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67 Cards in this Set
- Front
- Back
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What is visible light?
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It is one part of a spectrum of electromagnetic radiation.
It includes:- X rays Microwaves Radio waves |
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How might beams of radiation be referred to?
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They are referred to as either waves or rays.
These forms will differ by wavelength. |
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Define wavelength.
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It is the distance between two corresponding parts of a wave.
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Does the human eye discriminate between different wavelengths?
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Yes. As a result patterns of nerve impulses are sent to the brain. These impulses are interpreted as different colors.
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What awould be the numerical length of violet as it is seen?
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About 400nm represent the color violet.
650 nm would be interpreted as red. |
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What is white light?
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White light is composed of many colors or wavelengths. Its average wavelength is about 550 nm.
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Do moving electrons act as waves with wavelengths?
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Yes. This is dependent however on the potential voltage of the electron beam.
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What would the wavelength of electrons be at 10,000 volts?
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At 10,000 volts it would be 0.01 nm.
The wavelength of electrons at 1,000,000 V is 0.001 nm. |
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define magnification.
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It is the increase in size of an observed object. It is indicated by a number and an "X" which means "times".
16,000X means 16,000 times. |
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What actually causes magnification to result?
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It results when a beam of radiation bends or refracts as it is passing through a lens.
Curved glass lenses tend to refract light. Magnetic fields (magnetic lenses) tend to bend or refract electron beams. |
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Why exactly does a lens refract light?
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It refracts light because it is considered optically dense when compared to the immediate surrounding medium (air).
The light travels more slowly through the lens than it does through air. |
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How does the lens focus light rays on a so called focal point?
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Due to lens curvature, a lens tends to refract light rays that pass through its periphery quickly than light rays through the center.
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The leading edge of a light beam slows as it enters glass, and the beam bends. True/False
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True. Light will also bend as it leaves the glass and re-enters the air medium.
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How do light rays behave once they pass the focal point?
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They spread apart as they travel as they travel past the focal point and produce an enlarged image.
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The degree to which the image can be enlarged is based on what?
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This will be determined by the thickness of the lens, its curvature, and the speed of the traveling light.
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What are the two important properties that can determine the clarity of an image?
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Resolution and contrast.
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Define resolution.
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It is the innate ability to distinguish between objects that are close together.
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It is said that Leewenhoek's microscopes had a resolving power of about 1um. What does this mean?
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At 1um, he could distinguish between objects as long as they were more than 1um apart. Objects closer than 1um would appear as a single object.
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What kind of resolution do modern microscopes possess?
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They can distinguish between objects as close as 0.2um.
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Regarding modern microscopy, resolution distance is dependent on what?
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It is dependent of the wavelength of the electromagnetic radiation, the numerical aperture of the lens.
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What is Numerical aperture suggest?
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It refers to the ability of a lens to gather light.
Microscopes are better today because they use shorter wavelength radiation, such as blue light or electron beams, and they utilize lenses with larger numerical apertures. |
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What does the term contrast refer to?
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It refers to differences regarding intensity between two objects, or between an object and its surroundings.
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What is an acceptable mode for increasing the contrast between microorganisms and their background?
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Staining them would increase contrast.
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What are the most common types of microscopes employed?
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Bright field microscopes. The background is illuminated.
dark field microscopes, the specimen is made to appear light against a dark background. |
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How do Fluorescent microscopes work?
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They use invisible ultraviolet light to cause specimens to radiate visible light, this is termed fluorescence.
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What is a confocal microscope?
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These are microscopes that employ lasers to illuminate fluorescent chemicals in a thin or narrow plane of a specimen.
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How is magnification achieved in a basic compound microscope?
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It is achieved as light rays pass through a specimen and into an objective lens.
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Is an objective lens considered one lens/
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No. It is a series of lenses, that create a magnified image, and are intentionally designed to lessen the effect of aberrations in the shape and the color of the image.
Most light microscopes have 3-4 objective lens, that mount on a revolving nosepiece. |
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Describe the objective lenses found on basic light microscopes.
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Scanning lens =4X
Low power objective lens= 10X High power lens= 40X Oil immersion lens= 100X |
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What is the advantage of employing immersion oil?
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Placing immersion oil between the slide and the oil immersion objective lens, gives the lens the ability to capture this light, because light travels through immersion oil at the same relative speed as it does through glass.
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Does immersion oil increase the numerical aperture?
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Yes. This increases resolution as well, because more light rays are gathered into the lens to produce the image.
The space between the slide and the lens is called the working distance. |
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What effect does the objective lens have on light rays?
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It bends the light rays, which then pass up through one or two ocular lenses, which are the lenses closest to the eyes.
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Ocular lenses have the ability to further magnify specimens by how much?
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They can magnify the image created another 10X.
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How does one determine the total magnification value of a compound microscope?
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This is calculated by multiplying the magnification of the objective lens by the magnification of the ocular lens
10X ocular by 10X objective yields a total magnification of 100 times.. |
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Why is 2,000X magnification considered a limit, employing magnification oil immersion?
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In light microscopes, their resolution is restricted by the wavelength of visible light.
The objective lens forms the initial image of the specimen. This is the real image. When the real image is projected to the plane of the eyepiece (ocular), the ocular lens magnifies it to produce a second image, called the virtual image. |
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What is the function of the condenser of a compound microscope?
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The condenser or condenser lenses direct light through the plane of the specimen.
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What is the function of the diaphragm?
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This will control the amount of light enetring the condenser.
