ECSE6963, BMED 6961 Cell

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ECSE-6963, BMED 6961Cell Tissue Image Analysis

• Lecture 4 Bio-molecular Imaging - II
• Badri Roysam
• Rensselaer Polytechnic Institute, Troy, New York
  12180.

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Recap

• Molecular imaging systems
• Produce spatial maps capturing distributions/locat
  ions of specific molecules
• Interactions of molecules with light
• Intrinsic imaging
• Imaging with contrast agents, especially
  fluorophores
• Fluorescence is a hugely important phenomenon
• Imaging genes and gene activity
• FISH fluorescence in-situ hybridization
• Imaging proteins the products of gene activity
• Immunofluorescence The use of fluorescently
  conjugated antibodies to tag specific proteins
  of interest
• Multiplexing Simultaneous use of multiple tags
  to image multiple proteins preserving relative
  context

Non-radiative Transition / Loss
Radiative Transition
Absorption
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Proteins and Antibodies

• Proteins have specific shapes
• They bind to other molecules with great
  specificity
• The other molecule is called a ligand
• The part of the protein that has the
  complementary shape is called a binding site
• Antibodies
• Y shaped proteins with antigen binding sites
  (grabbers)
• The shapes of the grabbers are variable, and
  match the shapes of antigens (x)
• Immunofluorescnece
• If we can attach a fluorescent entity to the
  antibody, we can effectively image a molecule of
  interest (antigen x) by proxy.

Antigen
Fluorescent entity
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Poly- and Mono-clonal Antibodies

• Polyclonal
• a mixture of antibodies
• Will recognize multiple epitopes provide robust
  detection
• Tolerant to changes in antigen, can work with
  denatured proteins
• Can be used when the antigen is not fully
  characterized
• Produced from intact living animals

• Monoclonal
• only one type of antibody
• Highly specific, great as a primary antibody
• Vulnerable to loss of epitope
• Will attach to one antigen in a mixture
• Less background response
• Produced from animal cells in culture

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Direct Immunofluorescence

• Choose an antibody that attaches to the molecule
  of interest
• A fluorochrome is chemically attached to the
  antibody to form a conjugate
• The molecule of interest is considered to exist
  wherever fluorescence from the attached
  fluorochrome is detected!
• Advantage simplicity
• Disadvantage
• Good for the most common applications, not
  flexible enough for general lab use
• Expensive we need N conjugations to see N
  antigens
• The fluorescent signal could be too weak
• Need an amplification method!

Fluorochrome
Primary Antibody
Molecule of Interest (antigen)
http//www.ihcworld.com/_books/Dako_Handbook.pdf
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Indirect Immunofluorescence
Fluorochrome

• A second antibody is attached to the complex
  composed of the antigen and the primary
  antibody
• The fluorochrome is attached to the secondary
  antibody
• The secondary antibody must be generated against
  the immunoglobulins of the primary antibody
  source,
• e.g., if the primary antibody is raised in
  rabbit, then the secondary antibody could be goat
  anti-rabbit.
• Advantages
• Flexibility No need to stock large numbers of
  labeled antibodies
• Can result in greater fluorescence if more than
  one secondary antibodies stick to a primary
  antibody.
• Especially important for multiple-fluor imaging

Secondary Antibody
Primary Antibody
Molecule of Interest (antigen)
http//www.ihcworld.com/_books/Dako_Handbook.pdf
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Poly-conjugated Secondary Antibody

• There are many ways to achieve signal
  amplification
• One way is to look for antibodies with multiple
  fluorochromes attached

Fluorochrome
Secondary Antibody
Primary Antibody
Molecule of Interest (antigen)
http//www.ihcworld.com/_books/Dako_Handbook.pdf
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Multiple Secondary Antibodies
Fluorochrome
Secondary Antibody
Primary Antibody
Molecule of Interest (antigen)

• Polyclonal secondary antibodies can attach to
  multiple epitopes on the primary antibody

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Another way to Amplify Use Enzymes!

