1. Microscopy and Classification
Microscopy, Staining and Classification - Chapter 4
Most of these sections will be covered in lab
-- Principles of Staining
-- Taxonomic and Identifying Characteristics
Relevant metric units
mm = 10-3 meter
μm (micrometer)
nm (nanometer)
Resolution vs
Magnification
Microscopy and Classification

2. Microscopy and Classification
How does a lens work?
Optical properties
refraction
magnification
resolution
distortion
Limitations of a single lens
illumination
aberration
Wikipedia link
Microscopy and Classification

3. Microscopy and Classification
Some principles of a compound microscope
Ocular lens
Objective lens
Condenser lens
How is magnification calculated?
Limitations to a compound
microscope
Microscopy and Classification

4. Microscopy and Classification
What were the key advances
in light microscopy?
Oil immersion lens
Fused lenses
Advanced optics
How do oil immersion and
fused lenses improve resolution?
Refractive index =
Borosilicate glass = 1.52
Air = 1.00
Water = 1.33
Immersion oil = 1.52
Fused lenses combine different types of glass
-- compensate for aberrations
Speed of light in vacuum
Speed of light in medium
Microscopy and Classification

5. Microscopy and Classification
Advanced optics only improve contrast
Theoretical limit of resolution
= 0.2 uM
Dark field
manipulates the
path of light
Phase contrast
and DIC
manipulate
phase of light
Dark field
Bright field
DIC
Microscopy and Classification

6. Microscopy and Classification
Fluorescence Microscopy allows precise
staining of cells and cellular components
Standard fluorescence
manipulates the
path of light
Confocal
-- puts everything in focus
Standard Fluorescence vs Confocal image of Tetrahymena
Microscopy and Classification

7. Microscopy and Classification
How does electron microscopy yield
higher resolution?
Resolution
- Eye ~ 0.2 mm
- LM ~ 0.2 uM
- EM ~ 0.2 nM ( uM)
Wavelengths of
… of electrons ~ nm
… visible light ~ 550 nm
Microscopy and Classification

8. Microscopy and Classification
What are types of electron microscopy?
Scanning vs Transmission
STM is different
Driscoll et al
Nature, 346, 6281
Microscopy and Classification

9. Microscopy and Classification
Biological classification
Biological taxa
-- Muddled in the middle
-- Blurry at the bottom
Binomial naming
Writing conventions
Staphylococcus aureus
Staphylococcus aureus
S. aureus
Common names and strains
Pneumoncoccus
E. coli O157 H7
Domain: Bacteria
Kingdom: Eubacteria
Phylum: Firmicutes
Class: Bacilli
Order: Bacillales
Family: Staphylococcaceae
Genus: Staphylococcus
Species: aureus
Microscopy and Classification

10. Microscopy and Classification
What are the 3 domains of life?
Eukarya – Archaea – Bacteria
What traits do Archaea
and Bacteria share?
-- prokaryotic cell structure
-- circular chromosome
and Eukaria share?
-- Nucleosomes
-- Multiple RNA Polymerases
-- Ribosome structure
Some Archaea have unique properties
-- Genetically distinctive
-- Cell membrane
-- Cell wall structure
The Antiquity of Microbes
Adam
Had ‘em
-- Strickland Gillilan
Microscopy and Classification

11. Microscopy and Classification
What are the characteristics of
Eukaryotes vs Prokaryotes?
-- all “cellular organisms”
Basic similarities?
membrane
genetic information
biochemistry
Distinctive properties of eukaryotes
‘ eu’ = true ‘karyo’ = nucleus
size
organelles
mitochondria, chloroplasts, etc
chromosomes
…vs prokaryotes ‘pre-nucleus’
Microscopy and Classification

12. Microscopy and Classification
Archaea Extremophiles
-- some notable ones…
Pyrolobus fumarii -- “Fire lobe of the chimney”
Hyper-thermophile -- 90OC – 113OC (106OC)
hydrothermal vents
Strain 121:
Picrophilus spp.
Extreme acidophiles -- pH <4 (opt 0.7)
Volcanic steam vents
Halobacterium spp.
Extreme halophiles – 5 M salt
Dead sea; great salt lake;
Salted foods
Microscopy and Classification