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Correlative Light and Electron Microscopy using Immunolabelled Ultrathin Sections

Heinz Schwarz

Max-Planck-Institut für Entwicklungsbiologie, Spemannstr. 35, D-72076 Tübingen, Germany

e-mail: [email protected]

Specific localization of moleculeswithin a complex biological structure

Actually the best approach in light microscopy is direct visualization with reporter molecules like GFP.

To detect GFP on EM level either photooxidation or antibodies against GFP are used

So far for correlative light and electron microscopy onlyfew reports exist on tags expressed in living cellsusing fluorophores which can photooxidize DAB:

ReAsh and GFP

ReAsH, GFP and GFP-tetracysteine

ReAsH is a membrane-permeant nonflourescent biarsenical derivative of the red fluorophore resorufin which becomes strongly fluorescent upon binding to tetracysteine motifs in recombinant proteins expressed in living cells. Gaietta, G. et al. (2002) Multicolor and electron microscopic imaging of connexin trafficking. Science 296, 504-507

GFP: GFP recognition after bleaching (GRAB)Grabenbauer, M. et al. (2005) Correlative microscopy and tomography of GFP through photooxidation. Nature Meth. 2, 857-862.

GFP-4C: Live observation of mannosidase II-GFP-4C and labelling with ReAsH for subsequent photoconversion for EMGaietta, G. et al. (2006) Golgi twins in late mitosis by genetically encoded tags forlive cell imaging and correlated electron microscopy. PNAS 103, 17777-17782.

ReAsH, a photooxidizable biarsenical fluorophore to image tetracysteine-tagged connexins in gap junctions

Gaietta, G. et al. (2002) Multicolor and electron microscopic imaging of connexin trafficking. Science 296, 504-507. Fig. 4.

Detection of a GFP tagged Golgi resident glycosylation enzyme, N-acetylgalactosaminyltransferase-2 (GalNAc-T2)

Grabenbauer, M. et al. (2005) Correlative microscopy and tomography of GFP through photooxidation. Nature Meth. 2, 857-862.2005. Fig. 2

0 min

2 min

4 min

8 min

25 µm

2 µm

0.5 µm10 µm

25 µm

However, still in most studies localizing molecules on cellular level affinity labelling with antibodies and

lectins is used and performed either with permeabilized samples or on

sections of embedded material

Hereby, of course, probes have to be visualized indirectly using coupled marker molecules e.g.

enzymes, fluorochromes, gold particles

Affinity localization of intracellular structures:Labelling of permeabilized samples and thick sections

(Pre-embedding techniques)

Identical labelling conditions for electron microscopy as for confocal light microscopy

3D access to remaining epitopes

Drawbacks of pre-embedding techniquesPermeabilization destroys some fine structure

Loss of soluble material is an intrinsic property

Penetration problems for antibodies and markersas extraction may be uneven as well as accessibility

Affinity localization of intracellular structures:Labelling of ultrathin sections (<0.3 µm)

(Post-embedding / On-section techniques)

Thawed cryosections of chemically fixed and sucrose-infiltrated samples according to Tokuyasu

Ultrathin resin sections of chemically fixed or cryofixed samples embedded in methacrylates or epoxy resins

General drawback of post-embedding techniques:

Only a low number of antigen copies is accessible on the section surface

Benefits and drawbacks of post-embedding labelling using thawed cryosections:

Antigens are always in an aqueous medium prior to labelling

Prefixation is the only potential denaturation step

Bad retention of soluble proteins and small molecules

using ultrathin resin sections:

Good structural preservation particularly in combinationwith cryoimmobilization and freeze-substitution

Resin monomers are potential skin irritants and sensitizers

Fixation, dehydration and embedding in resin may destroythe antigenicity

Visibility and sensitivity of different protein A-gold sizes

OmpA in E. coli wild type cells (Lowicryl K4M sections)

Label density vs preparation: OmpA labelling

Fixation Dehydration Resin Gold/µm

FA/GA prefixed PLT K4M 10.60

FA/GA prefixed FS methanol K4M 15.49

Cryofixed FS FA/GA methanol K4M 19.75

Cryofixed FS UA methanol K4M 22.95

Cryofixed FS Methanol K4M 24.52

Cryofixed FS Methanol HM20 23.37

Schwarz, H. and Humbel, B.M. (1989) Influence of fixatives and embedding media on immunolabelling of freeze-substituted cells. In Science of Biological Specimen Preparation 1988

(Albrecht, R.M. and Ornberg, R.L., eds.) Scanning Microscopy Supplement 3, 35-46.

