{ "componentChunkName": "component---src-templates-article-page-js", "path": "/journals/biology/micropub-biology-000566", "result": {"data":{"article":{"manuscript":{"id":"ae698010-f449-477e-ba63-6e6432ccbe28","submissionTypes":["new method"],"citations":["10.1007/s00253-022-11967-2","10.1021/acs.analchem.2c04921","10.21203/rs.3.rs-2637060/v1","10.1242/jcs.260240","10.1002/yea.3848","10.1002/advs.202302249","10.1016/j.celrep.2026.116948"],"doi":"10.17912/micropub.biology.000566","dbReferenceId":"","pmcId":"9132724","pmId":"35647499","proteopedia":"","reviewPanel":null,"species":["s. cerevisiae"],"integrations":[],"corrections":null,"history":{"received":"2022-03-21T18:26:26.097Z","revisionReceived":"2022-04-28T10:04:35.199Z","accepted":"2022-05-03T16:42:38.993Z","published":"2022-05-04T21:45:40.953Z","indexed":null},"versions":[{"id":"e59d08c9-1fb3-48a3-aaa4-8c0d8cc076a3","decision":"revise","abstract":"

The unicellular eukaryote S. cerevisiae is an invaluable resource for the study of basic eukaryotic cellular and molecular processes. However, due to its small size compared to other eukaryotic organisms the study of subcellular structures is challenging. Expansion microscopy (ExM) holds great potential to study the intracellular architecture of yeast, especially when paired with pan-labelling techniques visualising the full protein content inside cells. ExM allows to increase imaging resolution by physically enlarging a fixed sample that is embedded and cross-linked to a swellable gel followed by isotropic expansion in water. The cell wall present in fungi – including yeast – and Gram-positive bacteria is a resilient structure that resists denaturation and conventional digestion processes usually used in ExM protocols, resulting in uneven expansion. Thus, the digestion of the cell wall while maintaining the structure of the resulting protoplasts are crucial steps to ensure isotropic expansion. For this reason, specific experimental strategies are needed, and only a few protocols are currently available. We have developed a modified ExM protocol for S. cerevisiae, with 4x expansion factor, which allows the visualisation of the ultrastructure of the cells. Here, we describe the experimental procedure in detail, focusing on the most critical steps required to achieve isotropic expansion for ExM of S. cerevisiae.

","acknowledgements":"","authors":[{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["investigation","visualization","writing_originalDraft"],"email":"akorovesi@igc.gulbenkian.pt","firstName":"Artemis G.","lastName":"Korovesi","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":"","orcid":"https://orcid.org/0000-0003-1410-6757"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["investigation","visualization","writing_originalDraft"],"email":"lmorgado@igc.gulbenkian.pt","firstName":"Leonor","lastName":"Morgado","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":true,"WBId":"","orcid":"https://orcid.org/0000-0003-4510-8456"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["conceptualization","writing_reviewEditing"],"email":"mfumasoni@igc.gulbenkian.pt","firstName":"Marco","lastName":"Fumasoni","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":null,"WBId":"","orcid":"https://orcid.org/0000-0002-4507-7824"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal","MRC-Laboratory for Molecular Cell Biology. University College London, London, United Kingdom"],"departments":["",""],"credit":["conceptualization","writing_reviewEditing"],"email":"rjhenriques@igc.gulbenkian.pt","firstName":"Ricardo","lastName":"Henriques","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":null,"WBId":"","orcid":"https://orcid.org/0000-0002-2043-5234"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["supervision","dataCuration","writing_reviewEditing","conceptualization"],"email":"hsheil@igc.gulbenkian.pt","firstName":"Hannah S.","lastName":"Heil","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"https://orcid.org/0000-0003-4279-7022"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["supervision","dataCuration","writing_reviewEditing","conceptualization"],"email":"mrosario@igc.gulbenkian.pt","firstName":"Mario","lastName":"Del Rosario","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"https://orcid.org/0000-0002-0430-1463"}],"awards":null,"conflictsOfInterest":null,"dataTable":null,"extendedData":[{"description":"The video contains footage and instructions showing how to proceed throughout the protocol, focusing on the most critical steps. ","doi":null,"resourceType":"Audiovisual","name":"YeastExp_SuppVideo.mp4","url":"https://portal.micropublication.org/uploads/3e096e556e96bb41b3419ce8cd17a1aa.mp4"},{"description":"Supplementary figures of custom tools and troubleshooting table.","doi":null,"resourceType":"Text","name":"ExM_yeast_Supp.docx","url":"https://portal.micropublication.org/uploads/a512f2d5c8a9017f881bc69a9ab1c9d3.docx"}],"funding":"This work was supported by the Gulbenkian Foundation (AGK, LM, RH, MF, HSH, MDR) and received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. [101001332] to RH), the European Molecular Biology Organization (EMBO) (Installation Grant EMBO-2020-IG-4734 to RH and the postdoctoral fellowship ALTF 499-2021 to HSH) and the Wellcome Trust (203276/Z/16/Z to RH). MF is supported by The European Horizon 2020 Framework Programme, H2020-MSCA-IF-2020 - 101030203/ GENMAINEVO. LM is kindly supported by a joint-collaboration between Abbelight and the Instituto Gulbenkian de Ciência. AGK would like to acknowledge support from the Integrative Biology and Biomedicine (IBB) PhD programme from Instituto Gulbenkian de Ciência.","image":{"url":"https://portal.micropublication.org/uploads/6d92a01310d0edeee28f7d2b9bff5b16.png"},"imageCaption":"

a) Scheme of expansion microscopy protocol workflow. b) Maximum intensity projection of DIC/DAPI composite image of yeast, acquired with a Nikon HCS using a 100x/1.45 oil objective. c) Maximum intensity projection of fluorescence image of post-expanded yeast, acquired with a Zeiss LSM 980 using a 40x/1.1 water objective and airyscan detection. Signals of DAPI and DIC/NHS-Ester are represented in magenta and grey, respectively. Scale bars: 5 µm.

","imageTitle":"

Yeast expansion microscopy overview

","methods":"

Preparation of stock solutions:

Sorbitol buffer:

· 1.2M sorbitol solution in 0.1M KH2PO4.

Linking solution:

● 0.1 mg/mL acryloyl X-SE in PBS. Store in aliquots at -20°C.

Monomer solution:

Stocks:

● 38% sodium acrylate (w/w, diluted with ddH2O): 25 g in 65,79 ml, store at -20°C.

● 40% acrylamide stock: 20 g in 50 ml ddH2O, store at -20°C.

● 2% N,N’-Methylenebisacrylamide : 0.2 g in 10 ml ddH2O, store at -20°C.

Reagent

Final concentration

PBS

1x

Sodium acrylate*

19 g/100mL

Acrylamide

10 g/100mL

N,N’-Methylenebisacrylamide

0.1 g/100mL

Store in 493µl aliquots at -20 ºC.

*Sodium acrylate is provided with variable quality levels. One should test it before using it: the reagent should be fully dissolved and not show impurities in the solution, in the form of precipitates or discolouring.

Denaturation buffer:

Reagent

Final concentration

ddH2O

-

SDS

200 mM

NaCl

200 mM

Tris*

50 mM

Store in falcon tubes at -20 ºC.

*Adjust pH to 9.

Coverslip cleaning

Notes: i) This procedure should be performed under a fume hood, and one should use safety gloves when handling chloroform. ii) The chloroformand NaOH solutions can be reused. iii) The preparation of NaOH is an exothermic reaction, thus the NaOH stock solution should be prepared on an ice bath.

  1. Arrange single coverslips in a coverslip rack.

  2. Place a glass container in an ultrasonic bath and fill the bath with water.

  3. Place the rack in the glass container.

  4. Fill the glass container with chloroform and sonicate for 1 hour.

  5. Remove the rack with a tweezers/forceps and place it in an empty glass container. The coverslips will dry in a few minutes as the chloroform evaporates quickly.

  6. Pour the remaining chloroform back into the bottle through a funnel.

  7. Reinstall the rack in the glass container in the ultrasonic bath.

  8. Fill the glass container with a 5 M NaOH solution and sonicate for 1 hour.

  9. Pass the coverslips into a beaker with ddH2O and transfer the NaOH solution back into the bottle.

  10. Wash the coverslips three times in a volume of 500 mL fresh ddH2O.

  11. Pre-dry the coverslips with a nitrogen or filtered air gun and then place them in a drying cabinet until they are completely dried.

  12. Store in 100 % ethanol in a glass Petri dish.

Coverslip poly-L-lysine coating

  1. Dry the previously cleaned coverslips.

  2. Place the coverslips in a clean plastic plate, for example, in a 6-well plate.

  3. Add 300 µl of 0.1 mg/ml poly-L-lysine on each coverslip and incubate at room temperature (RT) for 10 minutes.

  4. Add water to each well until the coverslip is covered and incubate at RT for 5 minutes.

  5. Dry the coverslips.

  6. Store coverslips on a rack at 4 °C until needed for cell/gel mounting in needed.

Day 1

Yeast cells cultivation and fixation

The Saccharomyces cerevisiae BY4741 strain (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was used in this protocol.

  1. Culture yeast cells in the conditions required to assess the relevant biological question.

  2. Count yeast cells with a hemocytometer and calculate the volume required to collect 6x107 cells

  3. Centrifuge cells at 4,000 rpm for 2 minutes in a Falcon tube.

  4. Discard the supernatant and resuspend in 3mL of PBS 1X.

  5. Centrifuge cells at 4,000 rpm for 2 minutes in a Falcon tube.

  6. Resuspend the cells in 3mL of 4% paraformaldehyde (PFA), and fix them at 4 °C for 30 minutes in a multi-rotator.

  7. Centrifuge the fixed yeast cells at 4,000 rpm for 2 minutes.

  8. Discard the supernatant and resuspend in 3mL of PBS 1X.

  9. Centrifuge the fixed yeast cells at 4,000 rpm for 2 minutes.

  10. Discard the supernatant and resuspend in 6mL of sorbitol buffer.

Cell wall digestion

  1. Treat 200μl of fixed cells solution (2x106 cells) with 0.0075U/µl of the zymolyase enzyme and incubate the reaction at 30ºC for 3 minutes*.

  2. Place the samples on ice to block the enzymatic reaction.

* The length of the reaction provided is optimised for a population of exponentially growing cells. Samples collected in other conditions may require an adjusted treatment to optimise the cell wall digestion.

Mounting and fixating the cells on the coverslip

  1. Place the poly-L-lysine coated coverslips on a 6-well plate.

  2. Add 200 µL of treated cells to each coverslip and incubate them at RT for 10 minutes to allow the sample to sediment.

  3. Under a fume hood, add enough of the 4% PFA/1% Glutaraldehyde (GA) fixative to cover the sample and incubate at RT for 10 minutes.

  4. Wash 3x with PBS.

  5. Inspect the sample under a microscope to confirm that the cells are attached (i.e., cells are immobile and not floating).

Linking

  1. Remove PBS, add 1 mL of 0.1 mg/mL acryloyl X-SE solution in PBS to each well and incubate for 12 hours (overnight) at RT.

Day 2

Gelation

Note: Proceed quickly through steps 3 to 5, as after the gelation solution is prepared, it will polymerise in minutes. For this reason, it is recommended to proceed with these steps with a maximum of four samples each time. Reagents and slides are maintained on ice to prevent premature polymerisation.

  1. Cool the reagents (monomer solution, TEMED, and APS) and the gelation chamber slides (see supplementary figure 1) on ice for at least 10 minutes.

