Electron Microscopy for Cellular Imaging

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Electron Microscopyfor Cellular Imaging

• Vincent Gattone Ph.D.
• Department of Anatomy and Cell Biology

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Specialized Techniques

• Immunocytochemistry (pre-embedment or
  post-embedment) (more next week)
• Cytochemistry (direct reactions for enzymes or
  other molecules)
• Negative staining (structural proteins or
  viruses)
• Rotary shadowing (structure of proteins/complexes)
  
• Freeze fracture (membrane bound proteins)
• Freeze etch (almost 3-D of cytoplasmic
  components)
• EM Autoradiography (receptors, or
  uptake/metabolism)
• High Pressure Freezing Freeze Substitution
  Processing (get away from initial chemical
  fixation)

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Immunocytochemistry Techniques

• Have antibody, will Try!
• Similar to Immunohistochemistry at the light
  level, only more difficult.
• New Problems
• Antigen abundance (TEM sections are about
  1/1000th the thickness of a paraffin or frozen
  section so abundance of the antigen is a critical
  issue)
• React before embedment or after, on sections.
  (pre vs. post)
• Localization with gold conjugated 2o antibody
  (penetration)
• Affinity and specificity for the antigen
• Same old problems
• Will it work on fixed tissue? (frozen sections
  are an option)
• Background staining (working out dilutions and
  conditions)

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For Example, Na/K ATPase

• If I wanted to study the alteration in renal
  epithelial cell Na/K ATPase in disease Z, how
  could I approach that problem?
• Enzyme Cytochemistry
• Immunocytochemistry
• Freeze Fracture with Immunocytochemistry
• Negative Staining
• Autoradiography

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Enzyme Cytochemistry

• Na/K ATPase enzyme cytochemistry-renal
  basolateral plasma membrane.

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Enzyme Cytochemistry

• Na/K ATPase enzyme cytochemistry- (or for many
  other enzymes (peroxidase), lectins bound to
  glycoconjugates or other molecules). There are
  many reactions that allow the deposition of an
  electron dense reaction product. Provide good
  direct localization.

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Immunocytochemistry

• Na/K ATPase Immunocytochemistry- There are many
  ways to get an electron dense reaction product.
  Here, the secondary antibody was conjugated to
  colloidal gold. This provide indirect
  localization.

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Immunocytochemistry

• Immunocytochemistry- But there are many ways to
  get an electron dense reaction product DAB, 10nm
  colloidal gold attached to either a secondary
  antibody (like above) or protein A, nanogold,
  silver enhanced etc. While it provide
  localization, the fixation and processing
  approach will ultimately dictate the quality of
  the tissue morphology.

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Freeze Fracture

• Freeze fracture allows you to visualize
  intra-membranous proteins. You now are looking
  at the protein (protein complex) directly. In
  renal basolateral membrane, Na-K ATPase makes up
  a large proportion of the intramembranous
  proteins.

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Freeze Fracture with ICC

• Freeze fracture or enzyme cytochemistry can be
  partnered with immunocytochemistry, note the
  labeling of the P face.

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Negative Staining

• Isolated basolateral membrane vesicles negatively
  stained demonstrates the Na/K ATPase.

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Looking at the Specialized Techniques

• For molecules
• Immunocytochemistry
• Negative staining
• Rotary shadowing
• Freeze fracture
• Freeze etch
• Autoradiography
• High Pressure Freezing

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Positive versus Negative Staining

• Is the object of interest on a white or dark
  background?

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Negative Staining

• 1. Put specimen down on support film,
• 2. Flood with stain
• 3. Draw off excess stain
• 4. Examine with TEM

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Negative Staining
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Negative Staining

• Negative staining is used extensively for
  evaluating viruses. Here is the classic
  bacteriophage.

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Negative Staining

• Adenovirus (left), Rotavirus (right). Some of
  the viruses substructure is evident. Can be used
  in the diagnosis of herpes lesions.

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Negative Staining

• Negative staining can be used to evaluate the
  substructure of proteins. Here is collagen type
  VI.

