Bio 227 Lecture 6 Marvels of Microscopic Methods

1
Bio 227 Lecture 6Marvels of Microscopic Methods

• Have your clickers available
• Have the information about culturing your adopted
  bacteria available
• Be ready to test your knowledge on types of
  microscopy
• Take notes on todays material

2
Can your adopted bacteria grow on general purpose
media such as nutrient agar or trypic soy agar
(TSA)?

• Yes
• No, it requires more special media.
• No. It is viable, non-culturable (VNC) with no
  known culture conditions.
• I couldnt find culture information.
• I didnt look for culture information.

3
Does your adopted bacteria require enriched media
such as blood agar plates (BAP)?

• My adopted bacteria is fastidious and requires
  BAP for growth.
• It may grow on BAP but doesnt require.
• No known culture conditions.
• I couldnt find culture information.
• I didnt look for culture information.

4
Did you run across information about other types
of media that we discussed last time?

• Yes.
• No, only general purpose and enriched media
  information.
• No known culture conditions.
• I couldnt find culture information.
• I didnt look for culture information.

5
Live samples without staining

• () Live moving
• (-) Live and moving, may be hard to see
• () Quick easy
• () Negative staining without heat-fixing
  preserves live features

6
Fixed samples with staining

• (-) Dead not moving
• () Dead not moving
• () Stable on slide can be safer
• () Staining like film developing, highlights
  features
• () Simple differential stains possible

7
Stains

• Simple/Basic stains
• Differential
• As weve done
• Our spore stain will yield green spores pink
  vegetative
• Special
• C. neoformans
• To enhance flagella, we will not do, but will
  observe (may see in negative staining)

8
Stains

• Simple/Basic stains
• Differential
• As weve done
• Our spore stain will yield green spores pink
  vegetative
• Special
• C. neoformans
• To enhance flagella, we will not do, but will
  observe (may see in negative staining)

9
Stains

• Simple/Basic stains
• Differential
• As weve done
• Our spore stain will yield green spores pink
  vegetative
• Special
• C. neoformans
• To enhance flagella, we will not do, but will
  observe (may see in negative staining)

10
Now its time to play Name that Microscopic
Method
11
What type of microscopy?
Note the level of image detail lack of
staining. Size bar 5 ?m.

• Atomic force microscopy
• Bright-field microscopy
• Confocal microscopy
• Dark-field microscopy
• Differential interference contrast (DIC)
  microscopy
• Fluorescence microscopy
• Phase-contrast microscopy
• Scanning electron microscopy (SEM)
• Transmission electron microscopy (TEM)

C. neoformans budding fungal cell from Pierini,
L.M. and Doering, T.L. Spatial and temporal
sequence of capsule construction in Cryptococcus
neoformans. Molecular Microbiology (2001)
41(1)105-115
12
The image in the previous slide was made by

• Atomic force microscopy
• Bright-field microscopy
• Confocal microscopy
• Dark-field microscopy
• Differential interference contrast (DIC)
  microscopy
• Fluorescence microscopy
• Phase-contrast microscopy
• Scanning electron microscopy (SEM)
• Transmission electron microscopy (TEM)

13
Differential interference contrast (DIC)

• Method
• Polarized light (some wavelengths blocked) sent
  through specimen
• enhances differences in the refractive index of
  different sample segments
• Disadvantages
• Have to have microscope capacity
• Only 200 nm resolution
• Advantages
• High contrast of cell features
• Can use live and unstained samples

14
What type of microscopy?
The white in this image was originally red.
Making it white increases contrast. Notice how
the appear as clean slices. Size bar 5 ?m.

• Atomic force microscopy
• Bright-field microscopy
• Confocal microscopy
• Dark-field microscopy
• Differential interference contrast (DIC)
  microscopy
• Fluorescence microscopy
• Phase-contrast microscopy
• Scanning electron microscopy (SEM)
• Transmission electron microscopy (TEM)

A. Normal and B/C treated C. neoformans cells
from Reese, A.J. and Doering, T.L. Cell wall
a-1,3-glucan is required to anchor the
Cryptococcus neoformans capsule. Molecular
Microbiology (2003) 50(4)1401-1409.
15
The image in the previous slide was made by

• Atomic force microscopy
• Bright-field microscopy
• Confocal microscopy
• Dark-field microscopy
• Differential interference contrast (DIC)
  microscopy
• Fluorescence microscopy
• Phase-contrast microscopy
• Scanning electron microscopy (SEM)
• Transmission electron microscopy (TEM)

16
Confocal microscopy

• Method
• Tagged specimens emit light of one color when
  excited by another
• Scanning laser
• Disadvantages
• Special microscope
• Fluorescent samples
• Need antibody to structure of interest
• Need fluorescent tag on antibody (or secondary
  antibody)
• Only 200 nm resolution
• Advantages
• Generates clean slices or 3D images
• Can localize structures in or on cell

