preparation and labeling techniques for light microscopy · • formaldehyde • glutaraldehyde •...
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Center for Microscopy and Image Analysis University of Zurich
Preparation
and labeling
techniques
for
light microscopy
Urs Ziegler
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Center for Microscopy and Image Analysis University of Zurich
Preparation
and labeling
Preparation Labeling
Cells Tissue
Living - Fixed
Genetically encoded probes
Dye based probes
Microscopy
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Center for Microscopy and Image Analysis University of Zurich
Example
DNA
Bax
Mitochondria
DNA
Mitochondria
Cytochrome
C
DNA
Bax
Mitochondria
Cytochrome
C
Cell death
investigation
in Hela
cells: mitochondrial
damage
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Center for Microscopy and Image Analysis University of Zurich
Why Preparation
• Tissues / organisms observed under microscopes with transillumination are too thick
→ fixation of samples → preparation of thin slices→ embedding of samples
• Thin sections / isolated cells are colorless
→ staining of samples→ microscopy with suitable
contrast generation
• Identification of tissue / cells / components
→ staining of samples
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Center for Microscopy and Image Analysis University of Zurich
Fixation
reducing solubility of components in solutionfixation of proteins, carbohydrates, lipids
Ultimate aim:1.
preserve cell and tissue organization as near as possible to the
native organization
2.
protect the tissue against all later stages of preparation with minimal deterioration
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Center for Microscopy and Image Analysis University of Zurich
Fixation
• chemical fixation• formaldehyde• glutaraldehyde• alcohols (miscellaneous)• osmiumtetroxide• salts (miscellaneous)
• physical fixation• freezing• drying
Parameters leading
to stronger
fixation:
Longer
incubation
times
Higher
concentration
Glutaraldeyde
> formaldehyde
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Center for Microscopy and Image Analysis University of Zurich
Formaldehyde
First use in 1893 by Blum who noticed hardening of his fingers!MW: 30
CH
HO
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Center for Microscopy and Image Analysis University of Zurich
Formaldehyde -
Solutionscommercially available:
37 % formaldehyde solution (wt/wt) plus ≈
10 % methanol (stabilizer): formalin
35 % formaldehyde solution without methanol (> 1 %): tends to form polymers especially at 4°C
solid polymer termed paraformaldehyde = polyoxymethylen glycols containing 8 to 100 formaldehyde units per molecule
→ dissoves by adding water at 60°C and drops of 1 M NaOH until solution clears
C
O
HH CH3
O
CH2
O
CH2
O
CH
O
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Center for Microscopy and Image Analysis University of Zurich
Formaldehyde -
Reactions
+ OH2
R
NH
H
CH2OH
OH
CH2
O
CH2
OH
OH
+OH2
R
NH CH2 OH
R
NH CH2 OH +R
NH CH2 OHOH2
R
NH CH2 N
R
CH2 OH
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Center for Microscopy and Image Analysis University of Zurich
Formaldehyde -
Reactions
+CH2
O
CH2 CH2OH OH
OH2
CH2
CH2CH2
O
O
+CH2
ORSH CH2
OH
RS
+CH2
O
CCH
O
CH2
NH2
CCH
CHCH
CH
CH
OH
CH2
CCH
O
CH2
NH
CCH
CCH
CH
CH
OHOH2
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Center for Microscopy and Image Analysis University of Zurich
Alcohols
and Aceton
Fixation by
dehydration: shell
of water
around
proteins
is
removed
– precipitation
of proteins
Advantages: Quick –
relatively
good antigen
preservation
(in many
cases)
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Center for Microscopy and Image Analysis University of Zurich
Fixation -
Summary
Fixation aims
to keep
the
structure
and organization
as close
to the native state
as possible
In reality
structural
and organization
changes
occur
not
only
below
the detection
level!
