preparation and labeling techniques for light microscopy · • formaldehyde • glutaraldehyde •...

Center for Microscopy and Image Analysis University of Zurich

Preparation

and labeling

techniques

for

light microscopy

Urs Ziegler


Preparation

and labeling

Preparation Labeling

Cells Tissue

Living - Fixed

Genetically encoded probes

Dye based probes

Microscopy


Example

DNA

Bax

Mitochondria

DNA

Mitochondria

Cytochrome

C

DNA

Bax

Mitochondria

Cytochrome

C

Cell death

investigation

in Hela

cells: mitochondrial

damage


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


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


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


Formaldehyde

First use in 1893 by Blum who noticed hardening of his fingers!MW: 30

CH

HO


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


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


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


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)


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


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


Generation of fluorescence


Common Fluorochromes

-

FITC


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


Fluorescence

filters


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


Fluorescent

dyes: examples Ion sensitive dyes

• Fura-2: popular Ca2+ sensitive dye • Measurement: ratio imaging excitation 340 / 380 nm


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.:


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)


Fluorescent

Proteins –

GFP and Variants


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.


Fluorescent

Proteins –

GFP and Variants

GFP

CFP


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


Putting a shine on new fluorescent proteins

Chemistry & Biology 15, 1116–1124, 2008Nature Methods 5 (5), 2008, 401Nature Methods 5 (6), 2008, 545


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


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


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


Fluorescent

proteins

-

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


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.


FRAP –

Fluorescence

recovery

after

photobleaching

Beta adrenergic receptor expressing the SNAP tag was labeled with a cell impermeant Alexa 488 dye


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


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


Preparation

and labeling

Preparation Labeling

Cells Tissue

Living - Fixed

Genetically encoded probes

Dye based probes

Microscopy


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

preparation and labeling techniques for light microscopy · • formaldehyde • glutaraldehyde •...

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