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ECSE6963, BMED 6961 Cell

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Title: ECSE6963, BMED 6961 Cell


1
ECSE-6963, BMED 6961Cell Tissue Image Analysis
  • Lecture 4 Bio-molecular Imaging - II
  • Badri Roysam
  • Rensselaer Polytechnic Institute, Troy, New York
    12180.

2
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
3
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
4
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

5
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
6
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
7
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
8
Multiple Secondary Antibodies
Fluorochrome
Secondary Antibody
Primary Antibody
Molecule of Interest (antigen)
  • Polyclonal secondary antibodies can attach to
    multiple epitopes on the primary antibody

9
Another way to Amplify Use Enzymes!
  • Enzymes are match maker proteins

10
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

11
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
12
Tyramide Signal Amplifcation Example Mouse Brain
Imaging
Molecular Probes Handbook
13
(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

14
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
15
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
16
Summary of Direct Indirect Labeling
www.chemicon.com
17
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

18
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
19
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
20
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
21
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

22
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!
23
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
24
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
25
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).

26
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
27
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?

28
Homework 2
  • Using internet or library reference tools (e.g.,
    wikipedia) as needed, answer the following
    questions
  • An optical microscope has a resolution of 0.2µm.
    If the pixel size on this instrument is equal to
    the resolution, what would be the diameter of a
    cell nucleus (in pixels) for pyramidal neurons
    in the rat brain? In other words, lookup the
    typical size of a cell nucleus, and express it in
    terms of pixels.
  • DNA transcription occurs at a speed of roughly 30
    base pairs per second. For a medium sized gene
    with 1,500 base pairs, it is possible for about
    15 transcription operations occurring
    concurrently. How may RNA transcripts can be
    produced per hour?
  • The average molecular weight of a protein is
    30,000 daltons, and the average molecular weight
    of an amino acid is 120 daltons. A few proteins
    can be much larger. For example a protein called
    titin is 3 million daltons. Estimate how long
    it would take to translate a titin molecule if
    the speed of translation is 2 amino acids per
    second.
  • Protein synthesis is accurate about one mistake
    is made for every 10,000 amino acids. What is the
    fraction of average-sized (1,500bp) and titin
    (3Mbp) molecules that are synthesized without any
    errors? Hint For n amino acids,

29
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 (518) 276-8067
  • Fax (518) 276-8715
  • 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
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