  2. Remove the excess solution from the coverslip with a lint-free wipe and place it next to the slide on which it will be mounted (Note: thecoverslip side with the sample should be facing up).

  3. Prepare the gelation solution: Add first the TEMED to the monomer solution, then APS and vortex. (Note: it is important to follow this order, as when APS is added the polymerisation reaction starts).

Volume

Reagents

Stock concentration

Final concentration

493 µL

Monomer solution

-

-

5 µL

APS

50%

0.5%

2.5 µL

TEMED

99%

0.5%

500 µL

Final Volume

-

-

  1. Add 120 µL of gelation solution in each gelation chamber, place the coverslip on top, with the side with the sample facing the solution, and gently press it. Carefully remove excess with a lint-free wipe.

  2. Keep the slides for 5 more minutes on ice.

  3. Incubate for 1 hour at 37 ºC in a humidified chamber.

Denaturation

  1. Preheat a heating block to 95°C.

  2. Thaw the denaturation buffer in a warm water bath and proceed only when the solution is clear.

  3. Carefully remove the coverslip from the slide and transfer the polymerised gel to a new 6-well plate (Note: sometimes the coverslip comes attached to the gel, and it can be transferred along).

  4. Add 2 mL of denaturation buffer to each well and incubate for 15 minutes at RT.

  5. Prepare an 1.5 mL Eppendorf tube for each sample and add 1 mL of fresh denaturation buffer in each of them.

  6. Transfer the gel into the Eppendorf tube with a metal spatula and incubate at 95 ºC for 90 minutes.

Expansion

  1. Carefully pour the gel from the Eppendorf tube to a big clean plastic plate (e.g., Petri dish with a diameter of at least 60 mm) and remove the excess of denaturation buffer solution.

  2. Add ddH2O to Petri dish until the gel is completely submerged.

  3. Change ddH2O 2x after 30 minutes.

  4. Expand in ddH2O overnight at RT.

Day 3

NHS and DAPI staining

  1. Remove ddH2O and measure the width and length of the gel after expansion (to then calculate the macro-expansion factor).

  2. Cut gel in small pieces using the custom cutter shape (see supplementary figure 2) and a razor blade.

  3. Select the pieces to be stained, cut the correct corner for reference of the gel orientation (see supplementary figure 1), and transfer them to a new 6-well plate with a metal spatula.

  4. Wash 2x for 15 minutes with PBS. (Note: the gels will shrink by 50%.)

  5. Prepare 10µg/mL NHS Ester and 300 µM DAPI in PBS (Note: confirm that the NHS and DAPI solution is completely homogenised).

  6. Add 2 mL to each well, wrap it in aluminium foil and incubate for 90 minutes at RT on a rocking platform.

  7. Wash 3x for 15 minutes with PBS+Tween-20 0.1%.

  8. Add ddH2O to each well until the gel is completely submerged.

  9. Change ddH2O 2x after 30 minutes.

  10. Expand in ddH2O overnight at RT.

Imaging day

Immobilisation of the gel

  1. Place a poly-L-Lysine coated coverslip in an Attofluor™ Cell Chamber (or another comparable mounting device).

  2. Carefully dry a piece of gel on a spatula using lint-free wipes and transfer it to the coverslip, with the sample facing the coverslip (use cut corner as reference of gel orientation).

  3. Incubate at RT for 7 minutes. Confirm if the gel is immobilised on the coverslip.

  4. Add ddH2O until the gel is completely submerged.

Information related to troubleshooting steps is provided in Supplementary Table 1.

","reagents":"

Compounds and materials

Supplier

Article number

Acrylamide

Sigma

A9099

Acryloyl-X SE

Invitrogen

A20770

APS - Ammonium Persulfate

Roth

9592.3

DAPI

Invitrogen

D1306

DMAA - N,N’-Methylenebisacrylamide

Sigma

M7279-25G

GA - Glutaraldehyde

Sigma

G5882-10X1ML

KH2PO4 - Potassium dihydrogen phosphate

Sangon Biotech

A100781

NaCl

Roth

HN00.2

NHS-Ester

Thermo Fisher Scientific

D2184

PFA - Paraformaldehyde

Thermo Fisher Scientific

43368

Poly-L-Lysine

Sigma

P8920

SDS - Sodium Dodecyl Sulfate

Sigma

L3771

Sodium acrylate

Sigma

408220

Sorbitol solution

Sangon Biotech

A100691

TEMED - N,N,N′,N′-Tetramethylethylenediamine

Sigma

T9281

Tris

Carl Roth

5429.1

Tween-20

Roth

9127.1

Zymolyase

Zymo research

E1004

AttofluorTM Cell Chamber

Thermofisher Scientific

A7816

Coverslip (24 mm round #1.5)

Marienfeld

117640

Coverslip rack

Diversified Biotech

WSDR-1000

Razor blade

Carl Roth

CK08.1

","patternDescription":"

The unicellular eukaryote S. cerevisiae represents an invaluable resource for the study of basic eukaryotic cellular and molecular processes. The combination of a high genetic amenability, numerous genetic tools, and vast genomic resources makes it one of the most versatile model organisms, employed in a wide range of basic research disciplines. However, its small size compared to other eukaryotic organisms has limited its use for the study of sub-cellular structures. The diameter of unbudded yeast cells ranges approximately between 4 μm in haploids and 6 μm in diploids (Milo and Phillips 2015), complicating conventional diffraction-limited light microscopy approaches.

The last decade witnessed the rise of super-resolution (SR) techniques that enable sub-diffraction resolution fluorescence imaging of cellular structures. Expansion microscopy (ExM), for instance, allows for increasing imaging resolution by physically enlarging a fixed sample that is embedded and cross-linked to a swellable gel and is then expanded isotropically in water (Wassie, et al. 2019). By doing so, fluorescently-labelled structures that previously would be too small or too close together to be distinguished can be resolved in 3 dimensions. The increased z-resolution can be exploited by z-sectioning, using confocal or light-sheet microscopy. In addition, ExM can also be used in combination with other super-resolutionmethods, such as airyscan confocal microscopy and Structured Illumination Microscopy (SIM), further enhancing resolution. ExM holds great potential to study the cellular structures of yeast, especially when paired with pan-labelling techniques to visualise total protein content inside cells. ExM protocols present a robust tool to study cellular organelles, obtaining super-resolution results with the advantage of employing inexpensive and straightforward equipment compared to other techniques that require more complex and expensive setups, such as optical SR approaches and EM.

ExM can be applied in various organisms and cellular models, offering outstanding outcomes with expansion factors ranging from 4x to 20x (Faulkner, et al. 2020; Truckenbrodt, et al. 2018). However, certain cellular organelles are resistant to these processes, such as the cell wall of fungi and Gram-positive bacteria. These organisms present a cell wall composed of peptidoglycans or glycoproteins that are highly resistant to denaturation and conventional digestion processes. The digestion of the cell wall and the maintenance of the structure of the resulting protoplasts are crucial steps to achieve isotropic expansion. For this reason, specific experimental strategies are needed, and only a few protocols are currently available. In particular, only one study describes an ExM protocol applied to S. cerevisiae, showing different yeast structures in high resolution (Chen, et al. 2021).

Here we describe an adapted and extended ExM protocol for S. cerevisiae (Figure 1) with a final expansion factor of 4x. Samples are stained with DAPI and the pan-labelling reagent NHS-Ester BODIPY FL, allowing an overview of the ultrastructure of the cells. Detailed step-by-step instructions are provided, supported by supplementary video documentation (Supplementary Video 1).

Briefly, the first day begins with the preparation and fixation of yeast cells, which includes the digestion of the cell wall with zymolyase. The generated protoplasts are mounted on the coverslip, and linking takes place overnight, during which linker molecules will attach to the sample’s proteins, acting as anchors for the next step. On the second day, the first stepisgelation, where the sample is embedded in a monomer solution that polymerases and forms a matrix to which the anchor molecules will be connected. This is followed by the denaturation step, where the sample structures are disintegrated. The samples are then left expanding overnight in water, swelling the polymerised gel, which will push the anchored molecules apart in an isotropic manner. Finally, on the third day, staining with DAPI and NHS-Ester takes place. Samples are left again expanding overnight, and they can be imaged from the next day onwards.

","references":[{"reference":"

Chen L, Yao L, Zhang L, Fei Yn, Mi L, Ma J. 2021. Applications of Super Resolution Expansion Microscopy in Yeast. Frontiers in Physics. 9:650353.

","pubmedId":"","doi":"10.3389/fphy.2021.650353"},{"reference":"Faulkner EL, Thomas SG, Neely RK. 2020. An introduction to the methodology of expansion microscopy. Int J Biochem Cell Biol 124: 105764.","pubmedId":"32407880","doi":""},{"reference":"Götz R, Panzer S, Trinks N, Eilts J, Wagener J, Turrà D, Di Pietro A, Sauer M, Terpitz U. 2020. Expansion Microscopy for Cell Biology Analysis in Fungi. Front Microbiol 11: 574.","pubmedId":"32318047","doi":""},{"reference":"

Milo, R., & Phillips, R. 2015. Cell Biology by the numbers (1st ed.). Garland Science.

","pubmedId":"","doi":"10.1201/9780429258770"},{"reference":"Truckenbrodt S, Maidorn M, Crzan D, Wildhagen H, Kabatas S, Rizzoli SO. 2018. X10 expansion microscopy enables 25-nm resolution on conventional microscopes. EMBO Rep 19: .","pubmedId":"29987134","doi":""},{"reference":"Wassie AT, Zhao Y, Boyden ES. 2019. Expansion microscopy: principles and uses in biological research. Nat Methods 16: 33-41.","pubmedId":"30573813","doi":""}],"title":"

Expansion Microscopy on Saccharomyces cerevisiae

","reviews":[{"reviewer":{"displayName":"Ulrich Terpitz"},"openAcknowledgement":true,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"8b67ae0a-336f-4be6-9bba-82a80539de00","decision":"edit","abstract":"

The unicellular eukaryote S. cerevisiae is an invaluable resource for the study of basic eukaryotic cellular and molecular processes. However, due to its small size compared to other eukaryotic organisms the study of subcellular structures is challenging. Expansion microscopy (ExM) holds great potential to study the intracellular architecture of yeast, especially when paired with pan-labelling techniques visualising the full protein content inside cells. ExM allows to increase imaging resolution by physically enlarging a fixed sample that is embedded and cross-linked to a swellable gel followed by isotropic expansion in water. The cell wall present in fungi – including yeast – and Gram-positive bacteria is a resilient structure that resists denaturation and conventional digestion processes usually used in ExM protocols, resulting in uneven expansion. Thus, the digestion of the cell wall while maintaining the structure of the resulting protoplasts are crucial steps to ensure isotropic expansion. For this reason, specific experimental strategies are needed, and only a few protocols are currently available. We have developed a modified ExM protocol for S. cerevisiae, with 4x expansion factor, which allows the visualisation of the ultrastructure of the cells. Here, we describe the experimental procedure in detail, focusing on the most critical steps required to achieve isotropic expansion for ExM of S. cerevisiae.