Relocation of OmpA during sectioning in cryofixed E. colifreeze-substituted in ethanol omitting any crosslinking fixatives

Lowicryl HM20 section labelled for OmpA

Relocation of OmpA during sectioning in cryofixed E. colifreeze-substituted in ethanol omitting any crosslinking fixatives

Cross section of a Lowicryl HM20 section labelled for OmpA

Visibility and sensitivity of different protein A-gold sizes

OmpA in E. coli wild type cells (Lowicryl K4M sections)

Outer membrane protein OmpA in E. coli

Immunofluorescence of an ultrathin Lowicryl K4M section labelled for OmpA using a 100x oil immersion objective

Examples for immunolabelling of ultrathin resin sections of the very same specimen block for light and electron microscopy

α−tubulin in trypanosomesβ-catenin in intestine and heart muscleRecent examples for correlative microscopyProlactin in zebrafish anterior pituitary gland

Chitin in Drosophila embryosα-tubulin, γ-COP in Arabidopsis pollenα-tubulin in Drosophila embryos and nematodes

Post-embedding labelling offers a good combinationof both, structural integrity and reliable signalfor correlative light and electron microscopy

v

500-1000 nm Section 50-100 nm Section 50-100 nm SectionCoverslip Coverslip EM Grid

Toluidine Blue Primary Antibody Primary Antibody

Fluorescent Marker Gold MarkerDAPI / PI

Epon Mowiol Uranyl AcetateLead Citrate

Bright-Field Fluorescence Electron Microscopy

Orientation and Localization and High-ResolutionDifferentiation Overview Localization

Strategy

Microtubules in Trypanosoma brucei

High-pressure frozen, freeze-substituted in osmium/acetone, embedded in EponMicrographs provided by Christoph Grünfelder

Tubulin in Trypanosoma brucei: Immunofluorescence

On-section labelling of α-tubulin in a Lowicryl HM20-embedded sample

Tubulin in Trypanosoma brucei: Immunogold

On-section labelling of α-tubulin in a Lowicryl HM20-embedded sample

The use of 500 nm thick resin sections in light microscopy

Toluidine blue stained Lowicryl K4M section of rat intestine

16x oil immersion objective 100x oil immersion objective

Correlative immunolabelling on methacrylate sections: β−catenin in epithelial cells of rat intestine

β−catenin in rat intestine (low mag)

PhD630

β−catenin in rat intestine (high mag)

PhD629

Correlative immunolabelling on methacrylate sections: β−catenin and F-actin in epithelial cells of rat intestine

β−catenin Cy3propidium iodide F-actin FITC Double exposure β−catenin 15 nm gold

Correlative immunolabelling on methacrylate sections: β−catenin and F-actin in guinea pig heart muscle

β−catenin: Cy3 (yellow) DNA: DAPI (blue)

Kurth, T., Schwarz, H., Schneider, S., and Hausen, P. (1996) Fine structure immunocyto-chemistry of catenins in amphibian and mammalian muscle. Cell Tissue Res. 286, 1-12.

Localization of β−catenin in the Z-line of heart muscle:the signal is associated with a structural correlative

Kurth, T., Schwarz, H., Schneider, S., and Hausen, P. (1996) Fine structure immunocytochemstry of catenins in amphibian and mammalian muscle. Cell Tissue Res. 286, 1-12.

