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Markus Sauer

Ralph Götz

Tobias Kunz

Julian Fink

Franziska Solger

Jan Schlegel

Jürgen Seibel

Vera Kozjak-Pavlovic

Thomas Rudel

Figure 1

Expansion microscopy of cellular and bacterial membranes by functionalized ceramides

Expansion microscopy (ExM) enables super-resolution imaging on standard fluorescence microscopes. By linking a protein of interest into a dense, cross-linked network of a swellable polyelectrolyte hydrogel, biological specimens can be physically expanded allowing ~70 nm lateral resolution by confocal laser scanning microscopy. Since its introduction by Boyden and co-workers in 20151, expansion microscopy (ExM) has shown impressive results including the magnified visualization of pre- or post-expansion labeled proteins and RNAs with fluorescent proteins, antibodies, and oligonucleotides, respectively, in cells, tissues, and human clinical specimen2. In order to be usable for ExM, the molecule of interest has to exhibit amino groups that can react with glutaraldehyde (GA)3, MA-NHS3, AcX4, or Label-X5 and be linked into the polyelectrolyte hydrogel. The plasma membrane of cells is mainly composed of glycerophospholipids, sphingolipids, and cholesterol. Due to the lack of primary amino groups, these lipids neither can be fixed by formaldehyde, glutaraldehyde and other chemical fixatives nor expanded using available ExM protocols. We used an unnatural short-chain sphingolipid functionalized with an azide and primary amino group to enable fluorescence labeling and chemical fixation as well as linking of the lipid into a swellable hydrogel. Our results demonstrate that the designed unnatural bifunctional sphingolipid is efficiently incorporated into membranes of cells and accumulates in bacterial membranes, which allowed us to investigate the distribution of lipids and interactions with proteins in cellular and bacterial membranes with high spatial resolution.

-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;α-NH2-ω-N3-C6-ceramide (homemade)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1x PBS (Thermo Fisher, 14190169)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4% Paraformaldehyde (PFA, Morphisto, 10303.01000)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Glutaraldehyde 25% (GA, Sigma Aldrich, G5882)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Triton X-100 (Thermo Fisher, 28314)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Hanks’ Balanced Salt solution (HBSS, Merck, H6648)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Click-IT Alexa Fluor® 488 DIBO alkyne dye (Jena Bioscience, CLK-1278-1)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Fetal bovine serum (FBS, Sigma-Aldrich, F7524)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Ammonium persulfate (APS, Sigma, A3678)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Tetramethylethylenediamine (TEMED, Sigma, T7024)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Protease K (Thermo Fisher, AM2548)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Lysozyme (BioChemika, 62970)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Poly-D-Lysin (PDL, Gibco, A3890401)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Sodium acrylate (Sigma, 408220)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2.5 % Acrylamide (Sigma, A9926)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;0.15 % N,N‘-Methylenbisacrylamide (Sigma, A9926)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2 M NaCl (Sigma, S5886)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;DMAA (Sigma, 274135)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;KPS (Sigma, 379824)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;EDTA (Sigma, ED2P)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Guanidine HCl (Sigma, 50933)Reagent setupFor preparation of 4x monomer solution mix: - 8.625 % Sodium acrylate (Sigma, 408220)- 2.5 % acrylamide (Sigma, A9926)- 0.15 % N, N‘-Methylenbisacrylamide (Sigma, A9926)- 2 M NaCl (Sigma, S5886)dissolved in 1x PBS.&nbsp;For preparation of 10x monomer solution mix:- 1 ml of 10x Monomer solution contains- 0.267 g DMAA (Sigma, 274135)- 0.064 g Sodium acrylate (Sigma, 408220)dissolved in 0.57 g ddH2O&nbsp;The digestion buffer consists of - 50 mM Tris pH 8.0- 1 mM EDTA (Sigma, ED2P)- 0.5 % Triton X-100 (Thermo Fisher, 28314)- 0.8 M Guanidine HCl (Sigma, 50933)

