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An in-depth exploration of microscopy, focusing on the pathway of light, magnification, and related concepts such as resolution, refraction, and numerical aperture. It covers various types of microscopes, including compound light microscopes and electron microscopes, and discusses the importance of oil immersion and the role of wavelength in resolution.
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Slide 1 References
Slide 2 References
Biol 240 S
Slide 3 References
Biol 240 S
3
This is where theimage if formed
-^
Detects light andcolour
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Light shone throughspecimen–
Specimen interfereswith light
The image you see ishow the specimen hasaffected the light
Slide 4 References
Biol 240 S
Light focused
-^
on specimen by
Describe the pathway that a beam of light follows from the source of illumination thatwould allow you to see an object on a microscope slide?
Slide 7 References
Biol 240 S
7
Each lens has a numerical aperture, what information does this figure tell you aboutthe resolution of the lens?
Slide 8 References
Biol 240 S
apparent sizeof object
Explain what magnification is and how it is achieved.
Slide 9 References
Biol 240 S
9
Provide a definition of the terms resolution and refraction.
Slide 10 References
Biol 240 S
The shorter thewavelength
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The smaller the objectthat can be seen
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The greater theresolution
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Wavelength of light
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400-700nm–
Bacteria ~ 2um(2000nm)
-^
Virus <200nm
Electron beam 2nm
Describe the relationship between the wavelength of light and resolution.
Slide 13 References
Biol 240 S
13
air
Explain why it is necessary to use oil to see specimens magnified by the 100x objectiveusing a compound light microscope?
Slide 14 References
Biol 240 S
-^
-^
Arrange the following measurements in decreasing order of size: 10 um, 100 nm, 10mm, 0.1mm, 0.001 um. Metric system
Slide 15 References
Biol 240 S
15
see in focushorizontally
magnificationreduces field ofview
focus vertically
magnificationreduces depth offield
Slide 16 References
Biol 240 S
Once one is in focusthe others are nearlyin focus
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Revolving Nosepiece–
Do not use lenses ashandles
Low power–
Scanning
High dry–
Observe eukaryote cells
Oil immerson–
Observation bacteria
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Fine detail eukaryotecells
Slide 19 References
Biol 240 S
19
microscope
cells
Slide 20 References
Biol 240 S
Fluorescence–
Absorb short wave light(UV) but emit longer wave(visible) light
Fluorochrome
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Dye that fluoresces
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Creates fluorescentmicrobe under UV light
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Immunofluorescence
Describe how immunoflourescence can be used diagnostically?
Slide 21 References
Biol 240 S
21
Objects < 0.2um can ONLY be observed by EM
-^
Beam of electrons focused by magnets
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Shorter wavelength–
~2nm
100,000 x magnification
-^
Greater resolving power 0.5 nm
What is the difference between a transmission electron microscope and a scanninglight microscope?
Slide 22 References
Biol 240 S
through specimen
Explain how an electron microscope can magnify objects more than a compound lightmicroscope?
Slide 25 References
Biol 240 S
25
Gram positive bacteria
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Purple
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Gram negative
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Pink
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Effectiveness
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False positive–
Dead Gram +ve bacteriaappear pink
What is the difference between a simple stain and a differential stain?
Slide 26 References
Biol 240 S
The Gram stain is a differential stain, at which stage in the procedure are Grampositive and Gram negative organisms differentiated?
Slide 27 References
Biol 240 S
27
M. tuberculosis
M. leprae
When would a microbiologist use an acidfast stain?
Slide 28 References
Biol 240 S
Describe an instance when a negative stain would be used?