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Specificly, how do dark field microscopes work/
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Pale objects are best observed with these microscopes.
They use a dark field stop in the condenser, that prevents light from entering the objective lens. Only the light rays scattered by the specimen enter the objective lens and are seen, so the specimen appears light against a dark background. |
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Why do microbiologists use phase microscopes?
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These are used to examine living microorganisms or specimens that would be damaged or altered by attaching them to slides or staining them.
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When are light rays considered to be in phase?
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They are in phase when their crests and troughs are aligned, and out of phase when when they are not.
Light rays that are out of phase tend to interfere with each other. Combining these two phase altered phenomena a contrast is created. |
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Phase contrast microscopes produce sharp images. True/False
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True. They are excellent for defining fine structures of living cells.
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U/V light increases resolution because it has a shorter wavelength. True/False
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True. This is relative to visible light and contrast is improved because fluorescing structures are visible against a black background.
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What causes the molecules to become fluorescent?
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Molecules that absorb energy from the invisible radiation such as U/V light and then radiate the energy back as a longer visible wavelength are said to be fluorescent.
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Pathogens such as Pseudomonas aeruginosa and chlorophyll tend to be naturally fluorescent. True/False
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True.
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Some fluorescent dyes are specific for certain cells. Give 2 examples.
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The dye fluroscein isothiocyanate attaches to cells of B. anthracis, and appears apple green.
Another dye auramine O that fluoresces yellow, stains Mycobacterium tbc. |
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What is immunofluorescence?
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Fluorescent dyes are covalently linked to antibodies. These dye tagged antibodies will bind specifically to complementary shaped antigens. A microbial specimen that has bound dye tagged antibodies becomes visible.
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Can structures closer together than 200nm be distinguished using a light microscope?
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No. Because the shortest visible radiation (violet) has a wavelength of about 400nm, structures closer together than 200nm cannot be distinguished.
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What is the potential magnification of electron microscopes?
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They can magnify objects 10,000X-100,00, through millions of times magnification with good resolution.
Cell's ultrastructures are only seen with electron microscopes. |
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What are the two basic types of electron microscopes?
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Transmission electron microscope (TEM) and Scanning electronn microscope (SEM).
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How does a TEM work?
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It generates a beam of electrons that produce an image on a fluorescent screen. The path of electrons is similar to a path of light.
Electerons pass through the specimen, through magnetic fields (instead of glass lenses). These maipulate and can focus the beam, and then onto a fluorescent screen that absorbs the electrons, changing some of their energy into visible light. |
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Will dense areas of th specimen block some of the electrons?
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Yes. This will result in a dark area on the screen (no electron absorption). Where the specimen is less dense, the screen will fluoresce more brightly.
The brighness of each region of the screen will correspond to the quantity of electrons striking it. |
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How are TEM images made?
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The screen can be bent ot folded out of the way to allow the electrons to strike a photographic film located in the base of the microscope.
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Why must the column of a TEM be a vacuum?
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Because matter, including air absorbs electrons.
The specimen must be very thin. |
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How are specimens prepared?
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They must be dehydrated, embedded in plastic and cut to a thickness of about 100nm with a diamond or glass knife called an ultramicrotome. They are placed onto a copper grid.
TEMs cannot be use to study living organisms. |
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Do scanning electron microscopes also use magnetic fields within a vacuum?
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Yes. This is in order to manipulate a beam of electrons, referred to as primary electrons.
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Do the electrons of a SEM pass through the specimen?
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No. The SEM quickly focuses the electrons back and forth across the specimen surface, which is previously coated with a metal such as platinum or gold.
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What immediately happens when the primary electrons contact the metal coat on the specimen?
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They displace the electrons off the surface of the coated specimen, and these as a result scatter secondary electrons, which pass through a detector and a photomultiplier, producing an amplified signal that is quickly displayed on a monitor.
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What is the average resolution potential with a scanning electron microscope?
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SEMs can magnify on average about 10,0000X with a resultant resolution of about 20 nm.
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Does an SEM have any advantages over a TEM?
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Yes. Whole specimens can be studied because sectioning is not a requirement. SEMs give a very realistic and three dimensional image.
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Do SEMs have any disadvantages?
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Yes. SEMs magnify only the outer or external surface of a specimen.
It can only be used to observe dead specimens. |
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How does a Scanning tunneling microscope function? (STM)
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It passes a metallic probe, which is sharpened to end in a single finite atom, back and forth across and slightly above the surface of the studied specimen.
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Does an STM scatter a beam of electrons into a detector much like an SEM?
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No. An STM measures the flow of electrons to and from the actual probe and the surface of the specimen.
The amount of electron flow employed is called the "tunneling current". It is directly proportional to the distance from the probe to the specimens surface. |
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What kind of distances can an STM measure?
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It can measure distances as small as 0.01nm and demonstrate details on the specimen surface at the atomic level.
All specimens however, must be electrically conductive. |
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Is their a difference in function between an STM and an Atomic force microscope (AFM)?
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An AFM also uses a pointed probe, but it is able to traverse the tip of the probe lightly on the surface of the specimen, rather than at a distance.
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What is a unique characteristic of the AFM?
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They can magnify specimens that do not conduct electrons.
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Can they magnify living specimens?
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Yes. This is because neither an electeronic beam nor a vacuum is necessary.
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What have AFMs been used for?
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They have been used to magnify the surfaces of bacteria, viruses, proteins and amino acids.
They have also found purpose in single living bacteria in three dimensional views while in the process of dividing. |
