tuberculosis (L. tuberculum, a small swelling + Gr. -osis, characterized by) is a human pathogen that causes tuberculosis. It is very slow-growing and not readily stained by Gram’s stain method. The cell is 1 to 4 ?m in length, straight or slightly curved, occurring singly and in occasional threads. This bacterium can be most readily identified after staining with fluorochromes or specifically labeling it with fluorescent antibodies using complicated immunofluorescence procedures, which are both times consuming and expensive. By using commercially prepared slides, the student is able to immediately examine a pathogenic bacterium, such as M. tuberculosis, and gain expertise in using the fluorescence microscope. In this exercise, the microscopic technique is more important than what is being observed.

Medical Applications

Fluorescence microscopy is commonly used in the clinical laboratory for the rapid detection and identification of bacterial antigens in tissue smears, sections, and fluids, as well as the rapid identification of many disease-causing microorganisms. For example, a sputum specimen can be quickly screened for M. tuberculosis by staining it's with a fluorescent dye that binds specifically to M. tuberculosis. Only the stained bacterium of interest will be visible when the specimen is viewed under the fluorescence microscope.

Principles

Fluorescence microscopy is based on the principle of removal of incident illumination by selective absorption, whereas light that has been absorbed by the specimen and re-emitted at an altered wavelength is transmitted. The light source must produce a light beam of the appropriate wavelength. An excitation filter removes that wavelengths that are not effective in exciting the fluorochrome used. The light fluoresced by the specimen is transmitted through a filter that removes the incident wavelength from the beam of light. As a result, the only light that has been produced by specimen fluorescence contributes to the intensity of the image being viewed (figure 5.1a,b).

Fluorescence Microscope

(a)

Fluorescence Microscope

(b)

Figure .1 Fluorescence Microscopy. 
(a) Escherichia coli stained with DAPI and propidium iodine. These fluorochromes bind DNA (×1,000). The blue cells are viable and the red cells are dead. 
(b) Giardia lamblia stained with IFA (×1,000).

Fluorescence Microscope

Fluorescence Microscope

Procedure

1. The UV light turns on the source at least 30 minutes before using the fluorescence microscope. THE UV LIGHT NEVER LOOK AT SOURCE WITHOUT PROTECTIVE GLASSES THAT FILTER UV LIGHT BECAUSE RETINAL BURNS AND BLINDNESS MIGHT RESULT.
2. Make sure that the proper excitation filter and barrier filter are matched for the type of fluorescence expected and are in place.
3. Place a drop of the low-fluorescing immersion oil on the condenser.
4. Place the prepared slide on the stage and position it so that the specimen is over the light opening. Raise the condenser so that the oil just touches the bottom of the slide.
5. After the mercury vapor arc lamp has been warmed up, turn on the regular tungsten filament light source and focus on the specimen.
6. Starting with the 10× objective, find and focus the specimen.
7. After finding the specimen, move to the 90× to 100× objective, switch to the mercury vapor arc and view the specimen.
8. Compare what you see in the bright-field microscope with what you see in the fluorescence microscope by sketching the organisms in the report for exercise 5.

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