","acknowledgements":"","authors":[{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["investigation","visualization","writing_originalDraft"],"email":"akorovesi@igc.gulbenkian.pt","firstName":"Artemis G.","lastName":"Korovesi","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":"","orcid":"https://orcid.org/0000-0003-1410-6757"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["investigation","visualization","writing_originalDraft"],"email":"lmorgado@igc.gulbenkian.pt","firstName":"Leonor","lastName":"Morgado","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":true,"WBId":"","orcid":"https://orcid.org/0000-0003-4510-8456"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["conceptualization","writing_reviewEditing"],"email":"mfumasoni@igc.gulbenkian.pt","firstName":"Marco","lastName":"Fumasoni","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":null,"WBId":"","orcid":"https://orcid.org/0000-0002-4507-7824"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal","MRC-Laboratory for Molecular Cell Biology. University College London, London, United Kingdom"],"departments":["",""],"credit":["conceptualization","writing_reviewEditing"],"email":"rjhenriques@igc.gulbenkian.pt","firstName":"Ricardo","lastName":"Henriques","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":null,"WBId":"","orcid":"https://orcid.org/0000-0002-2043-5234"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["supervision","dataCuration","writing_reviewEditing","conceptualization"],"email":"hsheil@igc.gulbenkian.pt","firstName":"Hannah S.","lastName":"Heil","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"https://orcid.org/0000-0003-4279-7022"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["supervision","dataCuration","writing_reviewEditing","conceptualization"],"email":"mrosario@igc.gulbenkian.pt","firstName":"Mario","lastName":"Del Rosario","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"https://orcid.org/0000-0002-0430-1463"}],"awards":null,"conflictsOfInterest":null,"dataTable":null,"extendedData":[{"description":"Supplementary figures of custom tools and troubleshooting table.","doi":null,"resourceType":"Text","name":"ExM_yeast_Supp.docx","url":"https://portal.micropublication.org/uploads/a512f2d5c8a9017f881bc69a9ab1c9d3.docx"},{"description":"Supplementary Video 1. The video contains footage and instructions showing how to proceed throughout the protocol, focusing on the most critical steps. ","doi":null,"resourceType":"Audiovisual","name":"YeastExM_SuppVideo.mp4","url":"https://portal.micropublication.org/uploads/f9a5a37aca86fa241279a8a1e6fa2b82.mp4"}],"funding":"This work was supported by the Gulbenkian Foundation (AGK, LM, RH, MF, HSH, MDR) and received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. [101001332] to RH), the European Molecular Biology Organization (EMBO) (Installation Grant EMBO-2020-IG-4734 to RH and the postdoctoral fellowship ALTF 499-2021 to HSH) and the Wellcome Trust (203276/Z/16/Z to RH). MF is supported by The European Horizon 2020 Framework Programme, H2020-MSCA-IF-2020 - 101030203/ GENMAINEVO. LM is kindly supported by a joint-collaboration between Abbelight and the Instituto Gulbenkian de Ciência. AGK would like to acknowledge support from the Integrative Biology and Biomedicine (IBB) PhD programme from Instituto Gulbenkian de Ciência.","image":{"url":"https://portal.micropublication.org/uploads/6d92a01310d0edeee28f7d2b9bff5b16.png"},"imageCaption":"

a) Scheme of expansion microscopy protocol workflow. b) Maximum intensity projection of DIC/DAPI composite image of yeast, acquired with a Nikon HCS using a 100x/1.45 oil objective. c) Maximum intensity projection of fluorescence image of post-expanded yeast, acquired with a Zeiss LSM 980 using a 40x/1.1 water objective and airyscan detection. Signals of DAPI and DIC/NHS-Ester are represented in magenta and grey, respectively. Scale bars: 5 µm. Samples were assessed by manual segmentation and measurement of the area and circularity between expanded (n=13) and non-expanded (n=27) specimens. The average expansion factor across all experiments was 3.52 ± 0.42 (mean ± standard deviation) while the circularity was retained within the level 20% of the population’s standard deviation.

","imageTitle":"

Yeast expansion microscopy overview

","methods":"

Preparation of stock solutions:

Sorbitol buffer:

● 1.2 M sorbitol solution in 0.1 M KH2PO4.

Linking solution:

● 0.1 mg/mL acryloyl X-SE in PBS. Store in aliquots at -20 °C.

Monomer solution:

Stocks:

● 38% sodium acrylate (w/w, diluted with ddH2O): 25 g in 65.79 mL, store at -20 °C.

● 40% acrylamide stock: 20 g in 50 mL ddH2O, store at -20°C.

● 2% N,N’-Methylenebisacrylamide : 0.2 g in 10 mL ddH2O, store at -20 °C.

Reagent

Final concentration

PBS

1x

Sodium acrylate*

19 g/100 mL

Acrylamide

10 g/100 mL

N,N’-Methylenebisacrylamide

0.1 g/100 mL

Store in 493 µL aliquots at -20 °C.

*Sodium acrylate is provided with variable quality levels. One should test it before using it: the reagent should be fully dissolved and not show impurities in the solution, in the form of precipitates or discolouring.

Denaturation buffer:

Reagent

Final concentration

ddH2O

-

SDS

200 mM

NaCl

200 mM

Tris*

50 mM

Store in falcon tubes at -20 °C.

*Adjust pH to 9.

YPD Media:

1. Dissolve 5 g of yeast extract and 10 g of peptone in 375 mL of ddH2O by manually stirring the flask and autoclave the solution.

2. Add 100 mL of ddH2O, 20 mL of autoclaved 50% Glucose (Dextrose) and 5 mL 1% Adenine + 1% Tryptophan.

Coverslip cleaning

Notes: i) This procedure should be performed under a fume hood, and one should use safety gloves when handling chloroform. ii) The chloroform and NaOH solutions can be reused. iii) The preparation of NaOH is an exothermic reaction, thus the NaOH stock solution should be prepared on an ice bath. iv) Other coverslip cleaning protocols are available, such as a chloroform-free protocol (Pereira, et al. 2015).

  1. Arrange single coverslips in a coverslip rack.

  2. Place a glass container in an ultrasonic bath and fill the bath with water.

  3. Place the rack in the glass container.

  4. Fill the glass container with chloroform and sonicate for 1 hour.

  5. Remove the rack with a tweezers/forceps and place it in an empty glass container. The coverslips will dry in a few minutes as the chloroform evaporates quickly.

  6. Pour the remaining chloroform back into the bottle through a funnel.

  7. Reinstall the rack in the glass container in the ultrasonic bath.

  8. Fill the glass container with a 5 M NaOH solution and sonicate for 1 hour.

  9. Pass the coverslips into a beaker with ddH2O and transfer the NaOH solution back into the bottle.

  10. Wash the coverslips three times in a volume of 500 mL fresh ddH2O.

  11. Pre-dry the coverslips with a nitrogen or filtered air gun and then place them in a drying cabinet until they are completely dried.

  12. Store in 100 % ethanol in a glass Petri dish.

Coverslip poly-L-lysine coating

  1. Dry the previously cleaned coverslips.

  2. Place the coverslips in a clean plastic plate, for example, in a 6-well plate.

  3. Add 300 µL of 0.1 mg/mL poly-L-lysine on each coverslip and incubate at room temperature (RT) for 10 minutes.

  4. Add water to each well until the coverslip is covered and incubate at RT for 5 minutes.

  5. Dry the coverslips.

  6. Store coverslips on a rack at 4 °C until needed for cell/gel mounting in needed.

Day 1

Yeast cells cultivation and fixation

The Saccharomyces cerevisiae BY4741 strain (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was used in this protocol.

  1. Culture yeast cells in the conditions required to assess the relevant biological question. (In our case, we incubate yeast cells in fresh YPD media supplemented with adenine and tryptophan at 30 °C and 220 rpm of agitation until the culture growth reaches the exponential phase).

  2. Count yeast cells with a hemocytometer and calculate the volume required to collect 6x107 cells.

  3. Centrifuge cells at 4,000 rpm for 2 minutes in a Falcon tube.

  4. Discard the supernatant and resuspend in 3 mL of PBS 1X.

  5. Centrifuge cells at 4,000 rpm for 2 minutes in a Falcon tube.

  6. Resuspend the cells in 3 mL of 4% paraformaldehyde (PFA), and fix them at 4 °C for 30 minutes in a multi-rotator.

  7. Centrifuge the fixed yeast cells at 4,000 rpm for 2 minutes.

  8. Discard the supernatant and resuspend in 3 mL of PBS 1X.

  9. Centrifuge the fixed yeast cells at 4,000 rpm for 2 minutes.

  10. Discard the supernatant and resuspend in 6 mL sorbitol buffer.

Cell wall digestion

  1. Add 0.3 μL of zymolyase (5 U/μL) to 200 μL of fixed cells solution (2x106 cells) for a final concentration of 0.0075 U/µL and incubate the reaction at 30 °C for 3 minutes*.

  2. Place the samples on ice to block the enzymatic reaction.

* The length of the reaction provided is optimised for a population of exponentially growing cells. Samples collected in other conditions may require an adjusted treatment to optimise the cell wall digestion.

Mounting and fixating the cells on the coverslip

  1. Place the poly-L-lysine coated coverslips on a 6-well plate.

  2. Add 200 µL of treated cells to each coverslip and incubate them at RT for 10 minutes to allow the sample to sediment.

  3. Under a fume hood, add enough of the 4% PFA/1% Glutaraldehyde (GA) fixative to cover the sample and incubate at RT for 10 minutes.

  4. Wash 3x with PBS.

  5. Inspect the sample under a microscope to confirm that the cells are attached (i.e., cells are immobile and not floating) and if the majority of cells present no cell wall.

  6. If needed to calculate the cell expansion factor, image the cells at this time point to measure dimensions such as area or diameter of cells before expansion.

Linking

  1. Remove PBS, add 1 mL of 0.1 mg/mL acryloyl X-SE solution in PBS to each well and incubate for 12 hours (overnight) at RT.

Day 2

Gelation

Note: Proceed quickly through steps 3 to 5, as after the gelation solution is prepared, it will polymerise in minutes. For this reason, it is recommended to proceed with these steps with a maximum of four samples each time. Reagents and slides are maintained on ice to prevent premature polymerisation.

  1. Cool the reagents (monomer solution, TEMED, and APS) and the gelation chamber slides (see supplementary figure 1) on ice for at least 10 minutes.

  2. Remove the excess solution from the coverslip with a lint-free wipe and place it next to the slide on which it will be mounted (Note: the coverslip side with the sample should be facing up).

  3. Prepare the gelation solution: Add first the TEMED to the monomer solution, then APS and vortex. (Note: it is important to follow this order, as when APS is added the polymerisation reaction starts).

Volume

Reagents

Stock concentration

Final concentration

493 µL

Monomer solution

-

-

5 µL

APS

50%

0.5%

2.5 µL

TEMED

99%

0.5%

500 µL

Final Volume

-

-

  1. Add 120 µL of gelation solution in each gelation chamber, place the coverslip on top, with the side with the sample facing the solution, and gently press it. Carefully remove excess with a lint-free wipe.

  2. Keep the slides for 5 more minutes on ice.

  3. Incubate for 1 hour at 37 °C in a humidified chamber.

Denaturation

  1. Preheat a heating block to 95 °C.

  2. Thaw the denaturation buffer in a warm water bath and proceed only when the solution is clear.

  3. Carefully remove the coverslip from the slide and transfer the polymerised gel to a new 6-well plate (Note: sometimes the coverslip comes attached to the gel, and it can be transferred along).

  4. Measure the width and length of the polymerised gel before expansion.

  5. Add 2 mL of denaturation buffer to each well and incubate for 15 minutes at RT.

  6. Prepare an 1.5 mL Eppendorf tube for each sample and add 1 mL of fresh denaturation buffer in each of them.

  7. Transfer the gel into the Eppendorf tube with a metal spatula and incubate at 95 °C for 90 minutes.

Expansion

  1. Carefully pour the gel from the Eppendorf tube to a big clean plastic plate (e.g., Petri dish with a diameter of at least 60 mm) and remove the excess of denaturation buffer solution.

