2054 Microscopes C2faculty.weber.edu/coberg/class/2054/Microscopy.pdf · Microbiology: An Evolving...
Transcript of 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
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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.
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Scanning EM
n Sample is coated with
heavy metal ¨ Not sliced ¨ Retains 3D structure ¨ Gives 3D image
n Only examines surface of sample
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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
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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
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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