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BIOL Exam I - Ex. 2

Basic Light Microscopy

QuestionAnswer
light/optical microscope - produces an image when light is passed through glass or other optically transparent lenses - can resolve up to 100 nanometers
What are compound microscopes? What do they consist of? - modern light microscopes - core consists of 3 lenses: condenser, objective, and ocular
condenser - shapes the direction of the light before it encounters the object - / gathers light and focuses it on the specimen
objective - collects the light after it passes through the object to produce a magnified real image - can magnify 4x (low power/scanning lens), 10x, 40x, 100x (high power/oil immersion)
ocular - produces a magnified virtual image of the real image - usually magnifies 10x
total magnification the product of the magnification of all the magnifying lenses through which the light travels after leaving the object to produce the image (M.total = M.objective * M.ocular)
(ocular) reticle - an arbitrary measuring ruler - part of the ocular - used to measure the relative size of an object
resolution - resolving power - the closest distance that two distinct parallel edges in the object can be separated, and still be perceived as separate edges in the image produced by the lens - eye can resolve up to 100 micrometers
Most biological specimens are transparent. How can contrast be improved? - by submerging the object in a dye solution - by using advanced microscopy techniques (such as darkfield, phase contrast, fluorescence microscopy)
stains - the most useful dyes for microscopic observations of cells - bind tightly to cellular components - most kill the cells they encounter immediately
lens paper a non-abrasive, special paper used to remove grease, oil, or aqueous liquid from optical surfaces
The Abbe Equation tells us about the ___ of an objective. The formula is ___. - resolving power - d = (0.612λ)/N.A. - d = minimum distance between objects when you can still see them as separate objects (smaller d = better) - λ = wavelength of light (use like 500 nm) - N.A. = numerical aperture
How do you improve resolution [decrease resolution distance (d)]? - use shorter wavelengths - increase numerical aperture
When focusing on an object under the microscope, it is important to start out with the low-power objective because it provides the greatest ___ and ___. - field of view - depth of focus
What do you have to do to improve contrast and resolution? What does it result in? - set up the light path properly (set Köhler) - results in an evenly illuminated field and a bright image without glare
How do you set Köhler? - focus on specimen - close field diaphragm - adjust height of condenser until leaves of the field diaphragm are in sharp focus - use the condenser centering screws to move the image of the field diaphragm to the center - open field diaphragm.
How do you calibrate the ocular reticle? - using a stage micrometer - align it to ocular reticle and note the conversions - examples: at 4x, 1 reticle = 25 micrometers
How do you measure an objects' size in reticle units? - line up the object at 0 - each little mark is 1 reticle unit (not the big number), so something that appears to be 1.6 is actually 16 reticle units long
For a particular microscope, one reticle unit represents 25 µm when using the 4X objective. You want to know the number of µm’s per r.u. when using the 40X objective on the same microscope. What equation do you use? What's the answer? - known µm/r.u. x (Mag. of original objective / Mag. of new objective) = ? µm/r.u. - 25 µm/1 r.u. x (4/40) = 2.5 µm/r.u.
How would you make a wet mount of an Elodea leaf? - use forceps to remove a young leaf from the tip of an Elodea stem - place piece on a slide - add a drop of dH2O - place coverslip over specimen (place one edge down first, then gently lower)
Oil has a refractive index that is similar to ___. glass
Using oil immersion allows for the capture of more image forming light waves, which improves ___. resolution
How do you do an oil immersion? - focus on a specimen with the 40X objective - rotate halfway between the 40X and 100X objectives - place immersion oil and slowly rotate 100X objective
When is it acceptable to focus using the course focus knob? Only when the 4X objective is in alignment.
What should the state of the microscope be when one wishes to remove a specimen slide after viewing? - the objective with the greatest working distance should be in alignment / the scanning objective should be in alignment - the mechanical stage should be lowered
How does the “field of view” change with magnification? Relate this to the rule that one should always use the low-power scanning objective first to locate a specimen. - as magnification increases, the field of view decreases - the fields of view in low-power objectives are useful for locating specimens, because we can search in larger areas
How does the “depth of focus” change as magnification increases? as magnification increases, the depth of focus decreases
Are thick (e.g., Elodea) or thin (e.g., prepared Paramecium) samples of specimens preferred at high magnifications? - thin samples of specimens are preferred at high magnifications (lower depth of focus) - thick samples would benefit from larger depths of focus (lower magnification)
Describe how red and white blood cells look (compared to each other too). - red blood cells appear small, circular, and pink - white blood cells appear purple (stained?), big, and irregularly-shaped - there are much more RBC than WBC because RBC are always needed; WBC count increases only when there is an infection to fight
Describe Paramecium and mention any important features. - macro and micro nucleus visible, along with cytoplasm, plasma membrane, small food vacuoles - everything stained various shades of purple
Describe Elodea and mention any important features. - basic plant cell, really - can see cell wall, cytoplasm/central vacuole, and chloroplasts - sometimes can see a tiny nucleus, plasma membrane right up against cell wall, not visible
Created by: jessica.gvc