  2. Add ddH2O to Petri dish until the gel is completely submerged.

  3. Change ddH2O 2x after 30 minutes.

  4. Expand in ddH2O overnight at RT.

Day 3

NHS and DAPI staining

  1. Remove ddH2O.

  2. Measure the width and length of the gel after expansion. Divide these values by the ones measured before expansion to calculate the macro-expansion factor, which should be approximately 4x.

  3. Cut gel in small pieces using the custom cutter shape (see supplementary figure 2) and a razor blade.

  4. Select the pieces to be stained, cut the correct corner for reference of the gel orientation (see supplementary figure 1), and transfer them to a new 6-well plate with a metal spatula.

  5. Wash 2x for 15 minutes with PBS. (Note: the gels will shrink by 50%.)

  6. Prepare 10 µg/mL NHS Ester and 300 µM DAPI in PBS (Note: confirm that the NHS and DAPI solution is completely homogenised).

  7. Add 2 mL to each well, wrap it in aluminium foil and incubate for 90 minutes at RT on a rocking platform.

  8. Wash 3x for 15 minutes with PBS+Tween-20 0.1%.

  9. Add ddH2O to each well until the gel is completely submerged.

  10. Change ddH2O 2x after 30 minutes.

  11. Expand in ddH2O overnight at RT.

Imaging day

Immobilisation of the gel

  1. Place a poly-L-Lysine coated coverslip in an Attofluor™ Cell Chamber (or another comparable mounting device).

  2. Carefully dry a piece of gel on a spatula using lint-free wipes and transfer it to the coverslip, with the sample facing the coverslip (use cut corner as reference of gel orientation).

  3. Incubate at RT for 7 minutes. Confirm if the gel is immobilised on the coverslip.

  4. Add ddH2O until the gel is completely submerged.

","reagents":"

Resources

Supplier

Article number

Acrylamide

Sigma

A9099

Acryloyl-X SE

Invitrogen

A20770

Adenine

Sigma

A9126

APS - Ammonium Persulfate

Roth

9592.3

DAPI

Invitrogen

D1306

DMAA - N,N’-Methylenebisacrylamide

Sigma

M7279-25G

GA - Glutaraldehyde

Sigma

G5882-10X1ML

Glucose (Dextrose)

Sigma

G7021

KH2PO4 - Potassium dihydrogen phosphate

Sangon Biotech

A100781

NaCl

Roth

HN00.2

NHS-Ester

Thermo Fisher Scientific

D2184

Peptone

Thermo Fisher Scientific

211820

PFA – Paraformaldehyde

Thermo Fisher Scientific

43368

Poly-L-Lysine

Sigma

P8920

SDS – Sodium Dodecyl Sulfate

Sigma

L3771

Sodium acrylate

Sigma

408220

Sorbitol solution

Sangon Biotech

A100691

TEMED – N,N,N′,N′-Tetramethylethylenediamine

Sigma

T9281

Tris

Carl Roth

5429.1

Tryptophan

Sigma

T0254

Tween-20

Roth

9127.1

Yeast extract

Thermo Fisher Scientific

288620

Zymolyase enzyme

Zymo research

E1004

Attofluor™ Cell Chamber

Thermo Fisher Scientific

A7816

Coverslip (24 mm round #1.5)

Marienfeld

117640

Coverslip rack

Diversified Biotech

WSDR-1000

Razor blade

Carl Roth

CK08.1

","patternDescription":"

The unicellular eukaryote S. cerevisiae represents an invaluable resource for the study of basic eukaryotic cellular and molecular processes. The combination of a high genetic amenability, numerous genetic tools, and vast genomic resources makes it one of the most versatile model organisms, employed in a wide range of basic research disciplines. However, its small size compared to other eukaryotic organisms has limited its use for the study of sub-cellular structures. The diameter of unbudded yeast cells ranges approximately between 4 μm in haploids and 6 μm in diploids (Milo and Phillips 2015), complicating conventional diffraction-limited light microscopy approaches.

The last decade witnessed the rise of super-resolution (SR) techniques that enable sub-diffraction resolution fluorescence imaging of cellular structures. Expansion microscopy (ExM), for instance, allows for increasing imaging resolution by physically enlarging a fixed sample that is embedded and cross-linked to a swellable gel and is then expanded isotropically in water (Wassie, et al. 2019). By doing so, fluorescently-labelled structures that previously would be too small or too close together to be distinguished can be resolved in 3 dimensions. The increased z-resolution can be exploited by z-sectioning, using confocal or light-sheet microscopy. In addition, ExM can also be used in combination with other super-resolution methods, such as airyscan confocal microscopy and Structured Illumination Microscopy (SIM), further enhancing resolution. ExM holds great potential to study the cellular structures of yeast, especially when paired with pan-labelling techniques to visualise total protein content inside cells. ExM protocols present a robust tool to study cellular organelles, obtaining super-resolution results with the advantage of employing inexpensive and straightforward equipment compared to other techniques that require more complex and expensive setups, such as optical SR approaches and EM.

ExM can be applied in various organisms and cellular models, offering outstanding outcomes with expansion factors ranging from 4x to 20x (Faulkner, et al. 2020; Truckenbrodt, et al. 2018). However, certain cellular organelles are resistant to these processes, such as the cell wall of fungi and Gram-positive bacteria. These organisms present a cell wall composed of peptidoglycans or glycoproteins that are highly resistant to denaturation and conventional digestion processes. The digestion of the cell wall and the maintenance of the structure of the resulting protoplasts are crucial steps to achieve isotropic expansion. For this reason, specific experimental strategies are needed, and only a few protocols are currently available. In particular, only one study describes an ExM protocol applied to S. cerevisiae, showing different yeast structures in high resolution (Chen, et al. 2021).

Here we describe an adapted and extended ExM protocol for S. cerevisiae (Figure 1) with a final expansion factor of ~3.5x. The major difference regarding to the protocol of Chen et al., is the staining strategy. While the previously published protocol is based on immunolabeling to visualise and super-resolve specific target proteins, here, samples are stained with DAPI and the pan-labelling reagent NHS-Ester BODIPY FL. This allows to provide an overview of the ultrastructural context of the whole cells. However, both protocols complement each other and could be combined, in principle. Here, we have compiled detailed step-by-step instructions for ExM to visualise the ultrastructure of S. cerevisiae, supported by supplementary video documentation (Supplementary Video 1).

Briefly, the first day begins with the preparation and fixation of yeast cells, which includes the digestion of the cell wall with zymolyase. The generated protoplasts are mounted on the coverslip, and linking takes place overnight, during which linker molecules will attach to the sample’s proteins, acting as anchors for the next step. On the second day, the first step is gelation, where the sample is embedded in a monomer solution that polymerases and forms a matrix to which the anchor molecules will be connected. This is followed by the denaturation step, where the sample structures are disintegrated. The samples are then left expanding overnight in water, swelling the polymerised gel, which will push the anchored molecules apart in an isotropic manner. Finally, on the third day, staining with DAPI and NHS-Ester takes place. Samples are left again expanding overnight, and they can be imaged from the next day onwards.

Assessing the expansion factor and specimen cellular integrity following sample preparation is a critical step that requires consideration. During sample expansion, the denaturation/digestion steps facilitate isotropic expansion of the sample but different organelle structures might possess specific requirements that need to be assessed during the experimental planning phase of the experiment. The simplest solution to assess specimen integrity and expansion factor is to acquire microscopy data of the same field of view and specimen before and after expansion. This process will allow the user to detect immediate changes in the sample but carries the complication of being difficult to execute since finding the same sample, particularly in yeast, can be difficult and time-consuming. Other options, such as the one included in this protocol, image multiple expanded and non-expanded cells and compare their features (area and circularity) as means to assess expansion (average circularity 0.892 ± 0.063 on expanded samples (mean ± standard deviation, n=13) and 0.906 ± 0.035 on non-expanded ones (n=27)). Individual organelle expansion should be assessed via specific labelling of the structure and assess before and after expansion sizes and compare it to the expansion factor of the whole cell.

","references":[{"reference":"

Chen L, Yao L, Zhang L, Fei Yn, Mi L, Ma J. 2021. Applications of Super Resolution Expansion Microscopy in Yeast. Frontiers in Physics. 9:650353.

","pubmedId":"","doi":"10.3389/fphy.2021.650353"},{"reference":"Faulkner EL, Thomas SG, Neely RK. 2020. An introduction to the methodology of expansion microscopy. Int J Biochem Cell Biol 124: 105764.","pubmedId":"32407880","doi":""},{"reference":"Götz R, Panzer S, Trinks N, Eilts J, Wagener J, Turrà D, Di Pietro A, Sauer M, Terpitz U. 2020. Expansion Microscopy for Cell Biology Analysis in Fungi. Front Microbiol 11: 574.","pubmedId":"32318047","doi":""},{"reference":"

Milo, R., & Phillips, R. 2015. Cell Biology by the numbers (1st ed.). Garland Science.

","pubmedId":"","doi":"10.1201/9780429258770"},{"reference":"Pereira PM, Almada P, Henriques R. 2015. High-content 3D multicolor super-resolution localization microscopy. Methods Cell Biol 125: 95-117.","pubmedId":"25640426","doi":""},{"reference":"Truckenbrodt S, Maidorn M, Crzan D, Wildhagen H, Kabatas S, Rizzoli SO. 2018. X10 expansion microscopy enables 25-nm resolution on conventional microscopes. EMBO Rep 19: .","pubmedId":"29987134","doi":""},{"reference":"Wassie AT, Zhao Y, Boyden ES. 2019. Expansion microscopy: principles and uses in biological research. Nat Methods 16: 33-41.","pubmedId":"30573813","doi":""}],"title":"

Expansion Microscopy on Saccharomyces cerevisiae

","reviews":[],"curatorReviews":[{"curator":{"displayName":"Stacia Engel"},"openAcknowledgement":false,"submitted":"1771972776439"}]},{"id":"fc5df250-95b9-4476-ac2b-f1d72751a59d","decision":"accept","abstract":"

The unicellular eukaryote S. cerevisiae is an invaluable resource for the study of basic eukaryotic cellular and molecular processes. However, due to its small size compared to other eukaryotic organisms the study of subcellular structures is challenging. Expansion microscopy (ExM) holds great potential to study the intracellular architecture of yeast, especially when paired with pan-labelling techniques visualising the full protein content inside cells. ExM allows to increase imaging resolution by physically enlarging a fixed sample that is embedded and cross-linked to a swellable gel followed by isotropic expansion in water. The cell wall present in fungi – including yeast – and Gram-positive bacteria is a resilient structure that resists denaturation and conventional digestion processes usually used in ExM protocols, resulting in uneven expansion. Thus, the digestion of the cell wall while maintaining the structure of the resulting protoplasts are crucial steps to ensure isotropic expansion. For this reason, specific experimental strategies are needed, and only a few protocols are currently available. We have developed a modified ExM protocol for S. cerevisiae, with 4x expansion factor, which allows the visualisation of the ultrastructure of the cells. Here, we describe the experimental procedure in detail, focusing on the most critical steps required to achieve isotropic expansion for ExM of S. cerevisiae.

