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Fluorescence
microscopy
Fluorescence methods in life scienses
Ctirad Hofr
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Specimen labeling direct fluorescence and immunofluorescence
Direct fluorescence - fluorophore binds directly
biomolecule of our interests (BOI), typically DNA
(DAPI, ethidium bromide, Hoechst).
Immunofluorescence is used for visualization of
most molecules and structures in cell.
Direct immunofluorescence uses antibody
raised against BOI. The antibody is labelled
fluorescently by a fluorophore.
Indirect immunofluorescence uses two
antibodies. We raise a primary antibody against
BOI in rabbit or other species. The primary
antibody is allowed to bind in our specimen to
BOI. After rinsing and washing out the excess of
the primary antibody, the fluorescently labeled
secondary antibody(raised in e.g. mouse against
rabbit antibodies) is allowed to bind to primary
antibody. Secondary antibody label primary
antibody and thus BOI. Naturally, indirect
immunofluoresce shows lower specificity then
direct methods of labeling. Biotin-avidin
immunofluorescence employs the strong affinity
between biotin and basic glycoprotein avidin.
Primary antibody is biotinylated. Fluorophore
linked with avidin labels primary antibody.
https://www.thermofisher.com/cz/en/home/life-science/cell-analysis/cell-analysis-
learning-center/molecular-probes-school-of-fluorescence/labeling-your-
samples/immunolabeling.html
Direct fluorescence
A
B
Biotin-avidin
immunofluorescence
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Fluorescence microscope
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Possible arrangements
Transmitted light microscopyEpi fluorescence microscopy
95% of all fluorescence microscopes
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Epi-fluorescence microscopy
(Reflected light fluorescence microscope)
•excitační světlo prochází objektivem, dopadá na preparát a emisní světlo se vrací
zpět do objektivu
•nutno použít zvláštní typ zrcadla, které odráží excitační světlo do objektivu a
propouští emisní světlo do okuláru
•používá se dichroické zrcadlo, které propouští a odráží světlo podle toho, jakou má
vlnovou délku. Používá se tedy vždy takový typ zrcadla, který maximum excitačního
světla odráží a maximum emisního světa propouští.
•epifluorescenční typ mikroskopu je v současnosti více oblíbený než transmisní typ
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Excitation and barrier (emission) filter
We use two filters to observe fluorescence emission whose intensity is
100-fold lower than the intensity of excitation light. Without filters our
eyes would not be able to see the florescence.
Excitation filter transmits only light wavelengths that are best for
excitation of our fluorophore. Additionally, EF blocks other light
wavelengths that could cause intrinsic fluorescence of specimen and
increase background fluorescence.
Barrier (emission) filter transmits only light wavelengths of fluorescence
emission coming from molecule of our interest. BF blocks excitation light
that might be reflected to the detectors.
http://micro.magnet.fsu.edu/primer/java/fluorescence/opticalpaths/index.html
Microscope anatomy
http://zeiss-campus.magnet.fsu.edu/tutorials/axioobserver/index.html
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Mysterious dichroic mirror
• Dichroic mirror reflect light with lower wavelengths but
transmit longer wavelengths. Thus, dichroic mirror reflects
maximum of excitation light let go through maximum of
emission light used for a particular fluorophore.
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3 filters make a filter cube
A proper combination of dichroic mirror, excitation and
emission filter for a given fluorophore makes a filter block
called as a filter cube. Two sides are filters and diagonal
is dichroic mirror. Cubes are in a carousel and are
exchanged by carousel rotation.
http://micro.magnet.fsu.edu/primer/java/lightpaths/fluorescence/index.html
Selection of a proper filter set
(cube) for your fluorophore
http://www.microscopyu.com/tutorials/flash/spectra
lprofiles/index.html12
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http://www.olympusmicro.com/primer/java/fluores
cence/matchingfilters/index.html
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Transmitted light fluorescence microscope
Excitation light goes
through excitation filter
and comes from below as
in case of a classical
wide-field microscope.
The most important part
here is darkfield
condenser that adjusts
excitation light path so
the light illuminate
specimen from sides.
Thus, only fluorescence
emission goes in
objective and is observed
through objective.
Excitation light is
reflected so does not
enter objective.
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Confocal microscopy
• High signal:noise ratio
• Light source is laser,
• Two pinholes in light path. Light goes
through the source pinhole. The beam of
light is focused into a single point.
• On the light path from specimen to
detector, there is the detector pinhole.
• Only light from focus plane is detected =>
sharp image
• One point in time is detected => scanning
microscopy
http://www.olympusconfocal.com/java/confocalsimulator/index.html
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Virtual microscopy
http://micro.magnet.fsu.edu/primer/virtual/fluorescence/index.html
http://www.microscopyu.com/tutorials/java/kohler/index.html
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Practical demonstration of
fluorescence microscopy