-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;15 mm glass cover slide (VWR, ECN 631-1579)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Costar 12 Well Cell Culture plate (Corning, 3513)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Parafilm (Hartenstein, PF10)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TC dish 150 (Sarstedt, 83.3903)-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Glass chambers (Merck, 734-2055)

Sample preparationDay 1:1.&nbsp;&nbsp;&nbsp;Grow cells overnight on a 15mm glass cover slide in a 12-well plate in a humidified atmosphere containing 5% (v/v) CO2 at 37°C.Day 2:2.&nbsp;&nbsp;&nbsp;Optional: For sphingolipid ExM of bacteria infect the cells according to standard protocols and follow the protocol after bacterial propagation.Note: Ceramides may exhibit toxic effects on some bacterial species.3.&nbsp;&nbsp;&nbsp;Exchange the media of the cells for fresh media prewarmed to 37°C4.&nbsp;&nbsp;&nbsp;Treat the cells with 10 μM α-NH2-ω-N3-C6-ceramide prewarmed to 37°C for 1 hour at 37°C in the exchanged medium5.&nbsp;&nbsp;&nbsp;Wash the sample with 1x PBS6.&nbsp;&nbsp;&nbsp;Fix the cells using 4% PFA and 0.1% GA for 10 minutes7.&nbsp;&nbsp;&nbsp;Wash the sample 3 times with 1x PBSClick-reaction8.&nbsp;&nbsp;&nbsp;Permeabilize the sample for 15 minutes in 0.2% Triton X-100 in 1x PBS9.&nbsp;&nbsp;&nbsp;Wash the sample 3 times with 1xPBS10.&nbsp;Exchange the PBS for 1x HBSS prewarmed to 37°C11.&nbsp;Incubate the sample in 5 μM Click-IT Alexa Fluor® 488 DIBO alkyne dye at 37°C for 30 minutes. Note: Other functionalized dyes can be used according to preferences.12.&nbsp;&nbsp;Wash the sample 3 times with 1x PBSOptional: ImmunostainingFor samples that need additional immunolabelling, e.g. for colocalization of cellular or bacterial proteins, follow the steps a-e before proceeding with step 13. a.&nbsp;&nbsp;&nbsp;Block the sample with 2% FBS in 1x PBS for 1 hour.b.&nbsp;&nbsp;&nbsp;Incubate the sample in the primary antibody diluted in blocking buffer for 1 hour in a humid chamberc.&nbsp;&nbsp;&nbsp;&nbsp;Wash the sample 3 times with 1x PBSd.&nbsp;&nbsp;&nbsp;Incubate the sample in the corresponding secondary antibody diluted in blocking buffer for 1 hour in a humid chamberNote: Not all secondary antibodies are suitable for expansion microscopy. Our preferred secondary antibodies are Alexa Fluor 488, CF568, Atto643 and Atto647N.e.&nbsp;&nbsp;&nbsp;Wash the sample 3 times with 1x PBSExpansion Microscopy13.&nbsp;Incubate the labelled sample with 0.25% GA at RT for 10 minutes.14.&nbsp;Wash the sample 3 times with 1x PBS&nbsp;Note: The monomer solutions for 4x and 10x Expansion are different.15.&nbsp;For 4x Expansion, add 0.2% ammonium persulfate (APS) and 0.2% tetramethylethylenediamine (TEMED) to the 4x monomer solution16.&nbsp;Immediately pipet 80 μl of the mixture on parafilm and turn the labeled 15 mm glass slide with the cells facing the bottom onto the drop17.&nbsp;Incubate for 1 hour at RT for gelation18.&nbsp;For 10x Expansion, degas 1 ml of the 10x monomer solution on ice for 45 minutes with nitrogen.19.&nbsp;Add 100 μl KPS (0.036 g/ml) to the monomer solution20.&nbsp;Degas on ice for another 15 minutes with nitrogen21.&nbsp;Add 4 μl of TEMED22.&nbsp;Immediately pipet 80 μl of the mixture on parafilm and turn the labeled 15 mm glass slide with the cells facing the bottom onto the drop23.&nbsp;Incubate for 30 minutes at RT for gelation, followed by 1.5 hours at 37°C24.&nbsp;After gelation, remove the gel from the glass cover slide and digest the samples between 3 hours and overnight with 8U/ml protease K in digestion bufferNote: The cell wall digestion of some bacteria requires additional treatment, e.g.1mg/ml lysozyme in the case of Neisseria gonorrhoeae.25.&nbsp;Coat glass chambers with PDL overnight at RT.Day 3:26.&nbsp;Incubate digested gels in hourly changed ddH2O for expansion until expansion is saturatedNote: Due to the swelling of the gel, make sure that the fully expanded gel has enough space in the dish used, e.g. TC dish 150.27.&nbsp;Chop expanded gels in pieces fitting the PDL-coated glass chambers for imaging