","acknowledgements":"","authors":[{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["investigation","visualization","writing_originalDraft"],"email":"akorovesi@igc.gulbenkian.pt","firstName":"Artemis G.","lastName":"Korovesi","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":"","orcid":"https://orcid.org/0000-0003-1410-6757"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["investigation","visualization","writing_originalDraft"],"email":"lmorgado@igc.gulbenkian.pt","firstName":"Leonor","lastName":"Morgado","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":true,"WBId":"","orcid":"https://orcid.org/0000-0003-4510-8456"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["conceptualization","writing_reviewEditing"],"email":"mfumasoni@igc.gulbenkian.pt","firstName":"Marco","lastName":"Fumasoni","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":null,"WBId":"","orcid":"https://orcid.org/0000-0002-4507-7824"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal","MRC-Laboratory for Molecular Cell Biology. University College London, London, United Kingdom"],"departments":["",""],"credit":["conceptualization","writing_reviewEditing"],"email":"rjhenriques@igc.gulbenkian.pt","firstName":"Ricardo","lastName":"Henriques","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":null,"WBId":"","orcid":"https://orcid.org/0000-0002-2043-5234"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["supervision","dataCuration","writing_reviewEditing","conceptualization"],"email":"hsheil@igc.gulbenkian.pt","firstName":"Hannah S.","lastName":"Heil","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"https://orcid.org/0000-0003-4279-7022"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["supervision","dataCuration","writing_reviewEditing","conceptualization"],"email":"mrosario@igc.gulbenkian.pt","firstName":"Mario","lastName":"Del Rosario","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"https://orcid.org/0000-0002-0430-1463"}],"awards":null,"conflictsOfInterest":null,"dataTable":null,"extendedData":[{"description":"Supplementary figures of custom tools and troubleshooting table.","doi":"10.22002/D1.20159","resourceType":"Text","name":"ExM_yeast_Supp.docx","url":"https://portal.micropublication.org/uploads/a512f2d5c8a9017f881bc69a9ab1c9d3.docx"},{"description":"Supplementary Video 1. The video contains footage and instructions showing how to proceed throughout the protocol, focusing on the most critical steps. ","doi":"10.22002/D1.20160","resourceType":"Audiovisual","name":"YeastExM_SuppVideo.mp4","url":"https://portal.micropublication.org/uploads/f9a5a37aca86fa241279a8a1e6fa2b82.mp4"}],"funding":"This work was supported by the Gulbenkian Foundation (AGK, LM, RH, MF, HSH, MDR) and received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. [101001332] to RH), the European Molecular Biology Organization (EMBO) (Installation Grant EMBO-2020-IG-4734 to RH and the postdoctoral fellowship ALTF 499-2021 to HSH) and the Wellcome Trust (203276/Z/16/Z to RH). MF is supported by The European Horizon 2020 Framework Programme, H2020-MSCA-IF-2020 - 101030203/ GENMAINEVO. LM is kindly supported by a joint-collaboration between Abbelight and the Instituto Gulbenkian de Ciência. AGK would like to acknowledge support from the Integrative Biology and Biomedicine (IBB) PhD programme from Instituto Gulbenkian de Ciência.","image":{"url":"https://portal.micropublication.org/uploads/6d92a01310d0edeee28f7d2b9bff5b16.png"},"imageCaption":"

a) Scheme of expansion microscopy protocol workflow. b) Maximum intensity projection of DIC/DAPI composite image of yeast, acquired with a Nikon HCS using a 100x/1.45 oil objective. c) Maximum intensity projection of fluorescence image of post-expanded yeast, acquired with a Zeiss LSM 980 using a 40x/1.1 water objective and airyscan detection. Signals of DAPI and DIC/NHS-Ester are represented in magenta and grey, respectively. Scale bars: 5 µm. Samples were assessed by manual segmentation and measurement of the area and circularity between expanded (n=13) and non-expanded (n=27) specimens. The average expansion factor across all experiments was 3.52 ± 0.42 (mean ± standard deviation) while the circularity was retained within the level 20% of the population’s standard deviation.

","imageTitle":"

Yeast expansion microscopy overview

","methods":"

Preparation of stock solutions:

Sorbitol buffer:

● 1.2 M sorbitol solution in 0.1 M KH2PO4.

Linking solution:

● 0.1 mg/mL acryloyl X-SE in PBS. Store in aliquots at -20 °C.

Monomer solution:

Stocks:

● 38% sodium acrylate (w/w, diluted with ddH2O): 25 g in 65.79 mL, store at -20 °C.

● 40% acrylamide stock: 20 g in 50 mL ddH2O, store at -20°C.

● 2% N,N’-Methylenebisacrylamide : 0.2 g in 10 mL ddH2O, store at -20 °C.

Reagent

Final concentration

PBS

1x

Sodium acrylate*

19 g/100 mL

Acrylamide

10 g/100 mL

N,N’-Methylenebisacrylamide

0.1 g/100 mL

Store in 493 µL aliquots at -20 °C.

*Sodium acrylate is provided with variable quality levels. One should test it before using it: the reagent should be fully dissolved and not show impurities in the solution, in the form of precipitates or discolouring.

Denaturation buffer:

Reagent

Final concentration

ddH2O

-

SDS

200 mM

NaCl

200 mM

Tris*

50 mM

Store in falcon tubes at -20 °C.

*Adjust pH to 9.

YPD Media:

1. Dissolve 5 g of yeast extract and 10 g of peptone in 375 mL of ddH2O by manually stirring the flask and autoclave the solution.

2. Add 100 mL of ddH2O, 20 mL of autoclaved 50% Glucose (Dextrose) and 5 mL 1% Adenine + 1% Tryptophan.

Coverslip cleaning

Notes: i) This procedure should be performed under a fume hood, and one should use safety gloves when handling chloroform. ii) The chloroform and NaOH solutions can be reused. iii) The preparation of NaOH is an exothermic reaction, thus the NaOH stock solution should be prepared on an ice bath. iv) Other coverslip cleaning protocols are available, such as a chloroform-free protocol (Pereira, et al. 2015).

  1. Arrange single coverslips in a coverslip rack.

  2. Place a glass container in an ultrasonic bath and fill the bath with water.

  3. Place the rack in the glass container.

  4. Fill the glass container with chloroform and sonicate for 1 hour.

  5. Remove the rack with a tweezers/forceps and place it in an empty glass container. The coverslips will dry in a few minutes as the chloroform evaporates quickly.

  6. Pour the remaining chloroform back into the bottle through a funnel.

  7. Reinstall the rack in the glass container in the ultrasonic bath.

  8. Fill the glass container with a 5 M NaOH solution and sonicate for 1 hour.

  9. Pass the coverslips into a beaker with ddH2O and transfer the NaOH solution back into the bottle.

  10. Wash the coverslips three times in a volume of 500 mL fresh ddH2O.

  11. Pre-dry the coverslips with a nitrogen or filtered air gun and then place them in a drying cabinet until they are completely dried.

  12. Store in 100 % ethanol in a glass Petri dish.

Coverslip poly-L-lysine coating

  1. Dry the previously cleaned coverslips.

  2. Place the coverslips in a clean plastic plate, for example, in a 6-well plate.

  3. Add 300 µL of 0.1 mg/mL poly-L-lysine on each coverslip and incubate at room temperature (RT) for 10 minutes.

  4. Add water to each well until the coverslip is covered and incubate at RT for 5 minutes.

  5. Dry the coverslips.

  6. Store coverslips on a rack at 4 °C until needed for cell/gel mounting in needed.

Day 1

Yeast cells cultivation and fixation

The Saccharomyces cerevisiae BY4741 strain (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was used in this protocol.

  1. Culture yeast cells in the conditions required to assess the relevant biological question. (In our case, we incubate yeast cells in fresh YPD media supplemented with adenine and tryptophan at 30 °C and 220 rpm of agitation until the culture growth reaches the exponential phase).

  2. Count yeast cells with a hemocytometer and calculate the volume required to collect 6x107 cells.

  3. Centrifuge cells at 4,000 rpm for 2 minutes in a Falcon tube.

  4. Discard the supernatant and resuspend in 3 mL of PBS 1X.

  5. Centrifuge cells at 4,000 rpm for 2 minutes in a Falcon tube.

  6. Resuspend the cells in 3 mL of 4% paraformaldehyde (PFA), and fix them at 4 °C for 30 minutes in a multi-rotator.

  7. Centrifuge the fixed yeast cells at 4,000 rpm for 2 minutes.

  8. Discard the supernatant and resuspend in 3 mL of PBS 1X.

  9. Centrifuge the fixed yeast cells at 4,000 rpm for 2 minutes.

  10. Discard the supernatant and resuspend in 6 mL sorbitol buffer.

Cell wall digestion

  1. Add 0.3 μL of zymolyase (5 U/μL) to 200 μL of fixed cells solution (2x106 cells) for a final concentration of 0.0075 U/µL and incubate the reaction at 30 °C for 3 minutes*.

  2. Place the samples on ice to block the enzymatic reaction.

* The length of the reaction provided is optimised for a population of exponentially growing cells. Samples collected in other conditions may require an adjusted treatment to optimise the cell wall digestion.

Mounting and fixating the cells on the coverslip

  1. Place the poly-L-lysine coated coverslips on a 6-well plate.

  2. Add 200 µL of treated cells to each coverslip and incubate them at RT for 10 minutes to allow the sample to sediment.

  3. Under a fume hood, add enough of the 4% PFA/1% Glutaraldehyde (GA) fixative to cover the sample and incubate at RT for 10 minutes.

  4. Wash 3x with PBS.

  5. Inspect the sample under a microscope to confirm that the cells are attached (i.e., cells are immobile and not floating) and if the majority of cells present no cell wall.

  6. If needed to calculate the cell expansion factor, image the cells at this time point to measure dimensions such as area or diameter of cells before expansion.

Linking

  1. Remove PBS, add 1 mL of 0.1 mg/mL acryloyl X-SE solution in PBS to each well and incubate for 12 hours (overnight) at RT.

Day 2

Gelation

Note: Proceed quickly through steps 3 to 5, as after the gelation solution is prepared, it will polymerise in minutes. For this reason, it is recommended to proceed with these steps with a maximum of four samples each time. Reagents and slides are maintained on ice to prevent premature polymerisation.

  1. Cool the reagents (monomer solution, TEMED, and APS) and the gelation chamber slides (see supplementary figure 1) on ice for at least 10 minutes.

  2. Remove the excess solution from the coverslip with a lint-free wipe and place it next to the slide on which it will be mounted (Note: the coverslip side with the sample should be facing up).

  3. Prepare the gelation solution: Add first the TEMED to the monomer solution, then APS and vortex. (Note: it is important to follow this order, as when APS is added the polymerisation reaction starts).

Volume

Reagents

Stock concentration

Final concentration

493 µL

Monomer solution

-

-

5 µL

APS

50%

0.5%

2.5 µL

TEMED

99%

0.5%

500 µL

Final Volume

-

-

  1. Add 120 µL of gelation solution in each gelation chamber, place the coverslip on top, with the side with the sample facing the solution, and gently press it. Carefully remove excess with a lint-free wipe.

  2. Keep the slides for 5 more minutes on ice.

  3. Incubate for 1 hour at 37 °C in a humidified chamber.

Denaturation

  1. Preheat a heating block to 95 °C.

  2. Thaw the denaturation buffer in a warm water bath and proceed only when the solution is clear.

  3. Carefully remove the coverslip from the slide and transfer the polymerised gel to a new 6-well plate (Note: sometimes the coverslip comes attached to the gel, and it can be transferred along).