17
What type of microscopy?
Note the extreme level of external detail and
lack of staining.

• Atomic force microscopy
• Bright-field microscopy
• Confocal microscopy
• Dark-field microscopy
• Differential interference contrast (DIC)
  microscopy
• Fluorescence microscopy
• Phase-contrast microscopy
• Scanning electron microscopy (SEM)
• Transmission electron microscopy (TEM)

Bacteria from a biofilm forming on a slide placed
in a dogs bowl from Madigan, M.T., Martinko,
J.M. Parker, J. (2003) Brock Biology of
Microorganisms 10th Ed. Upper Saddle River, NJ
Prentice Hall
18
The image in the previous slide was made by

• Atomic force microscopy
• Bright-field microscopy
• Confocal microscopy
• Dark-field microscopy
• Differential interference contrast (DIC)
  microscopy
• Fluorescence microscopy
• Phase-contrast microscopy
• Scanning electron microscopy (SEM)
• Transmission electron microscopy (TEM)

19
Atomic force microscopy

• Method
• Scan peaks and valleys of surfaces with a tiny
  stylus and records patterns
• Disadvantages
• Special equipment
• Advantages
• No fixatives or coatings
• Samples can be live
• 1 nm lateral resolution

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What type of microscopy?
Note the dark background and white colored cells.

• Atomic force microscopy
• Bright-field microscopy
• Confocal microscopy
• Dark-field microscopy
• Differential interference contrast (DIC)
  microscopy
• Fluorescence microscopy
• Phase-contrast microscopy
• Scanning electron microscopy (SEM)
• Transmission electron microscopy (TEM)

Treponema pallidum, the spirochete that causes
the syphilis STD. From http//depts.washington.edu
/nnptc/online_training/std_handbook/gallery/pages/
treponemapallidum.html
21
The image in the previous slide was made by

• Atomic force microscopy
• Bright-field microscopy
• Confocal microscopy
• Dark-field microscopy
• Differential interference contrast (DIC)
  microscopy
• Fluorescence microscopy
• Phase-contrast microscopy
• Scanning electron microscopy (SEM)
• Transmission electron microscopy (TEM)

22
Dark-field microscopy

• Method
• Light reaches sample from the sides
• By putting a light-blocking disc over the
  condenser
• Disadvantages
• Modified light-field microscope
• Only 200 nm resolution
• Advantages
• Specimen light on dark background, enhances
  contrast
• Can do on live and unstained samples

23
What type of microscopy?

• Atomic force microscopy
• Bright-field microscopy
• Confocal microscopy
• Dark-field microscopy
• Differential interference contrast (DIC)
  microscopy
• Fluorescence microscopy
• Phase-contrast microscopy
• Scanning electron microscopy (SEM)
• Transmission electron microscopy (TEM)

Note the level of image detail (some internal
vacuoles etc. are visible) lack of staining.
Size bar 5 ?m.
A-C C. neoformans cells D. S. cerevisiae cells
from Reese, A.J. and Doering, T.L. Cell wall
a-1,3-glucan is required to anchor the
Cryptococcus neoformans capsule. Molecular
Microbiology (2003) 50(4)1401-1409.
24
The image in the previous slide was made by

• Atomic force microscopy
• Bright-field microscopy
• Confocal microscopy
• Dark-field microscopy
• Differential interference contrast (DIC)
  microscopy
• Fluorescence microscopy
• Phase-contrast microscopy
• Scanning electron microscopy (SEM)
• Transmission electron microscopy (TEM)

25
Phase-contrast microscopy

• Method
• Microscope transforms subtle changes as light
  passes through objects with different densities
  and refractive index properties
• Disadvantages
• Requires microscope
• Only 200 nm resolution
• Advantages
• Improves contrast without staining
• Can use live samples

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What type of microscopy?

• Atomic force microscopy
• Bright-field microscopy
• Confocal microscopy
• Dark-field microscopy
• Differential interference contrast (DIC)
  microscopy
• Fluorescence microscopy
• Phase-contrast microscopy
• Scanning electron microscopy (SEM)
• Transmission electron microscopy (TEM)

Note the detail on the external features. Size
bar 1 ?m.
Image of C. neoformans courtesy of Arturo
Casadevall.
27
The image in the previous slide was made by

• Atomic force microscopy
• Bright-field microscopy
• Confocal microscopy
• Dark-field microscopy
• Differential interference contrast (DIC)
  microscopy
• Fluorescence microscopy
• Phase-contrast microscopy
• Scanning electron microscopy (SEM)
• Transmission electron microscopy (TEM)

28
Scanning Electron Microscopy

• Method
• Electron beam scanned over specimen
• Disadvantages
• Special microscope
• Sample coated in heavy metal film
• Coatings can distort image
• Advantages
• External features
• 10 nm resolution
• Cool pictures!

