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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
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2 µm
0.5 µm10 µm
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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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D
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
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.
Wild type
Plakoglobin null-mutant
β-catenin in the intestine of plakoglobin null-mutant mice
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.
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
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
10 µm
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
5 µm
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
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
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
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
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N
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*
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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
N
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
Chemical fixation HPF – FS – Rehydration
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
Chemical fixation HPF – FS – Rehydration
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
α-tubulin in the nematode Pristionchus pacificus
Cortical layer
Medial layer
Basal layer
Surface coat /Epicuticle
500 nm
α-tubulin in the nematode Pristionchus pacificus
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)