  4. Measure the width and length of the polymerised gel before expansion.

  5. Add 2 mL of denaturation buffer to each well and incubate for 15 minutes at RT.

  6. Prepare an 1.5 mL Eppendorf tube for each sample and add 1 mL of fresh denaturation buffer in each of them.

  7. Transfer the gel into the Eppendorf tube with a metal spatula and incubate at 95 °C for 90 minutes.

Expansion

  1. Carefully pour the gel from the Eppendorf tube to a big clean plastic plate (e.g., Petri dish with a diameter of at least 60 mm) and remove the excess of denaturation buffer solution.

  2. Add ddH2O to Petri dish until the gel is completely submerged.

  3. Change ddH2O 2x after 30 minutes.

  4. Expand in ddH2O overnight at RT.

Day 3

NHS and DAPI staining

  1. Remove ddH2O.

  2. Measure the width and length of the gel after expansion. Divide these values by the ones measured before expansion to calculate the macro-expansion factor, which should be approximately 4x.

  3. Cut gel in small pieces using the custom cutter shape (see supplementary figure 2) and a razor blade.

  4. Select the pieces to be stained, cut the correct corner for reference of the gel orientation (see supplementary figure 1), and transfer them to a new 6-well plate with a metal spatula.

  5. Wash 2x for 15 minutes with PBS. (Note: the gels will shrink by 50%.)

  6. Prepare 10 µg/mL NHS Ester and 300 µM DAPI in PBS (Note: confirm that the NHS and DAPI solution is completely homogenised).

  7. Add 2 mL to each well, wrap it in aluminium foil and incubate for 90 minutes at RT on a rocking platform.

  8. Wash 3x for 15 minutes with PBS+Tween-20 0.1%.

  9. Add ddH2O to each well until the gel is completely submerged.

  10. Change ddH2O 2x after 30 minutes.

  11. Expand in ddH2O overnight at RT.

Imaging day

Immobilisation of the gel

  1. Place a poly-L-Lysine coated coverslip in an Attofluor™ Cell Chamber (or another comparable mounting device).

  2. Carefully dry a piece of gel on a spatula using lint-free wipes and transfer it to the coverslip, with the sample facing the coverslip (use cut corner as reference of gel orientation).

  3. Incubate at RT for 7 minutes. Confirm if the gel is immobilised on the coverslip.

  4. Add ddH2O until the gel is completely submerged.

","reagents":"

Resources

Supplier

Article number

Acrylamide

Sigma

A9099

Acryloyl-X SE

Invitrogen

A20770

Adenine

Sigma

A9126

APS - Ammonium Persulfate

Roth

9592.3

DAPI

Invitrogen

D1306

DMAA - N,N’-Methylenebisacrylamide

Sigma

M7279-25G

GA - Glutaraldehyde

Sigma

G5882-10X1ML

Glucose (Dextrose)

Sigma

G7021

KH2PO4 - Potassium dihydrogen phosphate

Sangon Biotech

A100781

NaCl

Roth

HN00.2

NHS-Ester

Thermo Fisher Scientific

D2184

Peptone

Thermo Fisher Scientific

211820

PFA – Paraformaldehyde

Thermo Fisher Scientific

43368

Poly-L-Lysine

Sigma

P8920

SDS – Sodium Dodecyl Sulfate

Sigma

L3771

Sodium acrylate

Sigma

408220

Sorbitol solution

Sangon Biotech

A100691

TEMED – N,N,N′,N′-Tetramethylethylenediamine

Sigma

T9281

Tris

Carl Roth

5429.1

Tryptophan

Sigma

T0254

Tween-20

Roth

9127.1

Yeast extract

Thermo Fisher Scientific

288620

Zymolyase enzyme

Zymo research

E1004

Attofluor™ Cell Chamber

Thermo Fisher Scientific

A7816

Coverslip (24 mm round #1.5)

Marienfeld

117640

Coverslip rack

Diversified Biotech

WSDR-1000

Razor blade

Carl Roth

CK08.1

","patternDescription":"

The unicellular eukaryote S. cerevisiae represents an invaluable resource for the study of basic eukaryotic cellular and molecular processes. The combination of a high genetic amenability, numerous genetic tools, and vast genomic resources makes it one of the most versatile model organisms, employed in a wide range of basic research disciplines. However, its small size compared to other eukaryotic organisms has limited its use for the study of sub-cellular structures. The diameter of unbudded yeast cells ranges approximately between 4 μm in haploids and 6 μm in diploids (Milo and Phillips 2015), complicating conventional diffraction-limited light microscopy approaches.

The last decade witnessed the rise of super-resolution (SR) techniques that enable sub-diffraction resolution fluorescence imaging of cellular structures. Expansion microscopy (ExM), for instance, allows for increasing imaging resolution by physically enlarging a fixed sample that is embedded and cross-linked to a swellable gel and is then expanded isotropically in water (Wassie, et al. 2019). By doing so, fluorescently-labelled structures that previously would be too small or too close together to be distinguished can be resolved in 3 dimensions. The increased z-resolution can be exploited by z-sectioning, using confocal or light-sheet microscopy. In addition, ExM can also be used in combination with other super-resolution methods, such as airyscan confocal microscopy and Structured Illumination Microscopy (SIM), further enhancing resolution. ExM holds great potential to study the cellular structures of yeast, especially when paired with pan-labelling techniques to visualise total protein content inside cells. ExM protocols present a robust tool to study cellular organelles, obtaining super-resolution results with the advantage of employing inexpensive and straightforward equipment compared to other techniques that require more complex and expensive setups, such as optical SR approaches and EM.

ExM can be applied in various organisms and cellular models, offering outstanding outcomes with expansion factors ranging from 4x to 20x (Faulkner, et al. 2020; Truckenbrodt, et al. 2018). However, certain cellular organelles are resistant to these processes, such as the cell wall of fungi and Gram-positive bacteria. These organisms present a cell wall composed of peptidoglycans or glycoproteins that are highly resistant to denaturation and conventional digestion processes. The digestion of the cell wall and the maintenance of the structure of the resulting protoplasts are crucial steps to achieve isotropic expansion. For this reason, specific experimental strategies are needed, and only a few protocols are currently available (Götz, et al. 2020). In particular, only one study describes an ExM protocol applied to S. cerevisiae, showing different yeast structures in high resolution (Chen, et al. 2021).

Here we describe an adapted and extended ExM protocol for S. cerevisiae (Figure 1) with a final expansion factor of ~3.5x. The major difference regarding to the protocol of Chen et al., is the staining strategy. While the previously published protocol is based on immunolabeling to visualise and super-resolve specific target proteins, here, samples are stained with DAPI and the pan-labelling reagent NHS-Ester BODIPY FL. This allows to provide an overview of the ultrastructural context of the whole cells. However, both protocols complement each other and could be combined, in principle. Here, we have compiled detailed step-by-step instructions for ExM to visualise the ultrastructure of S. cerevisiae, supported by supplementary video documentation (Supplementary Video 1).

Briefly, the first day begins with the preparation and fixation of yeast cells, which includes the digestion of the cell wall with zymolyase. The generated protoplasts are mounted on the coverslip, and linking takes place overnight, during which linker molecules will attach to the sample’s proteins, acting as anchors for the next step. On the second day, the first step is gelation, where the sample is embedded in a monomer solution that polymerases and forms a matrix to which the anchor molecules will be connected. This is followed by the denaturation step, where the sample structures are disintegrated. The samples are then left expanding overnight in water, swelling the polymerised gel, which will push the anchored molecules apart in an isotropic manner. Finally, on the third day, staining with DAPI and NHS-Ester takes place. Samples are left again expanding overnight, and they can be imaged from the next day onwards.

Assessing the expansion factor and specimen cellular integrity following sample preparation is a critical step that requires consideration. During sample expansion, the denaturation/digestion steps facilitate isotropic expansion of the sample but different organelle structures might possess specific requirements that need to be assessed during the experimental planning phase of the experiment. The simplest solution to assess specimen integrity and expansion factor is to acquire microscopy data of the same field of view and specimen before and after expansion. This process will allow the user to detect immediate changes in the sample but carries the complication of being difficult to execute since finding the same sample, particularly in yeast, can be difficult and time-consuming. Other options, such as the one included in this protocol, image multiple expanded and non-expanded cells and compare their features (area and circularity) as means to assess expansion (average circularity 0.892 ± 0.063 on expanded samples (mean ± standard deviation, n=13) and 0.906 ± 0.035 on non-expanded ones (n=27)). Individual organelle expansion should be assessed via specific labelling of the structure and assess before and after expansion sizes and compare it to the expansion factor of the whole cell.

","references":[{"reference":"

Chen L, Yao L, Zhang L, Fei Yn, Mi L, Ma J. 2021. Applications of Super Resolution Expansion Microscopy in Yeast. Frontiers in Physics. 9:650353.

","pubmedId":"","doi":"10.3389/fphy.2021.650353"},{"reference":"Faulkner EL, Thomas SG, Neely RK. 2020. An introduction to the methodology of expansion microscopy. Int J Biochem Cell Biol 124: 105764.","pubmedId":"32407880","doi":""},{"reference":"Götz R, Panzer S, Trinks N, Eilts J, Wagener J, Turrà D, Di Pietro A, Sauer M, Terpitz U. 2020. Expansion Microscopy for Cell Biology Analysis in Fungi. Front Microbiol 11: 574.","pubmedId":"32318047","doi":""},{"reference":"

Milo, R., & Phillips, R. 2015. Cell Biology by the numbers (1st ed.). Garland Science.

","pubmedId":"","doi":"10.1201/9780429258770"},{"reference":"Pereira PM, Almada P, Henriques R. 2015. High-content 3D multicolor super-resolution localization microscopy. Methods Cell Biol 125: 95-117.","pubmedId":"25640426","doi":""},{"reference":"Truckenbrodt S, Maidorn M, Crzan D, Wildhagen H, Kabatas S, Rizzoli SO. 2018. X10 expansion microscopy enables 25-nm resolution on conventional microscopes. EMBO Rep 19: .","pubmedId":"29987134","doi":""},{"reference":"

Wassie AT, Zhao Y, Boyden ES. 2019. Expansion microscopy: principles and uses in biological research. Nat Methods 16: 33-41.

","pubmedId":"30573813","doi":""}],"title":"

Expansion Microscopy on Saccharomyces cerevisiae

","reviews":[{"reviewer":{"displayName":"Ulrich Terpitz"},"openAcknowledgement":true,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"9f39bf1c-73f0-48b1-aa5e-0cb6d8d173a6","decision":"publish","abstract":"

The unicellular eukaryote Saccharomyces cerevisiae is an invaluable resource for the study of basic eukaryotic cellular and molecular processes. However, its small size compared to other eukaryotic organisms the study of subcellular structures is challenging. Expansion microscopy (ExM) holds great potential to study the intracellular architecture of yeast, especially when paired with pan-labelling techniques visualising the full protein content inside cells. ExM allows to increase imaging resolution by physically enlarging a fixed sample that is embedded and cross-linked to a swellable gel followed by isotropic expansion in water. The cell wall present in fungi – including yeast – and Gram-positive bacteria is a resilient structure that resists denaturation and conventional digestion processes usually used in ExM protocols, resulting in uneven expansion. Thus, the digestion of the cell wall while maintaining the structure of the resulting protoplasts is a crucial step to ensure isotropic expansion. For this reason, specific experimental strategies are needed, and only a few protocols are currently available. We have developed a modified ExM protocol for S. cerevisiae, with 4x expansion factor, which allows the visualisation of the ultrastructure of the cells. Here, we describe the experimental procedure in detail, focusing on the most critical steps required to achieve isotropic expansion for ExM of S. cerevisiae.