29
What type of microscopy?
Note the staining.

• Atomic force microscopy
• Bright-field microscopy
• Confocal microscopy
• Dark-field microscopy
• Differential interference contrast (DIC)
  microscopy
• Fluorescence microscopy
• Phase-contrast microscopy
• Scanning electron microscopy (SEM)
• Transmission electron microscopy (TEM)

Yogurt culture sample photographed by Stephen
Durr. From http//www.btinternet.com/stephen.durr
/microbes.html
30
The image in the previous slide was made by

• Atomic force microscopy
• Bright-field microscopy
• Confocal microscopy
• Dark-field microscopy
• Differential interference contrast (DIC)
  microscopy
• Fluorescence microscopy
• Phase-contrast microscopy
• Scanning electron microscopy (SEM)
• Transmission electron microscopy (TEM)

31
Bright-field microscopy

• Method
• Standard light passing through condenser and then
  specimen
• Different objective lenses available
• Disadvantages
• Only 200 nm resolution
• Advantages
• Availability of microscope
• Live and preserved samples
• Stained and unstained samples

32
What type of microscopy?
The white in this image was originally in red but
is in white for more contrast. Notice the fuzzy
look from cells at different levels in the
microscope view.

• Atomic force microscopy
• Bright-field microscopy
• Confocal microscopy
• Dark-field microscopy
• Differential interference contrast (DIC)
  microscopy
• Fluorescence microscopy
• Phase-contrast microscopy
• Scanning electron microscopy (SEM)
• Transmission electron microscopy (TEM)

Histoplasma capsulatum cells with C. neoformans
capsule from Reese, A.J. and Doering, T.L. Cell
wall a-1,3-glucan is required to anchor the
Cryptococcus neoformans capsule. Molecular
Microbiology (2003) 50(4)1401-1409.
33
The image in the previous slide was made by

• Atomic force microscopy
• Bright-field microscopy
• Confocal microscopy
• Dark-field microscopy
• Differential interference contrast (DIC)
  microscopy
• Fluorescence microscopy
• Phase-contrast microscopy
• Scanning electron microscopy (SEM)
• Transmission electron microscopy (TEM)

34
Fluorescence microscopy

• Method
• Tagged specimens emit light of one color when
  excited by another
• Disadvantages
• Special microscope
• Samples at various depths visible (vs. confocal)
• Fluorescent samples
• Need antibody to structure of interest
• Need fluorescent tag on antibody (or secondary
  antibody)
• Only 200 nm resolution
• Advantages
• Can localize structures in or on cell
• Useful diagnostic and research tool

35
What type of microscopy?
Notice the internal detail!

• Atomic force microscopy
• Bright-field microscopy
• Confocal microscopy
• Dark-field microscopy
• Differential interference contrast (DIC)
  microscopy
• Fluorescence microscopy
• Phase-contrast microscopy
• Scanning electron microscopy (SEM)
• Transmission electron microscopy (TEM)

Reese, A.J., Yoneda, A., Breger, J.A., Beauvais,
B., Griffith, C.L., Kim, M.-J., Liu, H., Yang,
S., Skau, C.T., Sefko, J.A., Osumi, M., Latge,
J.-P., Mylonakis, E.E., and Doering, T.L.. The
role of a-1,3-glucan synthase in Cryptococcus
neoformans capsule assembly. Molecular
Microbiology (2007) 63(5)1385-98.
36
The image in the previous slide was made by

• Atomic force microscopy
• Bright-field microscopy
• Confocal microscopy
• Dark-field microscopy
• Differential interference contrast (DIC)
  microscopy
• Fluorescence microscopy
• Phase-contrast microscopy
• Scanning electron microscopy (SEM)
• Transmission electron microscopy (TEM)

37
Electron microscopy Immunoelectron microscopy

• Method
• Electrons under vacuum sent through thin
  specimens
• Disadvantages
• Special microscope
• Need thin sections of specially coated (uranium,
  lead, etc) samples
• Need antibodies with gold beads and target for
  immunoelectron methods
• Advantages
• Internal features
• 0.5 nm resolution
• Cool pictures!

38
What is resolution in microscopy?

• The process of enlarging the size of an optical
  image.
• The capacity to tell two adjacent objects apart.
• It relates to the objective lens that is in place.

39
Some light microscopes have a blue filter to
improve resolving power. Why does this work?

• It enhances the impact of using oil.
• The shorter wavelengths improve resolving power.
• It increases the numerical aperture, thus
  improving resolving power.

40
Resolving power

• Resolution RP light wavelength (nm)
• 2 x numerical aperture of obj. lens
• The lower the RP number, the closer 2 objects can
  be and still be distinguished
• Shorter (bluer) wavelengths improve RP
• Can get closer to object (flea image in book)
• Higher numerical aperture (light gathering
  ability) improves RP
• Better light gathering in electron than
  bright-field microscopy