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M

Appearence of the skin of plakoglobin null-mutant mice

Bierkamp, C., McLaughlin, K.J., Schwarz, H., Huber, O., and Kemler, R. (1996) Embryonic heart and skin defects in mice lacking plakoglobin. Dev. Biol. 180, 780-785.

Appearence of desmosomes in the skin of plakoglobin null-mutant mice

Wild type (plakoglobin +/+) Null-mutant (plakoglobin -/-)

Bierkamp, C., Schwarz, H., Huber, O., and Kemler, R. (1999) Desmosomal localization of β -catenin in the skin of plakoglobin null-mutant mice. Development 126, 371-381.

β-catenin in the skin of plakoglobin null-mutant mice


Wild type

Plakoglobin null-mutant

β-catenin in the intestine of plakoglobin null-mutant mice


Wild type Plakoglobin null-mutant

Prolactin labelling in the pituitary of zebrafish

Nica, G., Herzog, W., Sonntag, C., Nowak, M., Schwarz, H., Zapata, A.G., and Hammerschmidt, M. (2006) Eya1 is required for lineage-specific differentiation, but not for cell survival in the zebrafish adenohypophysis. Developmental Biology 292, 189-204

Study with 7 day old fishes which were fixed with 4% FA, dehydrated in ethanol at progressively lower temperature

(PLT method) and embedded in Lowicryl K11M

Prolactin labelling in the pituitary of zebrafish wt 7 dpf

Schwarz, H. and Humbel B.M. (2007) Correlative light and electron microscopy using immunolabeled resin sections. In: Electron Microscopy: Methods and Protocols (Kuo, J.,

ed.) Methods Molecular Biology 369, 229-256. Humana Press, Totowa NJ, USA

Wild type

Toluidine blue stained 0.5 µm section Immunolabelled 50 nm sectionfor Prolactin (orange)

DAPI (blue)

10 µm




10 µm




5 µm




5 µm0.5 µm

1638

Spotting the region of interest on EM level

Search a 2 £ coin on a soccer field 90 x 45 m



Cuticle differentiation during Drosophilaembryogenesis

Moussian, B., Seifarth, C., Müller, U., Berger, J. and Schwarz, H. (2006) Cuticle dif ferentiat ion during Drosophila embryogenesis. Arthropod Structure & Development 35, 137-152

Study using high-pressure frozen fly embryos which were freeze-substituted in 2% OsO4, 0.5% UA, 0.5% GA in

acetone (containing 2.5% methanol) and embedded in Epon

Chitin labelling using wheat germ agglutinin (WGA)

Wild type Chitin synthase mutant CS-1/kkv (krotzkopf verkehrt)

CC

Mutant

Wild typeWGA -10 nm gold

Wild type

Mutant

MPL -10 nm gold

Labelling of α-tubulin and γ-COP in Arabidopsis pollenα-tubulin in Drosophila embryos and nematodes

Ripper, D., Schwarz, H., and Stierhof, Y.-D. (2008) Cryo-section immunolabelling of problematic specimens: Advantages of cryofixation, freeze-substitution and rehydration. Biol. Cell 10, 109-123.

Specimen were cryofixed by high-pressure freezing and in most casesfreeze-substituted in 0.1% OsO4, 0.2% UA, 0.5% GA in acetone. Samples were then washed at –35°C and rehydrated at 0°C in the presence of 0.5% and 0.25% GA.

For cryosectioning according to Tokuyasu rehydrated samples were infiltrated with sucrose/PVP and frozen in liquid nitrogen.All gold labelling was done with Nanogold or ultrasmall gold followed by silver enhancement

