References
Slide 1
Microscopy
Lecture 4
References
Slide 2
Biol 240 S06 2
Learning Outcomes
• Introduction to
microscopes
•Compound light
microscope
•Electron
microscopes
• Microscopy terms
•Preparing
specimens
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Microscopy: Understanding the Pathway of Light and Magnification in Biology - Prof. Barry , Study notes of Microbiology
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
Microscopy
Lecture 4
Slide 2 References
Biol 240 S
Learning Outcomes
- Introduction to
microscopes
- Compound light
microscope
- Electron
microscopes
- Microscopy terms• Preparing
specimens
Slide 3 References
Biol 240 S
3
Your Retina
•^
This is where theimage if formed
-^
Detects light andcolour
-^
Light shone throughspecimen–
Specimen interfereswith light
•^
The image you see ishow the specimen hasaffected the light
Slide 4 References
Biol 240 S
The Path of Light
•^
Light focused
-^
on specimen by
condenser
- Specimen alters beam of
light– Transmission– Reflection– Refraction
- Scattering
- Absorption
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
Numerical
Aperture
- Numerical
Aperture
- How much
scattered light isgathered by thelens
- Resolution for each
lens determined byNA and thewavelength of light
Each lens has a numerical aperture, what information does this figure tell you aboutthe resolution of the lens?
Slide 8 References
Biol 240 S
Magnification
- Magnification
- Increase in
apparent sizeof object
Explain what magnification is and how it is achieved.
Slide 9 References
Biol 240 S
9
Talking About Resolution…
- The ability of the
lenses todistinguishbetween two pointsa specifieddistance apart.
- Function of the
wavelength of light
Provide a definition of the terms resolution and refraction.
Slide 10 References
Biol 240 S
Wavelength and
Resolution
•^
The shorter thewavelength
-^
The smaller the objectthat can be seen
-^
The greater theresolution
-^
Wavelength of light
-^
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
Oil Immersion
- Light refracted when
it changes medium– water
Æ
air
- Immersion oil same
refractive index asglass
Æ
no refraction
- Maximum
magnification withlight
- 100x objective lens x
10x ocular = 1000x
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
Relative sizes
-^
Eukaryote cells~20um
-^
Bacteria ~ 2um
Mycoplasmas 100-200 nm
Escherichia coli,1.5 μm wide by 4.0μm long
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
MicroscopeTerminology
- Field of View
- How much you can
see in focushorizontally
- Increase
magnificationreduces field ofview
- Depth of field
- How much is in
focus vertically
- Increase
magnificationreduces depth offield
Slide 16 References
Biol 240 S
Parfocal Lenses
•^
Once one is in focusthe others are nearlyin focus
-^
Revolving Nosepiece–
Do not use lenses ashandles
•^
Low power–
Scanning
•^
High dry–
Observe eukaryote cells
•^
Oil immerson–
Observation bacteria
-^
Fine detail eukaryotecells
Slide 19 References
Biol 240 S
19
Illumination
- Brightfield
- Normal light
microscope
- Darkfield
- Opaque condenser
- Phase Contrast
- Living unstained
- Phase Contrast
- Opaque condenser
cells
Slide 20 References
Biol 240 S
Fluorescent-Antibody
Technique
•^
Fluorescence–
Absorb short wave light(UV) but emit longer wave(visible) light
•^
Fluorochrome
-^
Dye that fluoresces
-^
Creates fluorescentmicrobe under UV light
-^
Immunofluorescence
Describe how immunoflourescence can be used diagnostically?
Slide 21 References
Biol 240 S
21
Electron Microscopes
•^
Objects < 0.2um can ONLY be observed by EM
-^
Beam of electrons focused by magnets
-^
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
Electron Microscopes
- Transmission
- Electrons pass
through specimen
- Internal structure
- Scanning• electrons bounce
off surface– External structure
Explain how an electron microscope can magnify objects more than a compound lightmicroscope?
Slide 25 References
Biol 240 S
25
Gram Stain
•^
Gram positive bacteria
-^
Purple
-^
Gram negative
-^
Pink
-^
Effectiveness
-^
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
Acid Fast Stain
- Binds strongly to
waxy cell walls
Mycobacterium–
M. tuberculosis
M. leprae
Nocardia
When would a microbiologist use an acidfast stain?
Slide 28 References
Biol 240 S
Special Stains
- Capsule• Endospore• Flagella• Diagnosis• Classification
Describe an instance when a negative stain would be used?