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MicroscopyMicroscopy
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UNITS OF UNITS OF MEASUREMENTMEASUREMENT
1 m = 1000 mm (millimeters)1 m = 1000 mm (millimeters) 1000 mm = 1 µm (microns)1000 mm = 1 µm (microns)
Bacteria are about 1µm or smallerBacteria are about 1µm or smaller 1000 µm = 1nm (nanometers)1000 µm = 1nm (nanometers)
Viruses are about 1nmViruses are about 1nm 1000 viruses can fit into one bacterium1000 viruses can fit into one bacterium
Protozoa are fairly large single-celled animals. You can see them with the naked eye.
Bacteria are so small, they are measured in µm. They Bacteria are so small, they are measured in µm. They are the smallest things you can see under a are the smallest things you can see under a microscope with the oil immersion lens.microscope with the oil immersion lens.
Viruses are even smaller, so they are measured in nm.Viruses are even smaller, so they are measured in nm.
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3Figure 4.3
Limits of ResolutionLimits of Resolution
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VOCABULARY Immersion oil: keeps light from bending and allows lens to be
refracted. Resolution: ability of two lenses to distinguish two points. Parfocal: focused in all lenses. Depth of field: how much of the background is in focus at the
same time that the foreground is in focus. RESOLUTION: the ability of the lenses to distinguish two
points. A microscope with a resolving power of 0.4 nm can distinguish between two points greater than or equal to 0.4nm. When you go to the eye doctor, you look at the chart (Snellen chart) and read it from 20 feet away. If you can read what a normal sighted person can read from 20 feet away, it is called 20/20 vision. If you can’t read it well, your eyesight has less resolution than normal.
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TYPES OF MICROSCOPESTYPES OF MICROSCOPES
SIMPLE MICROSCOPESIMPLE MICROSCOPE: Has only : Has only one lens, like an ocular (eyepiece)one lens, like an ocular (eyepiece)
COMPOUND MICROSCOPECOMPOUND MICROSCOPE: More : More than one lens, like an ocular and an than one lens, like an ocular and an objective. An example is the objective. An example is the Brightfield microscope.Brightfield microscope. There are two main types of compound There are two main types of compound
microscopes: Light Microscopes and microscopes: Light Microscopes and Electron Microscopes.Electron Microscopes.
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SIMPLE MICROSCOPESIMPLE MICROSCOPE
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COMPOUND MICROSCOPE: COMPOUND MICROSCOPE: One EyepieceOne Eyepiece
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COMPOUND MICROSCOPE:COMPOUND MICROSCOPE:Two EyepiecesTwo Eyepieces
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Types of Compound Types of Compound MicroscopesMicroscopes
DissectingDissecting BrightfieldBrightfield DarkfieldDarkfield Phase-contrastPhase-contrast Differential Interference contrastDifferential Interference contrast FluorescenceFluorescence
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Dissecting Microscope:Dissecting Microscope:For looking at large For looking at large
objectsobjects
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BRIGHTFIELD BRIGHTFIELD ILLUMINATION:ILLUMINATION:
For live organisms with no For live organisms with no stainstain
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BRIGHTFIELD BRIGHTFIELD ILLUMINATION:ILLUMINATION:
Can also use for stained Can also use for stained tissuestissues
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DARKFIELD DARKFIELD ILLUMINATIONILLUMINATION
For live organisms with no For live organisms with no stainstain
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DARKFIELD DARKFIELD ILLUMINATIONILLUMINATION
For fluorescent organisms For fluorescent organisms
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Brightfield vs DarkfieldBrightfield vs Darkfield
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PHASE CONTRAST PHASE CONTRAST MICROSCOPYMICROSCOPY
For seeing organelles in live For seeing organelles in live organismsorganisms
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DIFFERENTIAL INTERFERENCE DIFFERENTIAL INTERFERENCE CONTRASTCONTRAST
For seeing organelles in live organisms in For seeing organelles in live organisms in three dimensionsthree dimensions
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DIFFERENTIAL DIFFERENTIAL INTERFERENCE CONTRASTINTERFERENCE CONTRAST
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DIFFERENTIAL DIFFERENTIAL INTERFERENCE CONTRASTINTERFERENCE CONTRAST
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Cells are Cells are stained with stained with fluorescent fluorescent dyes called dyes called fluorochromes.fluorochromes.
UV Light is shined on the specimen.
Fluorescent Fluorescent substances substances absorb UV absorb UV light and emit light and emit visible light. visible light.
Fluorescence Fluorescence MicroscopyMicroscopy
Figure 3.6b20
FLUORESCENCE FLUORESCENCE MICROSCOPYMICROSCOPY
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FLUORESCENCE FLUORESCENCE MICROSCOPYMICROSCOPY
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Transmission Electron Transmission Electron MicroscopeMicroscope
Much higher resolution than microscopes.
Can only be used on dead cells
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Transmission Electron Transmission Electron MicroscopeMicroscope
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Transmission Electron Transmission Electron Microscope:Microscope:
Inside of a Plant CellInside of a Plant Cell
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Scanning Electron Scanning Electron MicroscopeMicroscope
Very high resolution like a Very high resolution like a Transmission Electron Microscope Transmission Electron Microscope except makes images in three except makes images in three dimensions.dimensions.
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Scanning Electron Scanning Electron MicroscopeMicroscope
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Scanning Electron Scanning Electron Microscope:Microscope:
FleaFlea
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Scanning Electron Scanning Electron Microscope:Microscope:
PollenPollen
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Scanning Probe Scanning Probe MicroscopeMicroscope
A scan is passed over the specimen, A scan is passed over the specimen, line by line.line by line.
The surface dimensions are recorded The surface dimensions are recorded and sent to a computer, which shows and sent to a computer, which shows the image in false color.the image in false color.
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Scanning Probe Scanning Probe Microscope:Microscope:
Red Blood CellsRed Blood Cells
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Scanning Probe Scanning Probe Microscope:Microscope:
ChromosomesChromosomes
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COMPARISON OF MICROSCOPES
BRIGHTFIELD Dark objects are visible against a bright background.Light reflected off the specimen does not enter the objective lensNot for looking at live cellsMaximum resolution is 0.2µm and maximum magnification is 2000xStains are used on specimens
DARKFIELD Light objects are visible against dark backgroundUsed for live cells, cilia, flagellaEspecially good for spirochetesUses special condenser with an opaque disc that eliminates all light in the center
PHASE-CONTRAST
No staying requiredAccentuates diffraction of the light that passes through a specimenGood for live cells; good contrastMost sensitive; cilia shows upNot three-dimensional
DIFFERENTIAL INTERFERENCE CONTRAST
Uses two beams of lightShows three dimensionsHas a prism to get different colorsGood for live cells (unstained)Best resolution
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COMPARISON OF MICROSCOPES
FLUORESCENCE Uses ultraviolet lightStained cells with fluorescent dye; energizes electrons and creates visible lightNo live cellsQuick diagnosis of TB and syphilis
TRANSMISSION ELECTRON
Get flat imagesHave vacuum pumps to allow electrons to float betterStain with heavy metal saltsShows sections of cell, revealing organellesRequires an ultramicrotomeBest resolution of all microscopes
SCANNING ELECTRON
Surface view onlyNeeds a vacuumNo live cellsThree-dimensional view
SCANNING PROBE
Physical probe scans the specimenRaster scan: image is cut up into pixels and transmitted to computerNot limited by diffractionSlower in acquiring images Maximum image size is smaller
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