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Rotary Shadowing

• Start simple. Colloidal Ferritin .
• Individual or
• aggregates

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Rotary Shadowing of DNA

• Phage DNA by rotary shadowing

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Rotary Shadowing of Collagen

• Structural proteins can be evaluated by rotary
  shadowing.

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Rotary Shadowing of Aggrecan
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Rotary Shadowing of Nuclear Pores

• Isolate nuclei and shadow them for their
  structure

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Freeze Fracture

• Freeze
• Crack it with a knife
• Shadow with heavy metal, then Carbon
• Eat tissue away with bleach
• Wash the metal/carbon replica
• Pick replica on a grid
• Examine in TEM

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Freeze Fracture
Splits the bilaminar plasma membrane ( other
membranous organelles) such that the inside of
the outer lamina is called the E (Exterior) face
while the one still attached to the protoplasm is
the P-face.
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Freeze Fracture
As the bilaminar membranes split, the
intramembranous proteins tend to stick to one of
the faces and appear as globs on (actually in)
the P face generally, with holes (divits) in the
E face. However, some transmembranous proteins
may stay with E face rather than P face.
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Gap Junctions

• Transition from negative staining to freeze
  fracture.

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Freeze Fracture
A specialized piece of equipment is needed to do
freeze fracture and Freeze Etch processing. This
Balzers unit is the state-of-the-art for this
type of processing.
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Freeze Fracture
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Freeze Fracture
Freeze Fracture gives some intracellular data not
really available by other methodology, as well as
some very pretty pictures. Of course you only
see the fractured surface. I wonder if there is
any information just below the fractured surface
(i.e. Freeze Etch)
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Freeze Etch
An intercellular area where you can see the
cortical actin filaments and deep to them the
intermediate filaments. Etching (allow the
water to lyophilize briefly before shadowing )
allows a 3-D appreciation of the associated
structures.
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Freeze Etch
This Freeze Etched sperm flagella allows the
visualization of the core microtubules and
associated structures.
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EM Autoradiography
This rat was given 125I-albumin and it was
filtered and endocytosed by renal tubule cells.
Radioligand studies can look at receptors and
pulse chase evaluations for cell synthesis of
compounds or drug metabolism.
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High Pressure Freezing
Chemical fixation can alter cellular structure
and the coagulative fixation of tissues
cross-links proteins which can mask antigenic
sites. For some sensitive antigens and/or
antibodies, fresh frozen sections are used in
light immunohistochemistry. For EM, the method
of freezing is critical since ice crystal damage
could utterly destroy the ultrastructural
appearance of the tissue. There are a number of
specialized freezing systems that have been
developed for cryoEM. The latest and
state-of-the-art is the Leica high pressure
freezer. The system is designed to rapidly
freeze small pieces of tissue or cultured cells
rapidly (rate gt a 15,000 degree/second drop in
temperature). The faster you freeze, the
smaller the crystals. This rapid freezing leads
to minimal if any ice crystal damage.
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Freeze Processing of Tissues

You have your tissue frozen and sitting in liquid
nitrogen, now what. The object is to preserve
the structure and antigenicity as much as
possible. You can 1. Cut ultrathin frozen
sections. 2. Freeze substitution. This method
allows you to process the frozen tissues at
liquid nitrogen temperature (-196oC). Ethanol
and acetone do not freeze at this temperature and
OsO4 can be dissolved in these solvents to
provide fixation if desired. Once all of the
water is removed solvent changes and by prolonged
exposure at that temperature, the tissue can be
infiltrated and embedded in a plastic resin.
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HPF Freeze Substitution
Frozen thin sections are the most sensitive way
to detect some antigens, especially using some
types of monoclonal antibodies. In this
specimen, immunogold was performed on
cryosections (note the negative stain
appearance). The tissue may have been labelled
as easily after FSP and embeddment in a plastic
resin.
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Specialized Techniques

• Immunocytochemistry (pre-embedment or
  post-embedment)
• Negative staining (structural proteins or
  viruses)
• Rotary shadowing (structure of proteins/complexes)
  
• Freeze fracture (membrane bound proteins)
• Freeze etch (almost 3-D of cytoplasmic
  components)
• Autoradiography (receptors and cellular
  metabolism)
• High Pressure Freezing Freeze Substitution
  Processing (avoid chemical fixation)