2054 microscopes c2faculty.weber.edu/coberg/class/2054/microscopy.pdf · microbiology: an evolving...

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 1

Observing Microbes

n Human eyes have limited resolution

¨ 150 µµm (1/7 mm, 1/200 inch) n Microscope needed to see smaller objects

¨ Eukaryotic microbes n  Protozoa, algae, fungi n  10–100 µµm

¨ Prokaryotes n  Bacteria, Archaea n  0.4–10 µµm

10 µµm

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 2

Bacterial Shapes

n Cocci = spheres ¨ in bunches chains quartets

n Bacilli = sticks, rods ¨ alone in chains

n Vibrios = bent rods n  Irregular

n Spiral

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 3

Staining

n Fix cells to hold in position n Stain with dye

¨ Reacts with chemical structure of organism n  Gram stain reacts with thick cell wall

¨ Increases absorbance n  Easier to find in low-contrast conditions

Gram-positive cells Gram-negative cells

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 4

Bright-Field Microscopy

n  Increasing resolution ¨ Multiple lenses

n  Correct each other’s aberrations n  Compound microscope n  Need to focus two lenses

¨ Objective ¨ Condenser

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 5

Bright-Field Microscopy

n  Increasing resolution ¨ Use shorter wavelength light

n  UV, X-rays ¨ But images aren’t visible to human eye

¨ Lessen contrast n  Lenses with higher contrast give less resolution

¨ But need enough contrast to see object

¨ Immersion oil n  Collects more light from specimen

¨ Wider lens closer to specimen n  Higher numerical aperture (NA)

NA = n sinθθ

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 6

Dark-Field Microscopy

n  Light shines at oblique angle

¨ Only light scattered by sample reaches objective

¨ With enough light, some bounces off object n  Even objects smaller than wavelength of light

¨ Makes visible objects below resolution limit n  Flagella, very thin bacteria

Helical bundle of flagella

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 7

Phase-Contrast Microscopy

n  Light passes through and around sample

n  Light through sample is refracted ¨ Changes phase of light ¨ Light waves out of phase cancel

n Sample appears dark against light background

¨ Shows internal organelles of eukaryotes

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 8

Differential Interference Contrast (DIC) Microscopy

n Polarized light passes through specimen ¨ Sample boundaries bend light ¨ Second polarized lens blocks light ¨ Bent light affects brighter or darker than

background

Bacterium

Head of microscopic worm (C. elegans)

Cell nuclei

Pharynx (mouth) 10 µµm

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 9

Fluorescence Microscopy

n Fluorophores absorb high-energy light

¨ Short wavelength n Emit lower-energy light

¨ Longer wavelength n  Label molecules of interest in cell

¨ Marker for position of molecules within cell

UV blue green

blue green red

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 10

Fluorescently Labeling Molecules

n Attach directly to some molecules ¨ DAPI binds DNA

n Attach labeled antibody to molecules ¨ Antibody binds specific molecules

n  Fluor covalently bound to antibody

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 11

Electron Microscopy

n Electrons behave like light waves

¨ Very high frequency ¨ Allows very great resolution

n  A few nanometers

n Sample must absorb electrons ¨ Coated with heavy metal ¨ Electron beam and sample are

in a vacuum ¨ Lenses are magnetic fields

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 12

Transmission EM

n Sample is fixed to prevent protein movement ¨ Aldehydes to fix proteins ¨ Flash-freezing ¨ High-intensity microwaves

n Fixed sample is sliced very thin ¨ Microtome

n Sample is stained with metal ¨ Uranium ¨ Osmium

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 13

Transmission EM

n High resolution ¨ Can detect molecular

complexes n  Ribosomes n  Flagellar base n  Strands of DNA

n Need many slices to determine 3D structure

Figure 01: Metal shadowing.

Adapted from J. L. Ingraham and C. A. Ingraham. Introduction to Microbiology, Third edition. Brooks/Cole Publishing Company, 2003.

Figure 03: Negative contrast method of visualizing particles by electron microscopy.

Figure 04A: Image of negatively stained bacteriophage T7 helicase/primase in the presence of dTDP.

Part A reproduced from M. T. Norcum, et al., Proc. Natl. Acad. Sci. USA 102 (2005): 3623-3626. Photo courtesy of J. Anthony Warrington, Florida State University.

Figure 04B: Tomato bushy stunt virus particles

Part B courtesy of Robert G. Milne, Plant Virus Institute, National Research Council, Turin, Italy.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 18

Scanning EM

n Sample is coated with

heavy metal ¨ Not sliced ¨ Retains 3D structure ¨ Gives 3D image

n Only examines surface of sample

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 19

Visualizing Molecules

n X-ray crystallography

¨ Locates all atoms in a large molecular complex ¨ Sample must be crystallized

n Nuclear magnetic resonance (NMR) ¨ Measures resonance between chemical bonds ¨ Can locate all atoms in a small protein ¨ Shows atomic movement of proteins in solution

n  Proteins embedded in membranes

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 20

X-Ray Crystallography

n X-rays have tiny wavelength

¨ Resolution less than 1 Angstrom n  0.1 nm = width of a hydrogen ion

n No lenses to focus X-rays ¨ Shoot X-rays at crystallized sample

n  Many molecules in identical conformation n  X-rays diffract according to position of atoms n  Compute position of atoms from pattern of

scattered X-rays

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 21

X-Ray Crystallography

n Can detect position of thousands of atoms

in a complex of proteins

“Ribbon” shows position of “backbone” of amino acids in proteins