Day 9: Electron Microscopy, MB Lipids
• Friday, Sept. 4: Electron microscopy,
Ch. 9
• Spike protein assignment due, 5 PM
• Monday, Sept. 7: No Class – Labor
Day
• Wednesday, Sept. 9: Membrane
lipids, Ch. 10
• Quiz on Week 3 due by 1 PM
• Friday, Sept. 11: Membrane proteins,
Ch. 10
I. Microscopy
A. GFP and Ion indicators
B. Transmission electron microscopy
C. Scanning electron microscopy
II. Membrane lipids
D. Membrane lipid structure
E. Models of membrane structure
• After reading the text, attending lecture, and
reviewing lecture notes, you should be able to:
• Explain why EM has 1000X greater resolution than
LM.
• Compare and contrast TEM and SEM.
• Explain how TLC is used to separate lipids.
• Describe the structure of membrane lipids.
• Discuss spontaneous lipid structures and historical
models of membrane structure.
What is Green Fluorescent Protein (GFP)?

GFP used as a ”reporter”, showing activity of

specific neuronal regulatory DNA > neurons.
GFP used in vivo to follow the location and
dynamics of proteins

Tobacco plant cell w/ spliceosomal protein fused

to GFP in Cajal bodies over time > live dynamics.

Tobacco plant cell w/ mitochondrial protein

fused to GFP and red fluorescing chloroplasts.
Resolution limit of Immunofluorescence

MT actual width: 25 nm
MT apparent width w/IF: 0.2 um (200 nm)
D. Electron Microscopy 0.61
r
Comparison of resolving power NA

Light microscopy Electron microscopy C atoms in graphene

Why does electron microscopy have greater resolving power?

• Wavelength () of an electron (at 1,000,000 V) = 0.001 nm (versus 400 nm for LM).
• If NA = 0.004 (versus 1.4 for LM), r = 0.61(0.001nm)/0.004 = 0.2 nm (1000X better than LM).
 SARS-CoV-2 viruses from first U.S. case

CDC/ HANNAH A BULLOCK; AZAIBI TAMIN

https://www.forbes.com/sites/victoriaforster/2020/
04/18/what-does-coronavirus-look-like-cdc-releases
-images-from-first-american-covid-19-patient/#22e
859023577
, https://phil.cdc.gov/Details.aspx?pid=23336 Cynthia S. Goldsmith and A. Tamin
Hannah A Bullock; Azaibi Tamin/CDC
How are samples prepared for TEM?
• Fixation w/glutaraldehyde and OsO4
• Cross-links proteins; fixes and stains
lipids
• Embed in plastic resin
• Thin sectioning with ultramicrotome,
section placed on copper grid
• Stain with heavy metals to give
contrasting electron density
• Uranyl acetate, lead citrate

http://www.soest.hawaii.edu/AEMC/instruments/aemc_prep.htm
How does a TEM work?

Lenses are magnets

How are
proteins
localized at
the EM
level?
Immunogold labeling:
 JEOL JSM-840A SEM in CMU
Biology Microcopy Facility

• Electron beam scans across sample to produce image.

• Primary electrons hit sample and produce secondary electrons, which are collected and
converted to photons on a video screen.
• Large depth of field possible, good for 3-D.
• Used together with freeze-fracture technique to study membranes.
 SEM compared to DIC, TEM

< DIC

SEM >

< TEM

(A) A scanning electron micrograph of the stereocilia projecting from a hair cell in the inner ear of a
bullfrog. For comparison, the same structure is shown by (B) DIC light microscopy and (C) TEM.
II. Membrane Structure: How would you study the P.M.?

Lyse w/ dH2O, centrifuge

Plasma membrane “ghosts”
Extract w/ chloroform-MeOH
Centrifuge

pellet supernatant

SDS- Thin
PAGE: Layer
Chromatography:
Proteins separated by size: Lipids separated by polarity -
smallest travel farthest least travels farthest
Evidence of the phospholipid composition:
TLC of various membranes

Conclusion:
Membrane Lipid Components: Phospholipids
Membrane Lipid Components: Cholesterol,
Glycolipids

• Protection
Glycolipids: • Electrical
• Cell-recognition
Phospholipid interactions with water:
micelles, bilayers, and liposomes

Liposome resembles a cell:

B. Models of membrane
structure: Evidence for bilayer
• Gorter and Grendel (1925)
• Estimated red cell surface area and extracted lipid
from "ghosts."
• Predicted that area of RBC was 100 m2, found that
area covered by lipid was 200 m2 , indicating a bilayer
• Davson and Danielli Model (1935)
• How does differential permeability come about?
• Proposed lipid bilayer + protein lamellae on each side
(sandwich), pores allowed substances in or out.
• Robertson (1960)
• Viewed membranes with EM, seemed to agree with
Davson and Danielli model
• Suggested that all membranes of the same
composition (unit membrane).
• But unit MB model did not account for chemical
differences in membranes
Fluid Mosaic Model: Singer and Nicholson (1972) Science
175:720