preparation and labeling techniques for light microscopy · fixation • chemical fixation •...

Center for Microscopy and Image Analysis

University of Zurich

Preparation and labeling techniques for light

microscopy

Urs Ziegler

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


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 solution

fixation 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

C

H

H O


Formaldehyde - Solutions

commercially 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

CH2

OH

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

+CH 2

O

CH 2 CH 2

O H O H

OH 2

CH 2

CH 2CH 2

O

O

+CH 2

O

RSHCH 2

O H

RS

+

CH2

O

C

CH

O

CH2

NH2

C

CH

CH

CH

CH

CH

OH

CH2

C

CH

O

CH2

NH

C

CH

C

CH

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 - Synopsis

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, glutaraldehyed) 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 contrast

almost unlimited availability of colors

application to fixed and living systems

static 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 Microscopy Differential Interference Microscopy

Wollaston Prism

Wollaston Prism

Condenser

Objective

Phase Ring

Condenser

Objective with Phase Ring

Fluorescence Cube

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

FITC

FITC


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 samples

Gene expression

Reporter assays

Localisation studies

……

Fixation: Formaldehyd, Methanol, Ethanol, Aceton

Never: Glutaraldehyde


Fluorescent Proteins – GFP and Variants


Fluorescent Proteins – GFP and Variants

GFP

CFP


Fluorescent Proteins – GFP

GFP

Composed of 238 amino acids

Each monomer composed of a central -helix surrounded by an eleven

stranded cylinder of anti-parallel -sheets

Cylinder has a diameter of about 30Å and is about 40Å long

Fluorophore located on central helix inside cylinder

Fluorophore protected in very stable -can barrel structure

Autocatalytic formation of fluorophore


Fluorescent Proteins – DsRed

some fluorescent proteins tend to form

oligomers (DsRed!), size (GFP: 28 kDa)

Richard N. Day and Michael W. Davidson

Chem. Soc. Rev., 2009, 38, 2887-2921


Putting a shine on new fluorescent proteins

Chemistry & Biology 15, 1116–1124, 2008

Nature Methods 5 (5), 2008, 401

Nature Methods 5 (6), 2008, 545


Richard N. Day

and Michael W.

Davidson

Chem. Soc.

Rev., 2009, 38,

2887-2921


Rational to engineer new fluorescent proteins

Brighter

More photostable

No quenching in close proximity

FRET pairs

Monomeric forms

Blue variants

Understanding chromophore formation

High throughput screening

Rational design – no search for wild type forms


Optical highlighter FPs

in action


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


Transient and stable modification of mammalian cells with MultiLabel

Andrijana Kriz, Katharina Schmid, Nadia Baumgartner, Urs Ziegler, Imre Berger, Kurt Ballmer-Hofer & Philipp Berger, Nature

Communications, 2010


Fluorescent proteins - Synopsis

Reporter assays

Localization 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: 305

Shaner NC et al., Nat Methods. 2005; 2(12): 905


SNAP-tag and CLIP-tag system

SNAP-tag (gold) and CLIP-tag (purple) fused to protein of interest (blue) specifi

cally 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


Mini SOG system

Confocal fluorescence images of miniSOG-targeted endoplasmic reticulum (A), Rab5a (B), zyxin (C), tubulin

(D), β-actin (E), α-actinin (F), mitochondria (G), and histone 2B (H) in HeLa cells; scale bars, 10 µm.

Shu X, Lev-Ram V, Deerinck TJ, Qi Y, Ramko EB, et al. 2011 A Genetically Encoded Tag for Correlated Light

and Electron Microscopy of Intact Cells, Tissues, and Organisms. PLoS Biol 9(4): e1001041.

doi:10.1371/journal.pbio.1001041


Mini SOG system

MiniSOG produces correlated fluorescence and EM contrast with correct localization of

labeled proteins and organelles.


Mini SOG system

MiniSOG-tagged Cx43 forms gap junctions.


Preparation and labeling

Preparation Labeling

Cells Tissue

Living - Fixed

Genetically

encoded

probes

Dye based

probes

Microscopy

preparation and labeling techniques for light microscopy · fixation • chemical fixation •...

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