ELECTRON

MICROSCOPY
 Microscopy
Bacterium
Bacterium
Needle
Needle
Small
Blood
Blood
Mitochondria Virus
Mitochondria Virus molecule
cell
cell
1mm 1 um 1 nm
0.2
0.2 mm
mm 0.2
0.2 um
um 0.2
0.2 nm
nm
Eye Light Microscopy
Electron Microscopy
 Electron Microscopes
Transmission Electron
Microscope
Primarily images
internal structures
Scanning Electron
Microscope
Images surfaces
Transmission Electron
Microscope
 TEM
Needle
Needle
Small
Cells
Cells Organelles
Organelles Virus
Virus molecule
1mm 1 um 1 nm
0.2
0.2 mm
mm 0.2
0.2 um
um 0.2
0.2 nm
nm
Eye Light Microscopy
Electron Microscopy
Theory
 Visibility
Resolution Contrast
Difference
Difference in
in density
density or
or color
color
 Resolution Limit
Electrons, like light have a wave
nature
Resolution limited by
diffraction
Resolution
Diffraction Limited
Resolution
 Abbe
Equation
d = k (wavelength)
numerical aperture
NA= n (sine ½ collecting angle)
n = refractive index
TEM Resolution
d = k (wavelength)
numerical aperture
 TEM Resolution
Transmission Light Microscope……….. 0.2 micrometer
Transmission Electron Microscope…… 2 angstroms
 Design
Design of
of TEM
TEM
Photon Microscope Electron Microscope
Design
Design of
of TEM
TEM
Alterations from Photon
Microscope
• Electron Source
• Vacuum System
• Electromagnetic lenses
(Zoom)
• Translation of electrons to
photons
Contrast in TEM
Scanning Electron Microscope
Scanning Electron Microscope
(SEM)
Scanning Electron Microscopy
(SEM)
Primary
Primary
Electrons
Electrons
Secondary
Secondary
Electrons
Electrons
Similar in many respects to confocal image formation
SEM Image Formation
SEM Image Formation
SEM Image Formation
SEM Image Formation
SEM Image Formation
Scanning Electron Microscope
SEM Resolution: Probe Size not
Diffraction
SEM Resolution
SEM Contrast
 Why do TEM Study?
• Need to determine subcellular location
• Analyze changes in subcellular organelles
• Improve 3-D resolution
TEM provides improved resolution in all 3
dimensions for surface and internal
structures
 Why do SEM study?
• Need improved surface resolution
• Require analysis of changes in surface
• Complete view of surface at once
(interelationships)
Improved resolution and improved surface visualization
 Sample
Preparation
 TEM Specimen Preparation
• Maintain structural integrity
• Preserve specimen in fixative fluid (aldehyde)
• Cut thin section to allow viewing of internal
structures
• Embed in hard plastic
• Section to produce 6-10 nm slices
• Add contrast for improved visualization or
specific localization
• Stain material
 TEM specimen preparation is time
consuming, expensive, and sectioning
requires very special skills
 Preserving 3-D Structure:
Fixation
• In general crosslinking fixatives (Aldehydes:
glutaraldehyde or formaldehyde) are
required to maintain ultrastructure
– Use as mild a fixative as possible
– Use as brief a fixation as possible
– Time and concentration will vary,
particularly for immunochemistry
• Aldehydes good for proteins, OK for
carbohydrates, poor fixative for lipids and
nucleic acids.
 Glutaraldehyde
