Correlative Sample Protocols - FEI Company

Published Sample
Preparation Protocols
For Correlative Microscopy

Below is a list of protocols for the preparation of biological samples
for correlated fluorescence and transmission electron microscopy,
compatible with iCorr imaging.
Pages 2-8 list workflows for resin-embedded room temperature
specimens, while published protocols for correlative cryo-FM/EM are
summarized on pages 9-11.
Page 12 lists a non-exhaustive but representative selection of successful
approaches to cryo-TEM/ET of frozen hydrated biological samples. These
protocols can be directly adapted to iCorr imaging.
Abbreviations
FM fluorescence microscopy
(T)EM (transmission) electron microscopy
ET electron tomography
SEM scanning electron microscopy
PFA paraformaldehyde
GA glutaraldehyde
UA uranyl acetate
IgG Immunoglobulin G
2

Publication
Oberti, D., Kirschmann, M.A., and Hahnloser, R.H.R. Correlative microscopy of densely labeled projection neurons using neural tracers.
Frontiers in Neuroanatomy 2010; 4:24.
1 Material Bird brain
2 Fluorophores Injection of
• Lucifer yellow
• Alexa Fluor 647
• Tetramethylrhodamine
3 Fixation Chemical fixation (perfusion) with 2% PFA /0.075% GA in 0.1M phosphate buffer (pH7.4)
4 Confocal Imaging On 60 µm vibratome sections
5 Post-fixation 1% OsO + 1.5% potassium ferrocyanide, followed by
4
1%OsO , followed by
4
1% UA
NB Tracer fluorescence is quenched
6 Resin embedding Durcupan
7 Sectioning 60-90 nm sections
8 Labeling, fluorophores Primary: protein-specific IgG;
Secondary: IgG-Alexa 594
9 FM
10 Post stain 1% UA
Reynold’s lead citrate
11 TEM
12 Image correlation Registration of FM and EM images using Adobe Photoshop
3

Publications
Kukulski, W., Schorb, M., Welsch, S., Picco, A., Kaksonen, M., and Briggs J.A. Correlated fluorescence and 3D electron microscopy with high
sensitivity and spatial precision. J. Cell Biol. 2011; 192:111-119.
Kukulski W., Schorb M., Welsch S., Picco A., Kaksonen M., Briggs J.A. Precise, correlated fluorescence microscopy and electron tomography
of lowicryl sections using fluorescent fiducial markers. Methods Cell Biol. 2012;111:235-57.
4
1 Material Yeast and mammalian cells
2 Fluorophores fluorescence-tagged cellular proteins (RFP, (E)GFP, mCherry)
3 Fixation Cryo-fixation by high pressure freezing
4 Freeze substitution 0.1% UA in acetone, containing 0-3% water
5 Resin embedding Lowicryl HM20
6 Sectioning 300 nm or 50 nm sections
7 FM
8 Post stain Reynold’s lead citrate
9 TEM or ET
10 Image correlation Fiducial-based (fluospheres) precise correlation, using MatLab with Image Processing
Toolbox

Publications
Watanabe, S., Punge, A., Hollopeter, G., Willig, K.I., Hobson, R.J., Davis, M.W., Hell, S.W., Jorgensen, E.M. Protein localization in electron
micrographs using fluorescence nanoscopy. Nature Methods 2011; 8:80-84.
Watanabe S., Jorgensen EM. Visualizing proteins in electron micrographs at nanometer resolution. Methods Cell Biol. 2012;111:283-306.
1 Material Nematodes (C.elegans)
2 Fluorophores Fluorescence-tagged proteins (Citrine, tdEOS)
3 Fixation Cryo-fixation by high pressure freezing,
4 Fixation/ freeze substitution Acetone /0.1% potassium permanganate /0.1 ‰ OsO / 5% water, or acetone with
4
• 0.1-2% PFA ± 0.1-1% GA, or
• 0.1-1% GA, or
• 0.1% acrolein, or
• 0.1‰-0.5% OsO , and/or
4
• 0.1% KMnO4 5 Staining Acetone /0.1%UA / 5% water
replaced with 95% ethanol after staining
6 Resin embedding Lowicryl K4M /LR Gold /LR White /glycol methacrylate with2-5% water
7 Sectioning 70-100 nm ultrathin sections
8 FM Subdiffraction resolution imaging (STED, PALM)
9 Post stain 2.5% UA
10 SEM, TEM
11 Image correlation Gold fiducial-based image alignment, using Adobe Photoshop
5