","acknowledgements":"","authors":[{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["investigation","visualization","writing_originalDraft"],"email":"akorovesi@igc.gulbenkian.pt","firstName":"Artemis G.","lastName":"Korovesi","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":"","orcid":"https://orcid.org/0000-0003-1410-6757"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["investigation","visualization","writing_originalDraft"],"email":"lmorgado@igc.gulbenkian.pt","firstName":"Leonor","lastName":"Morgado","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":true,"WBId":"","orcid":"https://orcid.org/0000-0003-4510-8456"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["conceptualization","writing_reviewEditing"],"email":"mfumasoni@igc.gulbenkian.pt","firstName":"Marco","lastName":"Fumasoni","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":null,"WBId":"","orcid":"https://orcid.org/0000-0002-4507-7824"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal","MRC-Laboratory for Molecular Cell Biology. University College London, London, United Kingdom"],"departments":["",""],"credit":["conceptualization","writing_reviewEditing"],"email":"rjhenriques@igc.gulbenkian.pt","firstName":"Ricardo","lastName":"Henriques","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":null,"WBId":"","orcid":"https://orcid.org/0000-0002-2043-5234"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["supervision","dataCuration","writing_reviewEditing","conceptualization"],"email":"hsheil@igc.gulbenkian.pt","firstName":"Hannah S.","lastName":"Heil","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"https://orcid.org/0000-0003-4279-7022"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["supervision","dataCuration","writing_reviewEditing","conceptualization"],"email":"mrosario@igc.gulbenkian.pt","firstName":"Mario","lastName":"Del Rosario","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"https://orcid.org/0000-0002-0430-1463"}],"awards":null,"conflictsOfInterest":null,"dataTable":null,"extendedData":[{"description":"Supplementary figures of custom tools and troubleshooting table.","doi":"10.22002/D1.20159","resourceType":"Text","name":"ExM_yeast_Supp.docx","url":"https://portal.micropublication.org/uploads/a512f2d5c8a9017f881bc69a9ab1c9d3.docx"},{"description":"Supplementary Video 1. The video contains footage and instructions showing how to proceed throughout the protocol, focusing on the most critical steps. ","doi":"10.22002/D1.20160","resourceType":"Audiovisual","name":"YeastExM_SuppVideo.mp4","url":"https://portal.micropublication.org/uploads/f9a5a37aca86fa241279a8a1e6fa2b82.mp4"}],"funding":"This work was supported by the Gulbenkian Foundation (AGK, LM, RH, MF, HSH, MDR) and received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. [101001332] to RH), the European Molecular Biology Organization (EMBO) (Installation Grant EMBO-2020-IG-4734 to RH and the postdoctoral fellowship ALTF 499-2021 to HSH) and the Wellcome Trust (203276/Z/16/Z to RH). MF is supported by The European Horizon 2020 Framework Programme, H2020-MSCA-IF-2020 - 101030203/ GENMAINEVO. LM is kindly supported by a joint-collaboration between Abbelight and the Instituto Gulbenkian de Ciência. AGK would like to acknowledge support from the Integrative Biology and Biomedicine (IBB) PhD programme from Instituto Gulbenkian de Ciência.","image":{"url":"https://portal.micropublication.org/uploads/6d92a01310d0edeee28f7d2b9bff5b16.png"},"imageCaption":"

a) Scheme of expansion microscopy protocol workflow. b) Maximum intensity projection of DIC/DAPI composite image of yeast, acquired with a Nikon HCS using a 100x/1.45 oil objective. c) Maximum intensity projection of fluorescence image of post-expanded yeast, acquired with a Zeiss LSM 980 using a 40x/1.1 water objective and airyscan detection. Signals of DAPI and DIC/NHS-Ester are represented in magenta and grey, respectively. Scale bars: 5 µm. Samples were assessed by manual segmentation and measurement of the area and circularity between expanded (n=13) and non-expanded (n=27) specimens. The average expansion factor across all experiments was 3.52 ± 0.42 (mean ± standard deviation) while the circularity was retained within the level 20% of the population’s standard deviation.

","imageTitle":"

Yeast expansion microscopy overview

","methods":"

Preparation of stock solutions:

Sorbitol buffer:

● 1.2 M sorbitol solution in 0.1 M KH2PO4.

Linking solution:

● 0.1 mg/mL acryloyl X-SE in PBS. Store in aliquots at -20 °C.

Monomer solution:

Stocks:

● 38% sodium acrylate (w/w, diluted with ddH2O): 25 g in 65.79 mL, store at -20 °C.

● 40% acrylamide stock: 20 g in 50 mL ddH2O, store at -20°C.

● 2% N,N’-Methylenebisacrylamide : 0.2 g in 10 mL ddH2O, store at -20 °C.

Reagent

Final concentration

PBS

1x

Sodium acrylate*

19 g/100 mL

Acrylamide

10 g/100 mL

N,N’-Methylenebisacrylamide

0.1 g/100 mL

Store in 493 µL aliquots at -20 °C.

*Sodium acrylate is provided with variable quality levels. One should test it before using it: the reagent should be fully dissolved and not show impurities in the solution, in the form of precipitates or discolouring.

Denaturation buffer:

Reagent

Final concentration

ddH2O

-

SDS

200 mM

NaCl

200 mM

Tris*

50 mM

Store in falcon tubes at -20 °C.

*Adjust pH to 9.

YPD Media:

1. Dissolve 5 g of yeast extract and 10 g of peptone in 375 mL of ddH2O by manually stirring the flask and autoclave the solution.

2. Add 100 mL of ddH2O, 20 mL of autoclaved 50% Glucose (Dextrose) and 5 mL 1% Adenine + 1% Tryptophan.

Coverslip cleaning

Notes: i) This procedure should be performed under a fume hood, and one should use safety gloves when handling chloroform. ii) The chloroform and NaOH solutions can be reused. iii) The preparation of NaOH is an exothermic reaction, thus the NaOH stock solution should be prepared on an ice bath. iv) Other coverslip cleaning protocols are available, such as a chloroform-free protocol (Pereira et al. 2015).

  1. Arrange single coverslips in a coverslip rack.

  2. Place a glass container in an ultrasonic bath and fill the bath with water.

  3. Place the rack in the glass container.

  4. Fill the glass container with chloroform and sonicate for 1 hour.

  5. Remove the rack with a tweezers/forceps and place it in an empty glass container. The coverslips will dry in a few minutes as the chloroform evaporates quickly.

  6. Pour the remaining chloroform back into the bottle through a funnel.

  7. Reinstall the rack in the glass container in the ultrasonic bath.

  8. Fill the glass container with a 5 M NaOH solution and sonicate for 1 hour.

  9. Pass the coverslips into a beaker with ddH2O and transfer the NaOH solution back into the bottle.

  10. Wash the coverslips three times in a volume of 500 mL fresh ddH2O.

  11. Pre-dry the coverslips with a nitrogen or filtered air gun and then place them in a drying cabinet until they are completely dried.

  12. Store in 100 % ethanol in a glass Petri dish.

Coverslip poly-L-lysine coating

  1. Dry the previously cleaned coverslips.

  2. Place the coverslips in a clean plastic plate, for example, in a 6-well plate.

  3. Add 300 µL of 0.1 mg/mL poly-L-lysine on each coverslip and incubate at room temperature (RT) for 10 minutes.

  4. Add water to each well until the coverslip is covered and incubate at RT for 5 minutes.

  5. Dry the coverslips.

  6. Store coverslips on a rack at 4 °C until needed for cell/gel mounting.

Day 1

Yeast cells cultivation and fixation

The Saccharomyces cerevisiae BY4741 strain (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was used in this protocol.

  1. Culture yeast cells in the conditions required to assess the relevant biological question. (In our case, we incubate yeast cells in fresh YPD media supplemented with adenine and tryptophan at 30 °C and 220 rpm of agitation until the culture growth reaches the exponential phase).

  2. Count yeast cells with a hemocytometer and calculate the volume required to collect 6x107 cells.

  3. Centrifuge cells at 4,000 rpm for 2 minutes in a Falcon tube.

  4. Discard the supernatant and resuspend in 3 mL of PBS 1X.

  5. Centrifuge cells at 4,000 rpm for 2 minutes in a Falcon tube.

  6. Resuspend the cells in 3 mL of 4% paraformaldehyde (PFA), and fix them at 4 °C for 30 minutes in a multi-rotator.

  7. Centrifuge the fixed yeast cells at 4,000 rpm for 2 minutes.

  8. Discard the supernatant and resuspend in 3 mL of PBS 1X.

  9. Centrifuge the fixed yeast cells at 4,000 rpm for 2 minutes.

  10. Discard the supernatant and resuspend in 6 mL of sorbitol buffer.

Cell wall digestion

  1. Add 0.3 μL of zymolyase (5 U/μL) to 200 μL of fixed cells solution (2x106 cells) for a final concentration of 0.0075 U/µL and incubate the reaction at 30 °C for 3 minutes*.

  2. Place the samples on ice to block the enzymatic reaction.

* The length of the reaction provided is optimised for a population of exponentially growing cells. Samples collected in other conditions may require an adjusted treatment to optimise the cell wall digestion.

Mounting and fixating the cells on the coverslip

  1. Place the poly-L-lysine coated coverslips on a 6-well plate.

  2. Add 200 µL of treated cells to each coverslip and incubate them at RT for 10 minutes to allow the sample to sediment.

  3. Under a fume hood, add enough of the 4% PFA/1% Glutaraldehyde (GA) fixative to cover the sample and incubate at RT for 10 minutes.

  4. Wash 3x with PBS.

  5. Inspect the sample under a microscope to confirm that the cells are attached (i.e., cells are immobile and not floating) and if the majority of cells present no cell wall.

  6. If needed to calculate the cell expansion factor, image the cells at this time point to measure dimensions such as area or diameter of cells before expansion.

Linking

  1. Remove PBS, add 1 mL of 0.1 mg/mL acryloyl X-SE solution in PBS to each well and incubate for 12 hours (overnight) at RT.

Day 2

Gelation

Note: Proceed quickly through steps 3 to 5, as after the gelation solution is prepared, it will polymerise in minutes. For this reason, it is recommended to proceed with these steps with a maximum of four samples each time. Reagents and slides are maintained on ice to prevent premature polymerisation.

  1. Cool the reagents (monomer solution, TEMED, and APS) and the gelation chamber slides (see supplementary figure 1) on ice for at least 10 minutes.

  2. Remove the excess solution from the coverslip with a lint-free wipe and place it next to the slide on which it will be mounted (Note: the coverslip side with the sample should be facing up).

  3. Prepare the gelation solution: Add first the TEMED to the monomer solution, then APS and vortex. (Note: it is important to follow this order, as when APS is added the polymerisation reaction starts).

Volume

Reagents

Stock concentration

Final concentration

493 µL

Monomer solution

-

-

5 µL

APS

50%

0.5%

2.5 µL

TEMED

99%

0.5%

500 µL

Final Volume

-

-

  1. Add 120 µL of gelation solution in each gelation chamber, place the coverslip on top, with the side with the sample facing the solution, and gently press it. Carefully remove excess with a lint-free wipe.