Chemical fixation
(formaldehyde, glutaraldehye) lead
to better
structural preservation
than
alcohol
fixation
Perfusion
fixation
leads
to better
tissue
fixation
than
immersion
fixation
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Center for Microscopy and Image Analysis University of Zurich
Staining
Fluorescent
dyes
are
by
far the
most
versatile
tool
fluorescence
has a very
high contrastalmost
unlimited
availability
of colors
application
to fixed
and living
systemsstatic
or
dynamic
some
dyes
can
be
switched
on / off
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Center for Microscopy and Image Analysis University of Zurich
Generation of fluorescence
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Center for Microscopy and Image Analysis University of Zurich
Common Fluorochromes
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FITC
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Center for Microscopy and Image Analysis University of Zurich
Light Path and Optical Elements in Different Microscopic Techniques
Bright Field Microscopy Phase Contrast Microscopy Fluorescence MicroscopyDifferential InterferenceMicroscopy
Wollaston Prism
Wollaston Prism
Condenser
Objective
Phase Ring
Condenser
ObjectivewithPhase Ring
FluorescenceCube
Objective
Condenser
Objective
Polarizer
Polarizer
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Center for Microscopy and Image Analysis University of Zurich
Fluorescence
filters
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Center for Microscopy and Image Analysis University of Zurich
Fluorescence
filters
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Center for Microscopy and Image Analysis University of Zurich
FITC
Direct Immunofluorescence
FITC
Indirect immunofluorescence
Antigen
Antigen
Number of dye molecules / antibody
Quenching if too many dye molecules / too dim if not enough
Labeling
with
antibodies
FITCFITC
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Center for Microscopy and Image Analysis University of Zurich
Fluorescent
dyes: examples Ion sensitive dyes
• Fura-2: popular Ca2+ sensitive dye • Measurement: ratio imaging excitation 340 / 380 nm
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Center for Microscopy and Image Analysis University of Zurich
Fluorescent
dyes: examples
Dyes
with
preferential
uptake
into
selective
cellular
compartments
Mitochondria: selective
dyes
that
stains mitochondria
in live cells
and its
accumulation
is
dependent
upon membrane
potential. Some
dyes
are
well-retained
after
aldehyde
fixation (e. g.: Mitotracker
(several
colors))
Lysosomes: Weakly basic amines selectively accumulate in cellular compartments with low internal pH and can be used to investigate the biosynthesis and pathogenesis of lysosomes.
(e. g.:
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Center for Microscopy and Image Analysis University of Zurich
Fluorescent
Proteins
Living
and fixed
samplesGene expressionReporter assaysLocalisation
studies
……
Fixation: Formaldehyd, Methanol, Ethanol, Aceton
Never: Glutaraldehyde
Disadvantage: some
fluorescent
proteins
tend
to form oligomers (DsRed!), size
(GFP: 28 kDa)
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Center for Microscopy and Image Analysis University of Zurich
Fluorescent
Proteins –
GFP and Variants
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Center for Microscopy and Image Analysis University of Zurich
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Center for Microscopy and Image Analysis University of Zurich
A plasmid-based multigene expression system for mammalian cells.Kriz A, Schmid K, Baumgartner N, Ziegler U, Berger I, Ballmer-Hofer K, Berger P.Nat Commun. 2010 Nov;1(8):120.
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Center for Microscopy and Image Analysis University of Zurich
Fluorescent
Proteins –
GFP and Variants
GFP
CFP
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Center for Microscopy and Image Analysis University of Zurich
Fluorescent
Proteins –
GFP
GFP
Composed of 238 amino acidsEach monomer composed of a central -helix surrounded by an eleven
stranded cylinder of anti-parallel -sheetsCylinder has a diameter of about 30Å
and is about 40Å
longFluorophore
located on central helix inside cylinderFluorophore
protected in very stable -can barrel structure Autocatalytic formation of fluorophore
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Center for Microscopy and Image Analysis University of Zurich
Putting a shine on new fluorescent proteins
Chemistry & Biology 15, 1116–1124, 2008Nature Methods 5 (5), 2008, 401Nature Methods 5 (6), 2008, 545
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Center for Microscopy and Image Analysis University of Zurich
Rational to engineer new fluorescent proteins
BrighterMore photostableNo quenching in close proximityFRET pairsMonomeric
forms
Blue variants
Understanding chromophore
formationHigh throughput screeningRational design –
no search for wild type forms
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Center for Microscopy and Image Analysis University of Zurich
