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Optimized sample preparation for single-molecule localization-based superresolution microscopy in yeast

Nature Protocols volume 10, pages 1007–1021 (2015)Cite this article

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Abstract

Single-molecule localization-based superresolution microscopy methods allow the resolution of cellular structures in the range of tens of nanometers. However, these techniques are of limited use in current yeast labeling protocols, owing to problems with structural preservation. Here we describe an optimized sample preparation protocol that enables single-molecule localization microscopy at high resolution combined with improved structural preservation in Saccharomyces cerevisiae. The protocol uses small binders called nanobodies and an enzymatic labeling strategy to deliver organic dyes to the target protein. These small binders readily penetrate through the yeast cell wall and thus eliminate the requirement for its prior degradation, and they allow structural preservation. In addition, the small size of the binders reduces the distance of the dye to the target protein, and thus it reduces the localization error. The preparation of S. cerevisiae cells for superresolution imaging takes 2–4 h to perform. Researchers should have skills in yeast molecular biology, immunolabeling techniques and access to a microscope equipped for single-molecule imaging.

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Figure 1: The use of small binders conjugated with organic fluorophores renders cell wall digestion unnecessary in yeast immunocytochemistry.
Figure 2: Schematic of sample preparation of budding yeast for single-molecule localization microscopy.
Figure 3: Budding yeast expressing Cdc11 linked to GFP fixed with different chemicals.
Figure 4: Results from single-molecule localization microscopy experiments in S. cerevisiae.

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Acknowledgements

We thank G. Shtengel (Janelia Farm, Howard Hughes Medical Institute, Ashburn, Virginia, USA) for sharing sample preparation knowledge and discussions. We thank the entire Ewers laboratory for discussions. We thank Y. Barral (Institute of Biochemistry, ETH Zurich, Zurich, Switzerland) for a plasmid. This work was supported by the Swiss National Centre of Competence in Research (NCCR) Neural Plasticity and Repair and Holcim, and by the Swiss National Competence Center for Biomedical Imaging (NCCBI).

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  1. Charlotte Kaplan & Helge Ewers

    Present address: Present addresses: Department of Molecular and Cell Biology, University of California, Berkeley, California, USA (C.K.); Institute for Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany (H.E.).,

Authors and Affiliations

  1. Institute of Biochemistry, ETH Zurich, Zurich, Switzerland

    Charlotte Kaplan & Helge Ewers

  2. Randall Division of Cell and Molecular Biophysics, King's College London, London, UK

    Helge Ewers

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C.K. and H.E. designed the research and analyzed the data. C.K. conducted the experiments. H.E. and C.K wrote the paper.

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Correspondence to Helge Ewers.

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Integrated supplementary information

Supplementary Figure 1 Quenching of glutaraldehyde by NaBH4 is required when using AF647-coupled anti-GFP nanobodies for immunostaining.

Budding yeast expressing Cdc11-GFP (strain YJR076C from GFP-collection) was treated with AF647-coupled anti-GFP nanobodies (Procedure until step 10| (A)). Shown is AF647 fluorescence. (a) No NaBH4 treatment was employed after fixation. The cell exhibits strong nonspecific anti-GFP nanobody staining and Cdc11-GFP staining is not very pronounced. (b) and (c) show that NaBH4 treatment for 30 min significantly reduces nonspecific binding. Longer treatment of the sample with NaBH4 (10 mg/ml) does not result in further improvements and therefore is not necessary. All scale bars are 10 μm.

Supplementary Figure 2 Optimization of blocking with different reagents.

Cdc11-SNAPf-tag expressing budding yeast cells were labeled as described (Procedure Step 10| (B)). Before application of BG-AF647, samples were treated with either Image-iT™ FX, bovine serum albumin (BSA) or horse serum (HS). After completion of the labeling procedure, images were acquired with identical settings and processed to identical brightness levels in ImageJ for a quantitative comparison of blocking efficiency. In general, all three blocking reagents are applicable to reduce the nonspecific background for superresolution imaging. Image-iT™ FX performs slightly better and might be considered under conditions when animal sera are not blocking efficiently. All scale bars are 5 μm.

Supplementary Figure 3 Saturation of labeling and the resulting fluorescent background.

An experiment was performed with budding yeast expressing septin Cdc11-SNAPf-tag and labeled with BG-AF647 (Procedures step 10| (B)). (a) Staining solution was exchanged after 30 min to PBS and cells were imaged. Subsequently, the labeling solution was applied a second time to the same sample. (b) After 90 min, the cells exhibit bright and nonspecific AF647 staining in the cytoplasm. Saturation of labeling is reached. Images of autofluorescence in the GFP channel are shown to demonstrate that signal in the cytoplasm is AF647-specific. All scale bars are 5 μm.

Supplementary Figure 4 Example of a less abundant protein in yeast, the spindle pole body protein Spc42.

Budding yeast (YKL042W from yeast clone GFP-collection) expressing Spc42-GFP was treated with AF647-coupled anti-GFP nanobodies (Procedure until step 10| (A)). (a) Left-hand image shows wide-field fluorescence of the GFP signal. Right-hand image shows wide-field fluorescence of the AF647 signal. Scale bars are 2 µm. (b) Superresolution image of right-hand image in (a). Scale bar is 2 µm. (c) Zoom of the insets in (b). Inset 1 shows a single spindle pole body. In inset 2, the spindle pole body starts to duplicate and in inset 3, two spindle pole bodies are detectable. Scale bars are 500 nm.

Supplementary Figure 5 Gold Nanorods as fiducials linked to the budding yeast cell wall.

Yeast cell preparations incubated with 200 x and 1,000 x dilutions from a stock solution of Gold Nanorods conjugated to streptavidin are shown to demonstrate the respective labeling density in a field of view. The phase contrast image merged with the AF647 channel demonstrates the attachment of the fiducials to the yeast cell wall and their bright fluorescence in the single-molecule localization microcopy channel. All scale bars are 5 μm.

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Kaplan, C., Ewers, H. Optimized sample preparation for single-molecule localization-based superresolution microscopy in yeast. Nat Protoc 10, 1007–1021 (2015). https://doi.org/10.1038/nprot.2015.060

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