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Unformatted text preview: Experiment 3-1:Brightfield Microscopy (pgs 63-68)
Purpose: To familiarize oneself with the components and the workings of a brightfield microscope
4 Types of Light Microscopes: Bright-field Microscope Dark-field Microscope Phase contrast Microscope Fluorescence Microscope Brightfield Microscope: A microscope that allows light rays to pass through a slide/specimen and then through various lenses to the eye. The lenses magnify the object. Microscopes are very expensive and must be handled with care.
-Transport: Microscopes should be transported to and from the cabinets with one hand on the arm and the other hand under the base -- Microsopes must never be held in only one hand -- Only one microscope can be transported at a time. Microscope Parts and Their Functions:
1. 2. 3. 4. 5. 6. Arm - all other parts are attached to it Base - bottom Stage - platform that holds the slide Mechanical stage - device used to clamp slides to the stage - allows the slide to be moved Light source - provides illumination and is located in the base Voltage control - varies the intensity of light; light will last longer if the voltage control is kept on low 7. Lens system: Microscopes have three lens systems a. Oculars => eyepieces (10X); has 2 or more lenses b. Objective => 4 objectives; attached to the nosepiece for movement 1. Scanning (red band) - 4X 2. Low (yellow band) - 10X 3. High Dry (blue band) - 40X 4. Oil immersion (white band) - 100X Total Magnification = magnificationocular X c. magnificationobjective Condenser => under the stage; collects and directs the light; concentrates the light for uniform illumination diaphragm - part of the condenser that controls the amount of light coming through 8. 9. Focusing knobs a. Coarse adjustment - major focus adjustment; use with low objectives (4X and 10X); moves the stage up and down b. Fine adjustment - minor focus adjustment; use to do the final focusing of an object Ocular adjustment - want to see one image pull apart or push together the oculars Diopter adjustment ring - focus 1st with right eye then use ring to focus the left 1 Resolution: The ability to distinguish two adjacent points as separate objects based upon
resolving power -Function of Three Things: -1. Numerical aperature -2. Wavelength of light -3. Design of condenser The optimum resolution of the best microscope with the oil immersion lens is 0.2m. To increase resolution: 1. A blue or green filter should be in place - decreases the wavelength => increase resolution 2. The condenser should be at its highest position which allows the maximum amount of light to enter the objective. 3. The diaphragm should be opened up a little => less contrast but higher numerical aperature 4. Immersion oil should be used between the slide and the 100X objective Numerical aperture (N A) the measure of a lens' ability to capture light coming from a specimen and use it to make the image; increases with the use of oil and the higher objectives Limit of Resolution: Wavelength D= N A condenser + N A of objective lens Focusing the Microscope
Procedure: 1. Start with the low objectives get object in focus with coarse focus adjustment and center object in field of view. The condenser should be in its highest position. 2. Move to high-dry (40X objective) use only fine focus adjustment - due to parfocalization, the object should stay in focus when moving between objectives 3. Move to Oil Immersion lens (100x) - oil has the same refractive index as glass thus the light rays will not diffract and bend light rays as it does upon hitting air (i.e. light is not lost due to the bending) - enhances resolving power - open diaphragm lighting is needed - At the end of lab, lens paper should be used to remove the immersion oil from the objective. Phase Contrast vs. Brightfield Microscopy - usually organelles and cells are transparent and difficult to differentiate without staining - staining kills the cells so you don't see living cell just artifacts - phase-contrast allows one to view living transparent organisms using phase (wavelength shifts) principles of light to produce bright objects in a dark background (like a negative of a photograph). 2 ...
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- Fall '08