Publication
Karreman, M.A., van Donselaar, E.G., Gerritsen, H.C., Verrips, C.T., and Verkleij, A.J. VIS2FIX: a high-speed fixation method for immunoelectron
microscopy. Traffic 2011; 12:806-814.
6
1 Material Mammalian cells
2 Fixation Cryo-fixation by high pressure freezing
3 Sectioning 60-80 nm CEMOVIS sections
4 A1 Freeze substitution 0.1% UA in acetone
0.01-0.5% GA in acetone
0.01%-0.5% OsO in acetone
4
4 A2 Rehydration Sequential steps of 0.01%-0.5% GA in water
OR
4 B1 Post-fixation 0.2% UA
0.01-0.5% GA
0.01%-0.5% OsO /2%-4% FA /1% acrolein
4
4 B2 Thawing and fixing as in 4 B1 5 Labeling, fluorophores Primary: protein-specific IgGs
Secondary: immuno-gold (10 nm) and IgG-Alexa 488
6 Post stain 2% UA, 2% Uranyl oxalate
7 Correlative microscopy Integrated light- and transmission electron microscope, custom-made correlation software

Publication
Karreman, M.A., Agronskaia, A.V., Van Donselaar, E.G., Vocking, C.E.M., Fereidouni, F. Humbel, B.M., Verrips, C.T., Verkleij, A.J., Gerritsen,
H.C. One protocol fits all: optimizing sample preparation for correlative microscopy on a single specimen. Submitted for publication.
1 Material Eukaryotic ells
2 Fixation Chemical fixation with aldehydes
3 Cryoprotection Sucrose
4 Freezing Liquid nitrogen
5 Freeze substitution In an organic solvent with added fixatives and/or heavy metal stains
6 Resin embedding Lowicryl
7 Sectioning 60-80 nm sections
8 Labeling, fluorophores Primary IgGs
Secondary immuno-gold (10 nm) and IgG-Alexa 488
9 Correlative microscopy Integrated light- and transmission electron microscope, custom-made correlation software
7

Publication
Fabig G, Kretschmar S, Weiche S, Eberle D, Ader M, Kurth T. Labeling of ultrathin resin sections for correlative light and electron
microscopy. Methods Cell Biol. 2012;111:75-93.
8
1 Material Mouse tissue (retina, testis)
2 Fixation Chemical fixation (perfusion) with 2-4% PFA +/- 0.05-0.5% GA
3 Dehydration Graded PLT series of ethanol (30%-100%, 0°C- 35°C)
4 Sectioning ultrathin sections
5 A Labeling, fluorophores
OR
Primary IgGs (+ bridging antibodies where needed)
(optional: postfixation)
Secondary: immunogold or proteinA-gold (10nm), IgG-Alexa 488 or -Alexa 555
5 B Labeling Primary IgGs
FluoroNanogold Fa,b fragments
6 FM
7 Post-staining Silver enhancement (of FluoroNanogold)
2-4% UA
8 TEM
9 Image correlation Not specified

