Transcript 7.1 Secondary fluorescence (fluorochromation) - Friedrich

7. Fluorescence microscopy
7.1 Fluorochromes
Fluorescence microscopy differentiates between two kinds of fluorochromes:
 Primary fluorescence (autofluorescence)
 Secondary fluorescence (fluorochromation)
 Fluorescence dyes
 Immunofluorescence (using Antibodies)
 Molecular tags (SNAP Tag, ...)
 Fluorescent Proteins
Applications of fluorochromes
 Identification of otherwise visible structures
 Localization and identification of otherwise invisible structures
 Monitoring of physiological processes
 Specific detection of a protein
 Using Photophysical properties of dyes (e.g. switching) for superresolution
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7. Fluorescence microscopy
7.1 Primary fluorescence (autofluorescence)
 Most samples fluoresce when excited with short-wave light
 Fluorescence very often occurs for systems containing many conjugated double
bonds:
 e.g. chlorophyll exhibits dark red fluorescence
when excited by blue or red light
N
H
N
N
H
N
Porphyrin ring –
central unit in Chlorophyll
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Moss reeds – green excitation
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7. Fluorescence microscopy
http://en.wikipedia.org/wiki/File:Chlorophyll_ab_spectra2.PNG
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7. Fluorescence microscopy
7.1 Primary fluorescence (autofluorescence)
 Further examples:
 Riboflavine (550nm)
 NAD(P)H (460nm, 400ps)
 Elastin und Collagen (305-450nm)
 Retinol (500nm)
 Cuticula (blue)
 Lignin (> 590nm)
Eucalyptus leaf section – UV excitation
 DNA (Ex @320nm, 390nm)
 Aminoacids:
 Tryptophane (348nm, 2.6ns)
 Tyrosin (303nm, 3.6ns, weak)
 Phenylalanine (282nm weak)
 Resins, Oils
http://en.wikipedia.org/wiki/Autofluorescence
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Nematode living sample – UV excitation
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7. Fluorescence microscopy
7.1 Secondary fluorescence (fluorochromation)
 Staining (labeling) specific structures with fluorescent labels (dyes):
fluorochromation
 Small dye concentrations are sufficient due to high fluorescence contrast
 fluorescence labels are superior than bright field dyes
 Single molecule sensitivity
 Fluorescence labels must selectively bind to structures
or selectively accumulate in specific compartments
 e.g. DAPI (= 4',6-diamidino-2-phenylindole) to label DNA (cell nuclei)
DAPI:
lexc = 358 nm
lem = 461 nm
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some dyes unquench upon binding
Fluorescence image of
Endothelium cells.
Microtubili are labeld in
green,
while actin filaments are
labeled red.
DNA within cell nuclei
are stained with DAPI.
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7. Fluorescence microscopy
7.1 Secondary fluorescence (fluorochromation): Immunofluorescence
 Immunofluorescence (antibody staining) is the labeling of specific proteins with an
antibody which is visualized by a dye
 Antibodies are proteins which are used by the immune system to identify and
neutralize foreign substances (antigens)
 Antibodies are made of two large heavy chains
(~440 amino acids) and two small light chains
(~220 amino acids)
 C-region is similar for all antibodies, while the V-region
is extremely variable and forms the specific binding site
for the antigen i.e. every antibody can recognize and
bind two antigens
 Specificity: antibody-antigen reaction.
The part of the antigen (protein) recognized by an antibody is called an epitope.
Highly specific interaction, called induced fit, allows antibodies to identify and bind
only their unique antigen in the midst of the millions of different molecules that
make up an organism
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7. Fluorescence microscopy
7.1 Secondary fluorescence (fluorochromation): Immunofluorescence
 Different groups of antibodies exist:
 Polyclonal antibodies
They are a mixture of antibodies secreted against a specific antigen, each
recognizing a different epitope i.e. bind to different areas of the protein.
The protein (e.g. tubulin) for which a special antibody should be generated
is injected into a suitable mammal (mostly rats, mice or goats).
Antibodies against the protein are produced by the mammalian immune
response and can be isolated from the blood serum
 Monoclonal antibodies
are all identical and bind to the same epitope
 Synthetic antibodies
are monoclonal antibodies which are produced in-vitro i.e. via
microorganism
 Other systems
scFv (M. Bruchez): single chain variable region anitbodies
nanobodies (H. Leohardt): small (from Camelidae) and not degrated
quickly inside a cell
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7. Fluorescence microscopy
7.1 Secondary fluorescence (fluorochromation): Immunofluorescence
Direct immunofluorescence
For the direct or primary labeling the specific antibody for
the investigated protein is labeled with the fluorochrome
The labeled antibodies are brought onto the sample and
only bind specifically to the wanted protein (antigene =
ligand); non bound antibodies are washed out
Detection of the bound antibodies via the attached
fluorochrome  Localization of the wanted protein
An interphase female human fibroblast cell.
Arrow points to the corresponding X
chromosome (right).
Labeling of a DNA-associated histone protein
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7. Fluorescence microscopy
7.1 Secondary fluorescence (fluorochromation): Immunofluorescence
Indirect immunofluorescence
 Two sets of antibodies;
Primary antibody detects antigen
A subsequent, secondary (indirect), dye-coupled antibody
recognizes the primary antibody.
 Signal amplification
(several secondaries bind to one primary)
 Color palette separating staining from target
 Example:
 1. Antibody / Rat – Anti Tubulin
Antibody against tubulin generated in a rat
 2. Antibody / Goat – Anti Rat
fluorescently labeled antibody against all rat antibodies
(generated in a goat)
 Negative test: Primary antibody is left out in order to test if the fluorescence
labeled secondary antibody binds unspecifically to the sample
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IPC Friedrich-Schiller-Universität Jena
7. Fluorescence microscopy
7.1 Secondary fluorescence (fluorochromation): Immunofluorescence
Indirect immunofluorescence
GPCR transfected HEK cells:
Double staining:
For an identification of single cells
the dye Hoechst 33342 was employed
(cell nuclei: blue);
Cell bound primary mouse anti-GPCR antibodies were
detected by secondary goat anti-mouse Ig(H+L) antibodies
labeled with Alexa Fluor 488
(GPCR-protein: green).
 The fluorescence labeled secondary antibodies can be employed for all
antibodies produced within one animal e.g. goat serum against rats reacts with
all primary antibodies produced in rats
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7. Fluorescence microscopy
7.1 Secondary fluorescence (fluorochromation)
Dye artefacts
 Bleaching:
Fluorescence dye is destroyed by irradiation with light
 Quenching:
Fluorescence can be quenched (reduced)
for large dye concentrations
 Cross-Talk:
Cross-Excitation:
Simultaneous excitation of two dyes if their excitation
wavelengths are too close to each other
Bleed through:
In case the emission spectra overlap too much
both dyes will be detected but to different amounts
Can be compensated by calibration and an inverse matrix technique
"Spectral unmixing"
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