• Excellent fixation
• Slow penetration
• Permanent
• Use stuff with relatively few impurities (it is
worth the extra expense).
• To avoid blebbing of membranes, use at
room temperature, pH 7.2-7.4, and slightly
hypotonic (<300 mOsm) for mammalian cells
(0.1M cacodylate is good buffer).
• Fluorescent
 Formaldehyde
• Not as good ultrastructure preservation
as glutaraldehyde
• Faster penetrator
• Effects can be partially reversed by
washing
• Make up fresh from paraformaldehdyde
• Include CaCl2 in fixative to prevent
membrane blebbing
 Osmium Tetroxide
• Very good lipid fixative
• Slow penetrator
• Usually used as secondary fixative
after aldehyde
• Contrasts phospholipid membranes
• Generally destroys antigenicity
 Analysis of “Good Fixation”
• Membranes are
intact.
• Cell and organelles,
particularly
mitochondria, not
swollen or distorted.
• No “empty spaces”
in cytoplasm
• Nuclear
membranes and
mitochondrial
membranes are
parallel.
Embedding and Sectioning
•Solid structure required for thin sectioning
•Plastic Embedding
•Remove water by dehydration
•Replace with liquid plastic
•Solidify plastic
Electron beam has limited penetration
ability, so need to cut thin slices
Electron Microscopy
Thin Sectioning
Plastic is not
removed
Paraffin sections ~ 5 um thick
Plastic sections ~ 8 nm thick
Contrast Specific Structures
with Stains
EM Stains are
heavy metals
TEM Staining
General Stains Uranyl Acetate:
•• uranium
uranium binds
binds
DNA
DNA proteins
proteins
primarily,
primarily, but
but also
also
stains
stains phosphate
phosphate
groups
groups and
and
carboxyl
carboxyl ends
ends of
of
some
some proteins.
proteins.
Lead Citrate:
• lead
lead precipitates
precipitates
on
on negatively
negatively
charged
charged polar
polar
groups
groups such
such as
as
membrane
membrane lipids,
lipids,
hydroxyl
hydroxyl groups
groups ofof
carbohydrates,
carbohydrates,
and
and RNA
RNA proteins.
proteins.
Cell
Cell with
with large
large lipid
lipid vacuole
vacuole
Tracers
15
10
5
0
0 80 160 240
TIME (minutes)
 Enzyme Cytochemistry
S
Suubbs
sttrra
attee
Visible
Visible
Product
Product
Product
Product
Beta-glycero- pH 4.5
Phosphate
phosphate Acid Phosphatase
Lead
Lead Phosphate
Acid Phosphatase Staining of
Golgi and Lysosomes
Immunohistochemistry
Gold Label
2 Step (Sandwich) Staining
Gold Label
Goat Anti-
Rabbit
Rabbit
Anti-X
X
X X
X
2 Step
 EM
Immuno-
Gold
labeling of
Lactoferrin
in PMN
 Pre-embed or Post-embed
Staining?
Pre-embed
Stain whole cells or View cells without
tissue sectioning
Embed and section cells
or tissue
Post-embed
Embed and section Stain sections
cells or tissue
Pre- or Postembed Staining
Pre-Embed Post-Embed
Less tissue preparation Embed procedure must
to alter Ag minimally alter Ag
Sample must be
Sectioning exposes
permeabilized unless
internal Ag
surface Ag
Can view live cells Cells must be preserved
Greater 3-D depth In EM, embedding
(whole mount) resins can be sticky
Permeabilization
146 A
30 x 40 x 50 A
If Pre-Embed, need to Permeabilize
Alcohols and Acetone (AVOID)
• Fix and extract