Publication
Cortese K, Vicidomini G, Gagliani MC, Boccacci P, Diaspro A, Tacchetti C. 3D HDO-CLEM: Cellular Compartment Analysis by Correlative
Light-Electron Microscopy on Cryosection. Methods Cell Biol. 2012;111:95-115.
1 Material Mammalian cells
2 Fixation Chemical fixation with 4% PFA /0.4% GA
3 Embedding Gelatin
4 Cryoprotection Sucrose
5 Freezing Liquid nitrogen
6 Sectioning Semithin (200-300 nm ) or ultrathin (60-65 nm) sections
7 Labeling, fluorophores On thawed sections.
Primary: protein-specific IgG
Secondary: Cy2- or Cy3-conjugated antibody (+DNA stain) followed by proteinA-gold
(10 nm or 15 nm)
Optional: postfixation with 1% GA, double labeling
8 FM
9 Post-staining 2% UA /0.15% oxalic acid
10 Contrasting, stabilization 0.4% UA /1.8% methylcellulose
11 TEM
12 Image correlation Registration using ImageJ with TurboReg plugin
9

Publication
Rigort A, Bäuerlein FJ, Leis A, Gruska M, Hoffmann C, Laugks T, Böhm U, Eibauer M, Gnaegi H, Baumeister W, Plitzko JM. Micromachining
tools and correlative approaches for cellular cryo-electron tomography.J Struct Biol. 2010 Nov;172(2):169-79.
10
1 Material Prion-infected yeast cells
2 Fluorophore GFP
3 Fixation Cryo-fixation by plunge freezing or high pressure freezing
4 Optional Cryo-planing of very thick samples
5 FM Liquid nitrogen-cooled
6 Sectioning Focussed ion beam (FIB) milling of areas of interest, targeted by FM
7 Cryo-EM SEM or TEM of FIB-milled areas

Publication
Rigort A, Villa E, Bäuerlein FJ, Engel BD, Plitzko JM. Integrative Approaches for Cellular Cryo-electron Tomography: Correlative Imaging and
Focused Ion Beam Micromachining. Methods Cell Biol. 2012;111:259-81.
1 Material Rat neurons
2 Fluorophore FM1-43 vital dye
3 Fixation Cryo-fixation by plunge freezing
4 FM Liquid nitrogen-cooled
5 Cryo-ET ET of thin specimen areas, targeted by FM
Publication
Gruska M, Medalia O, Baumeister W, Leis A. Electron tomography of vitreous sections from cultured mammalian cells. J Struct Biol. 2008
Mar;161(3):384-92.
1 Material Mouse heart muscle cells
2 Fluorophore Mitotracker Green FM dye
3 Fixation Cryo-fixation by plunge freezing (adherent cells) or high pressure freezing (detached cells)
4 Sectioning 50-150 nm vitreous sections
5 FM Liquid nitrogen-cooled
6 TEM or ET Areas of interest, identified by FM
11

Preparation of biological cryo-EM/ET specimens
Maurer UE, Sodeik B, Grünewald K. Native 3D intermediates of membrane fusion in herpes simplex virus 1 entry. Proc Natl Acad Sci U S A. 2008 Jul
29;105(30):10559-64
Cyrklaff M, Linaroudis A, Boicu M, Chlanda P, Baumeister W, Griffiths G, Krijnse-Locker J. Whole cell cryo-electron tomography reveals distinct
disassembly intermediates of vaccinia virus. PLoS One. 2007 May 9;2(5).
Carlson LA, de Marco A, Oberwinkler H, Habermann A, Briggs JA, Kräusslich HG, Grünewald K. Cryo electron tomography of native HIV-1 budding
sites. PLoS Pathog. 2010 Nov 24;6(11).
Cryoelectron microscopy of vitreous sections: a step further towards the native state. Bouchet-Marquis C, Fakan S. Methods Mol Biol.
2009;464:425-39.
CEMOVIS. Cryo-electron microscopy of vitreous sections. Dubochet, J., Al-Amoudi, A., Bouchet-Marquis, C., Eltsov, M., Zuber, B. In: Modern
cryo-preparation methods for electron microscopy. Eds. A. Cavalier, B. M. Humbel and D. Spehner, CRC Press.
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