Microbiology 201 PSU

8/11/2019 Microbiology 201 PSU

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Light microscopes


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To understand light microscopy, you need to

understand three concepts:

Light is a wave

Refraction

Diffraction

Three concepts


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Light is a wave

Ray of light

wavelength ()


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Refraction is the change in direction of a wave due

to a change in velocity at the interface between twotransmission media.

Refraction


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Refraction

http://www.youtube.com/watch?v=ybn-qr-Pvnw
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Magnification

Magnification is accomplished by refraction.


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Diffraction refers to various phenomena that occur

when a wave encounters an obstacle. The diffractionphenomenon is described as the apparent bending of

waves around small obstacles and the spreading out

of waves past small openings.

Diffraction
http://www.youtube.com/watch?v=BH0NfVUTWG4


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Diffraction

http://www.you

tube.com/watch?v=BH0NfVUTWG4
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When the aperture is comparable or smaller than the

wavelength, the distortion of the wave is large. Thislimits the ability to resolve nearby objects.

Diffraction


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d~

(dis the minimum distance that can be resolved)

( is the wavelength)

(~ means roughly)

Resolution


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d~

d~ 100 nm1000 nm

~ 100 nm1000 nm

Resolution


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d= 0.5/NA

(NA is the numerical aperture of the objective

lens)

(NA is a measure of the objective lens ability

to gather light and depends on material and

shape of the lens)

(requires more physics to derive)

Resolution


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Parts of the microscope


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Parts of the microscope


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Fixation and staining of

specimen

1. Spread culture on slide

2. Air dry

3. Fix with heat (attach cells to

slide)

4. Apply stain, rinse, dry

5. Microscopy

Simple staining- use of a single

dyeDifferential staining- use of >1

dye to differentiate between

different types of cells

1) 2)

3) 4)

5)

Staining to improve contrast


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Staining to improve contrast


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Time-lapse microscopy


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Used to visualize specimens

that fluoresce

Emit light of one color when

illuminated with another color of

light

Can be used with live cells Some cells fluoresce naturally

(autofluorescence)

Stain cells with a fluorescent

dye Highlight particular cell

structures

Express a fluorescent protein

FM4-64

(membrane)

DAPI(nucleic acid)

YFP

(cytoplasm)

overlay

Fluorescence microscopy


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Time-lapse fluorescent microscopy

http://www.youtube.com/watch?v=rBYYpPisjEs


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Electron microscopes


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Electron microscopy (EM)

Involves the use of:

electromagnets instead of glass

lenses

electrons instead of light


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lenses


Provides much higher resolution than a

light microscope

Light microscope scope ~ 200 nm (0.2

m)

EM ~ 0.5 nm


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Resolution (d) = 0.5

/NA

visible light: ~ 450 nm

electrons: ~ 0.005 nm




lenses


Provides much higher resolution than a

light microscope

Light microscope scope ~ 200 nm (0.2

m)

EM ~ 0.5 nm

Electrons behave as waves with very

short wavelengths hence the

difference in resolution between LM's

and EM's


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Figure 2.9


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The history of the microscope


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Microbiology 201 PSU