• Do not preserve ultrastructure
Saponin
• Gentle
• Thought to work by solubilizing Cholesterol
• Transient
Triton, Brij, Tween, Lysolecithin
• Harsher
• Permanent
Different membranes will react differently to different
permeabilizing agents
 Silver Enhancement
Silver
Gold-labeled
Goat Anti-Rabbit
Rabbit Anti-X
X
X X
X
 Silver Enhancement
Silver
Gold-labeled
Goat Anti-Rabbit
Rabbit Anti-X
X
X X
X
EM Gold Post-embed Staining
of Lowicryl Sections
Tissue fixed in 2.5% Paraformaldehyde in Cacodylate buffer,
dehydrated and embedded in Lowicryl (no Osmium Postfix). Thin
sections cut and placed on Nickel grids.
 EM-Gold Staining
• Wash 2X in PBS + 0.1% glycine, pH 7.4
• Wash 2 X in PBS + 5 mg/ml Pre-immune Goat Serum
• Incubate with Rabbit Anti-X in PBS + Pre-immune Goat
Serum for 1 hour at room temp
• Wash 4 X in PBS + Pre-immune Goat Serum, 2 minutes each
• Incubate with Goat Anti-Rabbit conjugated to 10 nm colloidal
Gold (about 10 ug IgG/ml) for 1 hour at room temperature
• Wash 3 X with PBS
• Refix sample in fresh 1% Paraformaldehyde for 15 minutes
• Wash 3 X with distilled water, 5 minutes each
• Counterstain with aqueous Uranyl acetate (optional)
SEM Preparation
• Simpler than TEM
• No sectioning
• Need to dry
specimen
• Mount on stub
• Metal coat
specimen
 Drying Specimen
Vapor phase at transition from liquid to gas has high
pressure (miniscus in graduated cylinder)
• Air dry SEM Drying
• Simple Methods
• Harsh
• Freeze sublimation
• Gentler drying
• Expensive
• Freezing difficult
• Low surface tension fluids (HMDS)
• Critical Point Drying
• Gentle
• Effective
• Reasonably simple
 Critical Point Drying
Critical point: That temperature and pressure at
which fluid instantly becomes gas.
Critical Constants
Substance Temp (C) Pressure (PSI)
Hydrogen -234.5 294
Oxygen -118 735
CO2 31.1 1072
H2O 374 3212
 Critical Point Drying
• Dehydrate sample
in EtOH
• Exchange EtOH
with Acetone
• Exchange Acetone
with liquid CO22
• Raise temp &
pressure to Critical
Point
Analysis of “Good Fixation and Drying”
• Cells or tissue not squashed.
• Membranes are intact.
• Fine detail is maintained and well defined.
Mount Specimen on Stub and
Apply Metal Coat
 Reason for Coating:
Improved Signal to Noise
C: 2,4
Pd: 2,8,18,18
Au: 2,8,18,32,18,1
 Reason for Coating:
Reduction of Heat
Increased
Signal
Heat drained to
Microscope
Correlative Microscopy
 Quantitative
Microscopy for
Comparison with
Biochemistry
Observation: Glycogen synthesis and deposition in
liver occurs in areas rich in sER.
Questions: Does sER increase during stimulation
of glycogen synthesis?
Does sER precede increases in
glycogen deposition?
 sER and Glycogen Synthesis
Glucocorticoid
Synthase Glycogen
Phosphatases (sER)
Glycogen Glycogen
D I A B
Synthase Phosphorylase
UDP-Glucose alpha-D-Glucose-1-P
 Protocol
ADX Rats
Fast to Deplete Glycogen
Dexamethasone Injection
Measure Glycogen
Evaluate Volume of Glycogen in Cell
Evaluate Volume of rER and sER in Cell
Glycogen Synthesis
Synthesis Volume
1.50 0.15
1.25 0.12
mg/mg liver wet weight