Stability
of new
fluorescent
proteins: TagBFP
Chemistry & Biology 15, 1116–1124, 2008Nature Methods 5 (5), 2008, 401Nature Methods 5 (6), 2008, 545
http://www.sciencedirect.com/science?_ob=MiamiCaptionURL&_method=retrieve&_udi=B6VRP-4TPFCS2-G&_image=B6VRP-4TPFCS2-G-7&_ba=&_user=5294990&_coverDate=10%2F20%2F2008&_rdoc=15&_fmt=full&_orig=browse&_srch=doc-info%28%23toc%236240%232008%23999849989%23699838%23FLA%23display%23Volume%29&_cdi=6240&_isHiQual=Y&_acct=C000049009&_version=1&_urlVersion=0&_userid=5294990&md5=10a60c8ac5ab072e40043e6faa46c6a6http://www.sciencedirect.com/science?_ob=MiamiCaptionURL&_method=retrieve&_udi=B6VRP-4TPFCS2-G&_image=B6VRP-4TPFCS2-G-7&_ba=&_user=5294990&_coverDate=10%2F20%2F2008&_rdoc=15&_fmt=full&_orig=browse&_srch=doc-info%28%23toc%236240%232008%23999849989%23699838%23FLA%23display%23Volume%29&_cdi=6240&_isHiQual=Y&_acct=C000049009&_version=1&_urlVersion=0&_userid=5294990&md5=10a60c8ac5ab072e40043e6faa46c6a6
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Center for Microscopy and Image Analysis University of Zurich
fusion proteins consisting of a tandem of either mTagBFP and mTagGFP or EBFP2 and mTagGFP, each containing the caspase-3 cleavage sequence, DEVD, within the linker between fluorescent proteins
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Center for Microscopy and Image Analysis University of Zurich
Fluorescent
proteins
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Summary
Reporter assaysLocalization
and kinetic
behaviour
Used
as sensors
(camgaroos, pericams)
Literature:Zhang J et al., Nat Rev
Mol Cell Biol. 2002,; 3(12): 906
Ward TH et al., Methods
Biochem
Anal. 2006; 47: 305Shaner
NC et al., Nat Methods. 2005; 2(12): 905
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Center for Microscopy and Image Analysis University of Zurich
SNAP-tag
and CLIP-tag
system (New England Biolabs)
SNAP-tag (gold) and CLIP-tag (purple) fused to protein of interest (blue) specifically recognize their substrates based on benzylguanine (BG) or benzylcytosine (CT) and self-label with label X (green
1. Gautier A.,et al. 2008. „An engineered protein tag for multiprotein labeling in living cells“. Chem Biol., 15(2), 128-136.
2. Rubinfeld H.et al. 1999. „Identification of a cytoplasmic-retention sequence in ERK2“. J. Biol. Chem., 274, 30349-30352.
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Center for Microscopy and Image Analysis University of Zurich
FRAP –
Fluorescence
recovery
after
photobleaching
Beta adrenergic receptor expressing the SNAP tag was labeled with a cell impermeant Alexa 488 dye
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Center for Microscopy and Image Analysis University of Zurich
Biarsenical-tetracysteine
system: FlAsH
and ReAsH (Invitrogen)
Principle of FLAsH system
Machleidt et al.; Methods in Molecular Biology, 356, Chapter 15Science 10 July 1998:Vol. 281 no. 5374 pp. 269-272
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Center for Microscopy and Image Analysis University of Zurich
ReAsH
can photoconvert DAB to produce an electron dense reaction product allowing for tetracysteine-tagged connexins to be imaged in the same cells by both fluorescent and electron
microscopy.
G Gaietta et al. Science 2002;296:503-507
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Center for Microscopy and Image Analysis University of Zurich
Preparation
and labeling
Preparation Labeling
Cells Tissue
Living - Fixed
Genetically encoded probes
Dye based probes
Microscopy
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Center for Microscopy and Image Analysis University of Zurich
Roger Y. Tsien 2010. Fluorescence readouts of biochemistry in live cells and organisms. In Molecular Imaging: Principles and Practice, ed. by R. Weissleder, S.S. Gambhir, B.D. Ross, A. Rehemtulla. People’s Medical Publishing House – USA. Chapter 48, pp. 808-828
Preparation and labeling techniques for light microscopyPreparation and labelingExampleWhy PreparationFixationFixationFormaldehydeFormaldehyde - SolutionsFormaldehyde - ReactionsFormaldehyde - ReactionsAlcohols and AcetonFixation - SummaryStainingGeneration of fluorescenceCommon Fluorochromes - FITCSlide Number 16Fluorescence filtersFluorescence filtersLabeling with antibodiesFluorescent dyes: examples�Ion sensitive dyesFluorescent dyes: examples��Dyes with preferential uptake into selective cellular compartmentsFluorescent ProteinsFluorescent Proteins – GFP and VariantsSlide Number 24Slide Number 25Fluorescent Proteins – GFP and VariantsFluorescent Proteins – GFPPutting a shine on new fluorescent proteinsRational to engineer new fluorescent proteinsStability of new fluorescent proteins: TagBFPSlide Number 31Fluorescent proteins - SummarySNAP-tag and CLIP-tag system�(New England Biolabs)FRAP – Fluorescence recovery after photobleachingBiarsenical-tetracysteine system: FlAsH and ReAsH�(Invitrogen)�Slide Number 36Preparation and labelingSlide Number 38
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