• Enzymes are match maker proteins

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Enzymes Natures chemical amplifiers

• They control almost all chemical reactions in
  cells without being changed themselves
• They can speed up a reaction a million-fold or
  more with great specificity
• The ligand in this case is also called a
  substrate, and the binding site is also called
  an active site
• Two or more substrates attach to an enzyme, and
  become chemically modified
• They react in the microenvironment of the active
  site
• The reaction product no longer fits the shape of
  the active site, so it is released!
• The enzyme then moves on to broker another
  reaction

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Enzymatic Signal Amplification

• Basic idea attach an enzyme to the secondary
  antibody instead of a fluorochrome
• The enzyme can convert a given substrate into
  large amounts of colored/fluorescent molecules in
  the neighborhood of the antigen in situ.
• They bond to places near the antigen
• Horseradish peroxidase (HRP) is commonly used
• an enzyme extracted from the root of the
  horseradish plant
• Highly studied, and has lots of uses
• Alkaline phosphatase is another commonly used
  enzyme
• Downside
• The amplification factor is not
  reproducible/quantitative
• Loss of spatial localization

Molecular Probes Handbook
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Tyramide Signal Amplifcation Example Mouse Brain
Imaging
Molecular Probes Handbook
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(Strept)Avidin-Biotin Techniques

• Biotin is a vitamin
• A variety of biotin binding antibodies, both
  polyclonal and monoclonal are available
• Biotinylation attach a biotin molecule to
  something else. For example, a biotinylated
  antibody
• Avidin is a protein found in egg white (its a
  tetramer)
• Now largely replaced by the more effective
  streptavidin
• Streptavidin also binds strongly to biotin- it is
  isolated from a bacterium (Streptomyces
  avidinii).
• They have an extraordinary affinity for each
  other, and form the strongest-known non-covalent
  bond between a protein and a ligand

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Avidin-Biotin Complex (ABC)

• Basic idea You can attach multiple biotins to
  the secondary antibody
• Each biotin can attach tightly to an Avidin
• In the end you get an entire complex of labels
  near the antigen
• Major signal amplificaton!

Streptavidin
Biotinylated Secondary antibody
Primary Antibody
Antigen
http//www.ihcworld.com/_books/Dako_Handbook.pdf
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Biotinylated Quantum Dots
Biotin

• 10 - 35 nanometers in size
• 2 to 20 biotin molecules on the surface.
• Choice of emission wavelengths in the range 490nm
  to 900nm,

From Evident Technologies Inc. website
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Summary of Direct Indirect Labeling
www.chemicon.com
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Limitations of Fluorophores

• Toxicity of the fluorescent label
• Could change the function of molecule of interest
• Extreme case could be toxic to the cell(s)
• They can be affected by the light used for
  imaging
• Photo-bleaching
• Destruction of the fluorochrome by light
• Depends upon the total amount of light used
• Quantum dots do not photobleach (nice!)
• Photo-toxicity / photo-damage to tissue
• Many fluors require ultraviolet light excitation
• UV causes mutations and kills cells
• Infrared light causes thermal damage

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Dealing with Toxicity of the Fluorophore

• Simple Idea
• Make a cell produce proteins that are naturally
  fluorescent!
• Inspired by the discovery of green fluorescent
  proteins (GFP) in a jellyfish (aequoria victoria)
  by Osamu Shimomura and Frank Johnson in 1961

http//www.lifesci.ucsb.edu/biolum/organism/photo
.html
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Green Fluorescent Proteins

• Green fluorescent protein (GFP)
• Isolated in the 60s from a jellyfish Aequoria
  Victoria
• When excited, it glows green
• Turned out that it has a protein aequorin that
  produces blue light (470nm) which excites GFP
  molecule which produces green (508nm).
• The gene for this protein was sequenced in 1992
• The detailed structure and properties of this
  protein are now known
• This gene has been mutated to produce a large
  number of variants of the original GFP
• This has revolutionized biology!
• Especially, the study of live cells

http//www.lifesci.ucsb.edu/biolum/organism/photo
.html
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Exploiting the Process of Life!