N

NN

N

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*

*

*

* *

Generative cells

*10 µm

α-tubulin in Arabidopsis pollenLabelling of 300 nm cryosections after

Chemical fixation HPF – FS – Rehydration

20 µm

NGC

2 µm

α-tubulin in Arabidopsis

pollen

HPF – FS –Rehydration

Generative cell

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M

ER

G

1 µm

1 µm

500 nm

HPF – FS – Rehydration

α-tubulin in Arabidopsis pollen

10 µm

Labelling of 300 nm cryosections after


γ-COP in Arabidopsis pollen

500 nm

ER

G

G

M

M

500 nm

γ-COP in Arabidopsis pollen

Labelling of 300 nm cryosections after


DICCuticleBody-wall

muscles

Pharynx

10 µm

α-tubulin F-actin

α-tubulin in the nematode Pristionchus pacificusLabelling of cryosections after HPF, freeze-substitution and rehydration

Pharynx

Body-wallmuscles

Cuticle5 µm

α-tubulin in the nematode Pristionchus pacificus

PharynxCuticle

2.5 µm


Cortical layer

Medial layer

Basal layer

Surface coat /Epicuticle

500 nm


Reasons for using ultrathin sections in light microscopy

Signal is restricted to the surface of a resin section:No out-of-focus signal blurs the image

Signal is not increased in thicker sections, but autofluorescence is

Serial sections of the same structure can be collected alternately for light and electron microscopy

High magnification objectives can be used routinely for ultrathin sections, even those stained for histology

Thin sectioning conserves rare material

Advantages of immunofluorescence over immunogold

Rapid screening of sample areas – large field of view

Labelling of multiple antigens is much easier with different fluorochromes than with gold particles of different sizes

Even the smallest gold marker may influence the binding properties of an antibody more than a fluorochrome

Increased resolution of EM can be exploited

Specificity of labelling is much easier to determine due to ultrastructural information

Gold particles allow quantification – this also serves as an additional control

Advantages of immunogold over immunofluorescence

Conclusions

Excellent structural preservation

Ease of orientation in stained histological tissue sections

Fast overview on labelled structures

High z-resolution in light microscopy

Direct correlation of label and antigen-containing cellular ultrastructure

Correlative light and electron microscopy using immunolabelled ultrathin sections

Further reading:

Schwarz, H., and Humbel, B.M. (2007) Correlative light and electron microscopy using immunolabeled resin sections. In: Electron Microscopy: Methods and Protocols (Kuo, J., ed.) Methods Molecular Biology 369, 229-256. Humana Press, Totowa NJ, USA

Schwarz, H., and Humbel, B.M. (2008) Correlative light and electron microscopy. Chapter 21 in: Handbook of Cryopreparation Methods for Electron Microscopy (Cavalier, A., Spehner, D., and Humbel, B.M. eds.), pp. 537-565. CRC Press, Boca Raton FL, USA

Stierhof, Y.-D., Van Donselaar, E., Schwarz, H., and Humbel, B.M. (2008) Cryo-fixation, Rehydration and Tokuyasu cryo-sectioning.Chapter 14 in: Handbook of Cryopreparation Methods for Electron Microscopy (Cavalier, A., Spehner, D., and Humbel, B.M. eds.), pp. 343-365. CRC Press, Boca Raton FL, USA

Array Tomography Method:

Micheva, K.D., and Smith, S.J. (2007) Array Tomography: A new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron 55, 25-36. Fig. 1

0.5 µm

0.1 µm 0.2 µm

Improved resolution over confocal microscopy

3D volume imaging

Consecutive staining with antibody elution

Correlative Scanning EMusing back-scattered electrons

Automation of array tomo-graphy and puncta quantification

Array tomography

Array tomography of serial thin sections mounted on slides offers fluorescent and gold labelling to be inspected first by wide field fluorescence microscopy and then by SEM using back-scattered electrons.

Note: Automated serial block face imaging SEM (by Gatan3View/SEM or by focused ion beam milling) can so far not visualize labelled intracellular structures. (e.g. photo-oxidized DAB).

Acknowledgements:

Experiments were done jointly withMatthias Hammerschmidt (Köln)Bernard Moussian (Tübingen) York Stierhof (Tübingen)

Thanks for continous support and discussions:Gareth Griffiths (Oslo)Bruno Humbel (Lausanne) Martin Müller (Zürich)

correlative light and electron microscopy using ... · actually the best approach in light...

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