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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.

Typology: Study notes

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Uploaded on 08/18/2009

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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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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

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?