• Now a widely used minimally invasive method for
  studying protein dynamics and function
• Using GFP we can see when proteins are made and
  where they can go.
• Basic Idea Insert the gene for GFP gene into the
  gene of the protein of interest so that when the
  protein is made it will have GFP hanging off it.
• Still retains the fluorescent properties!
• Usually, has minimal impact on the protein
  molecule
• Since GFP fluoresces one can shine light at the
  cell and wait for the distinctive green
  fluorescence associated with GFP to appear!

http//www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1
.htm
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Reporter Gene Technology
Promoter For gene of interest
Artificially inserted
Gene expression
GFP
GFP cDNA
AAAA
DNA Fragment

• GFP is Produced whenever the factors triggering
  the gene of interest are ON

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Fusion Protein Technology
Promoter For gene of interest
Artificially inserted
Gene expression
GFP
GFP cDNA
AAAA
Gene of interest
Protein
DNA Fragment
Produces the protein of interest with a GFP
attached! If the GFP can be verified (by other
means) to not affect the behavior of the protein,
we have a way of following the activities of the
protein!
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Example See the Microtubules!

• Basic Idea
• Attach a GFP to each of the ?-tubulin protein
  molecules (red balls below)

http//www.olympusfluoview.com/applications/gfpint
ro.html
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Modern Palette of Fluorescent Proteins

• The classic GFP
• 395nm excitation (ultraviolet)/509nm response
• Works at 28 degrees (too cold for mammalian
  cells)
• EGFP enhanced GFP
• Brighter, more convenient 484nm excitation
• Works at 37 degrees for mammalian use
• EYFP (yellow), ECFP(cyan), EBFP(blue), mOrange,
  DsRed,
• Many of these are derived from other creatures
  including reef corals, and anemones.
• They have all been modified to work in warmer
  mammalian cells, and to make them brighter, and
  easier to excite, etc.
• Rapidly growing field newer variants being
  produced every year!

SEE HANDOUT
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Live-Cell Imaging Example

• Epithelial cell from an opossum kidney.
• A mixture of fluorescent proteins variants were
  fused to peptide signals that mediate transport
  to either the
• Nucleus (enhanced cyan fluorescent protein
  ECFP),
• Mitochondria (DsRed fluorescent protein
  DsRed2FP), or
• The microtubule network (enhanced green
  fluorescent protein EGFP).

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Intra-Cellular Transport

• Vesicles are miniature taxicabs carrying cargo
  within the cells
• They slide over cytoskeletal fibers as they go
  from one place to another
• They have molecular equivalents of address
  labels so there is considerable specificity

Vesicle transport in a neuron
http//www.ohsu.edu/croet/faculty/banker/bankerlab
.html
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Summary

• Techniques for Imaging proteins
• Immunofluorescence (classical stuff)
• Exploit a specific class of proteins antibodies
• Amplification methods are commonly necessary
• Fluorescent Proteins (cool and contemporary
  stuff)
• Exploit the cells core mechanisms to produce them
• Abundant, replenished, specific
• Can be multiplexed to some extent
• Next Class
• Multi-photon imaging Yet another way to minimize
  photo damage
• How does a 3-D microscope work?

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Instructor Contact Information

• Badri Roysam
• Professor of Electrical, Computer, Systems
  Engineering
• Office JEC 7010
• Rensselaer Polytechnic Institute
• 110, 8th Street, Troy, New York 12180
• Phone [phone]
• Fax [phone]
• Email roysam_at_ecse.rpi.edu
• Website http//www.ecse.rpi.edu/roysam
• Course website http//www.ecse.rpi.edu/roysam/CT
  IA
• Secretary Laraine Michaelides, JEC 7012, (518)
  276 8525, michal_at_.rpi.edu
• Grader Ying Chen (cheny9_at_rpi.edu, Office JEC
  6308, 518-276-8207)

Center for Sub-Surface Imaging Sensing