Correlative Light and Electron Microscopy using Immunolabelled

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 molecules
within 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 only
few reports exist on tags expressed in living cells
using 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 EM
Gaietta, G. et al. (2006) Golgi twins in late mitosis by genetically encoded tags for
live 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)
25 µm
2 min
25 µm
2 µm
4 min
0 min
8 min
10 µm
0.5 µm
Grabenbauer, M. et al. (2005) Correlative microscopy and tomography of GFP
through photooxidation. Nature Meth. 2, 857-862.2005. Fig. 2
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 techniques
Permeabilization destroys some fine structure
Loss of soluble material is an intrinsic property
Penetration problems for antibodies and markers
as 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 sucroseinfiltrated 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 combination
with cryoimmobilization and freeze-substitution
Resin monomers are potential skin irritants and sensitizers
Fixation, dehydration and embedding in resin may destroy
the 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. coli
freeze-substituted in ethanol omitting any crosslinking fixatives
Lowicryl HM20 section labelled for OmpA
Relocation of OmpA during sectioning in cryofixed E. coli
freeze-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
Post-embedding labelling offers a good combination
of both, structural integrity and reliable signal
for correlative light and electron microscopy
Examples for immunolabelling of ultrathin resin sections of the
very same specimen block for light and electron microscopy
α−tubulin in trypanosomes
β-catenin in intestine and heart muscle
Recent examples for correlative microscopy
Prolactin in zebrafish anterior pituitary gland
Chitin in Drosophila embryos
α-tubulin, γ-COP in Arabidopsis pollen
α-tubulin in Drosophila embryos and nematodes
Strategy
500-1000 nm Section
Coverslip
Toluidine Blue
50-100 nm Section
Coverslip
50-100 nm Section
EM Grid
Primary Antibody
Primary Antibody
Fluorescent Marker
DAPI / PI
Gold Marker
v
Epon
Mowiol
Bright-Field
Fluorescence
Uranyl Acetate
Lead Citrate
Electron Microscopy
Orientation and
Differentiation
Localization and
Overview
High-Resolution
Localization
Microtubules in Trypanosoma brucei
High-pressure frozen, freeze-substituted in osmium/acetone, embedded in Epon
Micrographs 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
16x oil immersion objective
100x oil immersion objective
Toluidine blue stained Lowicryl K4M section of rat intestine
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 Cy3
propidium 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 immunocytochemistry 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
D
Z
M
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.
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
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 intestine of plakoglobin null-mutant mice
Wild type
Plakoglobin null-mutant
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.
Prolactin labelling in the pituitary of zebrafish
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
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
Prolactin labelling in the pituitary of zebrafish wt 7 dpf
Wild type
10 µm
Toluidine blue stained 0.5 µm section
Immunolabelled 50 nm section
for Prolactin (orange)
DAPI (blue)
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
Prolactin labelling in the pituitary of zebrafish wt 7 dpf
10 µm
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
Prolactin labelling in the pituitary of zebrafish wt 7 dpf
5 µm
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
Prolactin labelling in the pituitary of zebrafish wt 7 dpf
50.5
µmµm
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
Spotting the region of interest on EM level
1638
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 Drosophila
embryogenesis
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
Moussian, B., Seifarth, C., Müller, U., Berger, J. and Schw arz, H.
(2006) Cuticle differentiation during Drosophila embryogenesis.
Arthropod Structure & Development 35, 137-152
Chitin labelling using wheat germ agglutinin (WGA)
Wild type
Chitin synthase mutant CS-1/
kkv (krotzkopf verkehrt)
Wild type
WGA -10 nm gold
CC
Mutant
MPL -10 nm gold
Wild type
Mutant
Labelling of α-tubulin and γ-COP in Arabidopsis pollen
α-tubulin in Drosophila embryos and nematodes
Specimen were cryofixed by high-pressure freezing and in most cases
freeze-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
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.
α-tubulin in Arabidopsis pollen
Labelling of 300 nm cryosections after
Chemical fixation
HPF – FS – Rehydration
*
*
N
*
N
*
*
N
N
*
*
Generative cells
10 µm
α-tubulin in
Arabidopsis
pollen
N
GC
20 µm
2 µm
HPF – FS –
Rehydration
500 nm
G
Generative cell
ER
N
1 µm
α-tubulin in
Arabidopsis pollen
HPF – FS – Rehydration
M
1 µm
γ-COP in Arabidopsis pollen
Labelling of 300 nm cryosections after
Chemical fixation
10 µm
HPF – FS – Rehydration
γ-COP in Arabidopsis pollen
Labelling of 300 nm cryosections after
Chemical fixation
HPF – FS – Rehydration
500 nm
G
M
ER
G
500 nm
M
α-tubulin in the nematode Pristionchus pacificus
Labelling of cryosections after HPF, freeze-substitution and rehydration
α-tubulin
F-actin
DIC
Body-wall
muscles
Cuticle
Pharynx
10 µm
α-tubulin in the nematode Pristionchus pacificus
Pharynx
Body-wall
muscles
5 µm
Cuticle
α-tubulin in the nematode Pristionchus pacificus
Cuticle
Pharynx
2.5 µm
α-tubulin in the nematode Pristionchus pacificus
Surface coat /
Epicuticle
Cortical layer
Medial layer
Basal layer
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
Advantages of immunogold over immunofluorescence
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
Correlative light and electron microscopy
using immunolabelled ultrathin sections
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
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:
Improved resolution over
confocal microscopy
3D volume imaging
0.5 µm
Consecutive staining with
antibody elution
Correlative Scanning EM
using back-scattered
electrons
0.1 µm
Automation of array tomography and puncta
quantification
0.2 µm
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
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 with
Matthias Hammerschmidt (Köln)
Bernard Moussian (Tübingen)
York Stierhof (Tübingen)
Thanks for continous support
and discussions:
Gareth Griffiths (Oslo)
Bruno Humbel (Lausanne)
Martin Müller (Zürich)