µm3/µm3 hepatocyte
1.00 0.10
0.75 0.07
0.50 0.05
0.25 0.02
0.00 0.00
0 1 2 3 4 5 6
Time (Hrs)
 sER Volume Changes
sER
5
µm2/µm3 hepatocyte

4
3
2
1
0
0 1 2 3 4 5 6 7
Time (hrs)
Correlation of Glycogen and
sER Increases
Glycogen sER
5 0.12
R2=0.96 0.10
4
Glycogen (µm3/µm3)

sER (µm2/µm3)
0.08
3
0.06
2
0.04
1
0.02
0 0.00
0 2 4 6
Time (hrs)
 ER Volume Changes
rER sER Total
9
8
µm2/µm3 hepatocyte

7
6
5
4
3
2
1
0
0 1 2 3 4 5 6 7
Time (hrs)
SER-RER Transition
SER Glycogen
RER
Phospholipid and Protein
Synthesis
Phospholipid Protein
0.80
% increase from time 0

0.60
0.40
0.20
0.00
0 1 2 3 4 5 6 7
Time (hrs)
 Hypothesis
1. Initially enzymes are present but inactive
2. Conversion of rER into sER activates enzymes
3. Only later does new synthesis produce larger capacity for
glycogen synthesis
Quantitative
comparison of
biochemical and
structural
information
provided important
insights.
 LM, SEM, TEM
Cellular Cholesterol Metabolism
 Cellular Cholesterol Metabolism
LIPOPROTEIN
Chol-E
Chol-E
Endocytosis
LYSOSOME/LATE
LYSOSOME/LATE
Chol-E ENDOSOME
ENDOSOME
LAL
Chol-E
Chol + FA
Plasma
Plasma Acetate FA
Membrane
Membrane ACAT
HMCoA-R
Chol Chol Chol-E
NCEH
Cholesterol
Cholesterol Efflux
Efflux Promoter
Promoter
 1. Load cells with Cholesteryl Ester
2. Allow Lysosomes to hydrolyze CE to Cholesterol
3. Determine effect of excessive lysosomal accumulation
• Live cell imaging showed
intracellular cholesterol crystals
formed
• The crystals were not apriori toxic
• We could pinpoint areas of
crystal formation for subsequent
EM analysis
 Intracellular cholesterol
crystals were in lysosomes
 Live cell imaging showed eventual
increases in cell death among cells
with crystals
 SEM showed changes in
crystal size after liberation
from macrophages
Changes in Crystal Volume
150
100

Volume (µm3)
50
0
0 2 4 6 8 12
Time (hrs)
 Hypothesis
Macrophages colonize extracellular crystals
which can provide a mechanism for cells to
efflux their excess cholesterol stores.
 LM, TEM,
Biochemistry
Cellular Cholesterol Metabolism
LIPOPROTEIN
Chol-E
Chol-E
Endocytosis
LYSOSOME/LATE
LYSOSOME/LATE
Chol-E ENDOSOME
ENDOSOME
LAL
Chol-E
Chol + FA
Plasma
Plasma Acetate FA
Membrane
Membrane ACAT
HMCoA-R
Chol Chol Chol-E
NCEH
Cholesterol
Cholesterol Efflux
Efflux Promoter
Promoter
 Cells
incubated
with agg-
LDL for 1-7
days
Lipid Accumulation from Agg-LDL
Nile Red Fluorescence: THP-1, 3 days with Agg-LDL
Electron Microscopy Provided
Details of Distribution
Distribution of Cholesterol between
Lysosomes and Inclusions
Inclusion Lysosome
50
40
% of Cell Volume

30
20
10
0
D0 D1 D3 D7
Biochemistry Provided Details of
Cholesterol Species
Where is the free cholesterol
and where is the cholesteryl
LIPOPROTEIN
ester?
Chol-E
Chol-E
Endocytosis
LYSOSOME/LATE
LYSOSOME/LATE
Important
Chol-E ENDOSOME
ENDOSOME question for
LAL determining
Chol + FA
Chol-E mechanism of
Plasma
Plasma Acetate FA lysosomal
Membrane
Membrane ACAT
HMCoA-R accumulation
Chol Chol Chol-E
NCEH
Cholesterol
Cholesterol Efflux
Efflux Promoter
Promoter
Light Microscopy shows that
significant FC is in
lysosomes/late endosomes
 Quantitative Microscopy
combined with Biochemical
Manipulation provided
evidence that much of the
Cholesteryl Ester was also in
Lysosomes.
Light Microscopy shows that that FC
is trapped in Lysosomes
Loaded CD-treated
Control CD-Treated
 Conclusions
1. Agg-LDL produces both FC and CE
accumulation in lipid swollen lysosomes
2. CE accumulation suggests that CE hydrolysis
is inhibited
Hypothesis
1. Now investigating whether failure to efflux
cholesterol from lysosomes is contributing
factor to inhibition of hydrolysis.