The described protocol enables nanoscale imaging of cellular and bacterial membranes and differentiation of both layers within double membranes.

1.&nbsp;&nbsp;&nbsp;Chen, F., Tillberg, P.W. &amp; Boyden, E.S. Optical imaging. Expansion microscopy. Science 347, 543-548 (2015).2.&nbsp;&nbsp;&nbsp;Wassie, A.T., Zhao, Y. &amp; Boyden, E.S. Expansion microscopy: principles and uses in biological research. Nature Methods 16, 33-41 (2019).3.&nbsp;&nbsp;&nbsp;Chozinski, T.J. et al. Expansion microscopy with conventional antibodies and fluorescent proteins. Nature methods 13, 485-488 (2016).4.&nbsp;&nbsp;&nbsp;Tillberg, P.W. et al. Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies. Nat Biotechnol 34, 987-992 (2016).5.&nbsp;&nbsp;&nbsp;Chen, F. et al. Nanoscale imaging of RNA with expansion microscopy. Nature Methods 13, 679-684 (2016).

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) GRK 2157 to VKP, TR and MS, and DFG FOR 2123 to TR and MS.

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Due to the lack of primary amino groups, lipids cannot be fixed by formaldehyde, glutaraldehyde and other chemical fixatives nor expanded using expansion microscopy (ExM). We present a protocol that uses unnatural short-chain azide- and amino-functionalized ceramides, which upon incorporation into cellular and bacterial membranes can be labeled by click chemistry and linked into hydrogels, followed by 4x to 10x expansion.

Method Article

Markus Sauer, Ralph Götz, Tobias Kunz, Julian Fink, Franziska Solger, Jan Schlegel, Jürgen Seibel, Vera Kozjak-Pavlovic, Thomas Rudel

Markus Sauer

University of Wuerzburg

Corresponding Author

Ralph Götz

University of Wuerzburg

Tobias Kunz

University of Wuerzburg

Julian Fink

University of Wuerzburg

Franziska Solger

University of Wuerzburg

Jan Schlegel

University of Wuerzburg

Jürgen Seibel

University of Wuerzburg

Vera Kozjak-Pavlovic

Vera Kozjak-Pavlovic

Thomas Rudel

Vera Kozjak-Pavlovic

Corresponding Author

DOI:

10.21203/rs.3.pex-1249/v1

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super-resolution imaging, expansion microscopy, membranes

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10.1038/s41467-020-19897-1

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Markus Sauer, Ralph Götz, Tobias Kunz, Julian Fink, Franziska Solger, Jan Schlegel, Jürgen Seibel, Vera Kozjak-Pavlovic, Thomas Rudel

Markus Sauer

University of Wuerzburg

Corresponding Author

Ralph Götz

University of Wuerzburg

Tobias Kunz

University of Wuerzburg

Julian Fink

University of Wuerzburg

Franziska Solger

University of Wuerzburg

Jan Schlegel

University of Wuerzburg

Jürgen Seibel

University of Wuerzburg

Vera Kozjak-Pavlovic

Vera Kozjak-Pavlovic

Thomas Rudel

Vera Kozjak-Pavlovic

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