  2. Keep the slides for 5 more minutes on ice.

  3. Incubate for 1 hour at 37 °C in a humidified chamber.

Denaturation

  1. Preheat a heating block to 95 °C.

  2. Thaw the denaturation buffer in a warm water bath and proceed only when the solution is clear.

  3. Carefully remove the coverslip from the slide and transfer the polymerised gel to a new 6-well plate (Note: sometimes the coverslip comes attached to the gel, and it can be transferred along).

  4. Measure the width and length of the polymerised gel before expansion.

  5. Add 2 mL of denaturation buffer to each well and incubate for 15 minutes at RT.

  6. Prepare an 1.5 mL Eppendorf tube for each sample and add 1 mL of fresh denaturation buffer in each of them.

  7. Transfer the gel into the Eppendorf tube with a metal spatula and incubate at 95 °C for 90 minutes.

Expansion

  1. Carefully pour the gel from the Eppendorf tube to a big clean plastic plate (e.g., Petri dish with a diameter of at least 60 mm) and remove the excess of denaturation buffer solution.

  2. Add ddH2O to Petri dish until the gel is completely submerged.

  3. Change ddH2O 2x after 30 minutes.

  4. Expand in ddH2O overnight at RT.

Day 3

NHS and DAPI staining

  1. Remove ddH2O.

  2. Measure the width and length of the gel after expansion. Divide these values by the ones measured before expansion to calculate the macro-expansion factor, which should be approximately 4x.

  3. Cut gel in small pieces using the custom cutter shape (see supplementary figure 2) and a razor blade.

  4. Select the pieces to be stained, cut the correct corner for reference of the gel orientation (see supplementary figure 1), and transfer them to a new 6-well plate with a metal spatula.

  5. Wash 2x for 15 minutes with PBS. (Note: the gels will shrink by 50%.)

  6. Prepare 10 µg/mL NHS Ester and 300 µM DAPI in PBS (Note: confirm that the NHS and DAPI solution is completely homogenised).

  7. Add 2 mL to each well, wrap it in aluminium foil and incubate for 90 minutes at RT on a rocking platform.

  8. Wash 3x for 15 minutes with PBS+Tween-20 0.1%.

  9. Add ddH2O to each well until the gel is completely submerged.

  10. Change ddH2O 2x after 30 minutes.

  11. Expand in ddH2O overnight at RT.

Imaging day

Immobilisation of the gel

  1. Place a poly-L-Lysine coated coverslip in an Attofluor™ Cell Chamber (or another comparable mounting device).

  2. Carefully dry a piece of gel on a spatula using lint-free wipes and transfer it to the coverslip, with the sample facing the coverslip (use cut corner as reference of gel orientation).

  3. Incubate at RT for 7 minutes. Confirm if the gel is immobilised on the coverslip.

  4. Add ddH2O until the gel is completely submerged.

","reagents":"

Resources

Supplier

Article number

Acrylamide

Sigma

A9099

Acryloyl-X SE

Invitrogen

A20770

Adenine

Sigma

A9126

APS - Ammonium Persulfate

Roth

9592.3

DAPI

Invitrogen

D1306

DMAA - N,N’-Methylenebisacrylamide

Sigma

M7279-25G

GA - Glutaraldehyde

Sigma

G5882-10X1ML

Glucose (Dextrose)

Sigma

G7021

KH2PO4 - Potassium dihydrogen phosphate

Sangon Biotech

A100781

NaCl

Roth

HN00.2

NHS-Ester

Thermo Fisher Scientific

D2184

Peptone

Thermo Fisher Scientific

211820

PFA – Paraformaldehyde

Thermo Fisher Scientific

43368

Poly-L-Lysine

Sigma

P8920

SDS – Sodium Dodecyl Sulfate

Sigma

L3771

Sodium acrylate

Sigma

408220

Sorbitol solution

Sangon Biotech

A100691

TEMED – N,N,N′,N′-Tetramethylethylenediamine

Sigma

T9281

Tris

Carl Roth

5429.1

Tryptophan

Sigma

T0254

Tween-20

Roth

9127.1

Yeast extract

Thermo Fisher Scientific

288620

Zymolyase enzyme

Zymo research

E1004

Attofluor™ Cell Chamber

Thermo Fisher Scientific

A7816

Coverslip (24 mm round #1.5)

Marienfeld

117640

Coverslip rack

Diversified Biotech

WSDR-1000

Razor blade

Carl Roth

CK08.1

","patternDescription":"

The unicellular eukaryote S. cerevisiae represents an invaluable resource for the study of basic eukaryotic cellular and molecular processes. The combination of a high genetic amenability, numerous genetic tools, and vast genomic resources makes it one of the most versatile model organisms, employed in a wide range of basic research disciplines. However, its small size compared to other eukaryotic organisms has limited its use for the study of sub-cellular structures. The diameter of unbudded yeast cells ranges approximately between 4 μm in haploids and 6 μm in diploids (Milo and Phillips 2015), complicating conventional diffraction-limited light microscopy approaches.

The last decade witnessed the rise of super-resolution (SR) techniques that enable sub-diffraction resolution fluorescence imaging of cellular structures. Expansion microscopy (ExM), for instance, allows for increasing imaging resolution by physically enlarging a fixed sample that is embedded and cross-linked to a swellable gel and is then expanded isotropically in water (Wassie et al. 2019). By doing so, fluorescently-labelled structures that previously would be too small or too close together to be distinguished can be resolved in 3 dimensions. The increased z-resolution can be exploited by z-sectioning, using confocal or light-sheet microscopy. In addition, ExM can also be used in combination with other super-resolution methods, such as airyscan confocal microscopy and Structured Illumination Microscopy (SIM), further enhancing resolution. ExM holds great potential to study the cellular structures of yeast, especially when paired with pan-labelling techniques to visualise total protein content inside cells. ExM protocols present a robust tool to study cellular organelles, obtaining super-resolution results with the advantage of employing inexpensive and straightforward equipment compared to other techniques that require more complex and expensive setups, such as optical SR approaches and electron microscopy.

ExM can be applied in various organisms and cellular models, offering outstanding outcomes with expansion factors ranging from 4x to 20x (Faulkner et al. 2020, Truckenbrodt et al. 2018). However, certain cellular organelles are resistant to these processes, such as the cell wall of fungi and Gram-positive bacteria. These organisms present a cell wall composed of peptidoglycans or glycoproteins that are highly resistant to denaturation and conventional digestion processes. The digestion of the cell wall and the maintenance of the structure of the resulting protoplasts are crucial steps to achieve isotropic expansion. For this reason, specific experimental strategies are needed, and only a few protocols are currently available (Götz et al. 2020). In particular, only one study describes an ExM protocol applied to S. cerevisiae, showing different yeast structures in high resolution (Chen et al. 2021).

Here we describe an adapted and extended ExM protocol for S. cerevisiae (Figure 1) with a final expansion factor of ~3.5x. The major difference regarding to the protocol of Chen et al., is the staining strategy. While the previously published protocol is based on immunolabeling to visualise and super-resolve specific target proteins, here, samples are stained with DAPI and the pan-labelling reagent NHS-Ester BODIPY FL. This allows to provide an overview of the ultrastructural context of the whole cells. However, both protocols complement each other and could be combined, in principle. Here, we have compiled detailed step-by-step instructions for ExM to visualise the ultrastructure of S. cerevisiae, supported by supplementary video documentation (Supplementary Video 1).

Briefly, the first day begins with the preparation and fixation of yeast cells, which includes the digestion of the cell wall with zymolyase. The generated protoplasts are mounted on the coverslip, and linking takes place overnight, during which linker molecules will attach to the sample’s proteins, acting as anchors for the next step. On the second day, the first step is gelation, where the sample is embedded in a monomer solution that polymerases and forms a matrix to which the anchor molecules will be connected. This is followed by the denaturation step, where the sample structures are disintegrated. The samples are then left expanding overnight in water, swelling the polymerised gel, which will push the anchored molecules apart in an isotropic manner. Finally, on the third day, staining with DAPI and NHS-Ester takes place. Samples are left again expanding overnight, and they can be imaged from the next day onwards.

Assessing the expansion factor and specimen cellular integrity following sample preparation is a critical step that requires consideration. During sample expansion, the denaturation/digestion steps facilitate isotropic expansion of the sample but different organelle structures might possess specific requirements that need to be identified during the experimental planning phase of the experiment. The simplest solution to assess specimen integrity and expansion factor is to acquire microscopy data of the same field of view and specimen before and after expansion. This process will allow the user to detect immediate changes in the sample but carries the complication of being difficult to execute since finding the same sample, particularly in yeast, can be difficult and time-consuming. Other options, such as the one included in this protocol, image multiple expanded and non-expanded cells and compare their features (area and circularity) as means to assess expansion (average circularity 0.892 ± 0.063 on expanded samples (mean ± standard deviation, n=13) and 0.906 ± 0.035 on non-expanded ones (n=27)). Individual organelle expansion should be obtained via specific labelling of the structure and determining before and after expansion sizes and compare it to the expansion factor of the whole cell.

","references":[{"reference":"

Chen L, Yao L, Zhang L, Fei Yn, Mi L, Ma J. 2021. Applications of Super Resolution Expansion Microscopy in Yeast. Frontiers in Physics. 9:650353.

","pubmedId":"","doi":"10.3389/fphy.2021.650353"},{"reference":"Faulkner EL, Thomas SG, Neely RK. 2020. An introduction to the methodology of expansion microscopy. Int J Biochem Cell Biol 124: 105764.","pubmedId":"32407880","doi":""},{"reference":"Götz R, Panzer S, Trinks N, Eilts J, Wagener J, Turrà D, Di Pietro A, Sauer M, Terpitz U. 2020. Expansion Microscopy for Cell Biology Analysis in Fungi. Front Microbiol 11: 574.","pubmedId":"32318047","doi":""},{"reference":"

Milo, R., & Phillips, R. 2015. Cell Biology by the numbers (1st ed.). Garland Science.

","pubmedId":"","doi":"10.1201/9780429258770"},{"reference":"Pereira PM, Almada P, Henriques R. 2015. High-content 3D multicolor super-resolution localization microscopy. Methods Cell Biol 125: 95-117.","pubmedId":"25640426","doi":""},{"reference":"Truckenbrodt S, Maidorn M, Crzan D, Wildhagen H, Kabatas S, Rizzoli SO. 2018. X10 expansion microscopy enables 25-nm resolution on conventional microscopes. EMBO Rep 19: .","pubmedId":"29987134","doi":""},{"reference":"

Wassie AT, Zhao Y, Boyden ES. 2019. Expansion microscopy: principles and uses in biological research. Nat Methods 16: 33-41.

","pubmedId":"30573813","doi":""}],"title":"

Expansion Microscopy on Saccharomyces cerevisiae

","reviews":[],"curatorReviews":[]},{"id":"174e0888-0ed3-4f84-9fbb-3a7c508dedaa","decision":"publish","abstract":"

The unicellular eukaryote Saccharomyces cerevisiae is an invaluable resource for the study of basic eukaryotic cellular and molecular processes. However, its small size compared to other eukaryotic organisms the study of subcellular structures is challenging. Expansion microscopy (ExM) holds great potential to study the intracellular architecture of yeast, especially when paired with pan-labelling techniques visualising the full protein content inside cells. ExM allows to increase imaging resolution by physically enlarging a fixed sample that is embedded and cross-linked to a swellable gel followed by isotropic expansion in water. The cell wall present in fungi – including yeast – and Gram-positive bacteria is a resilient structure that resists denaturation and conventional digestion processes usually used in ExM protocols, resulting in uneven expansion. Thus, the digestion of the cell wall while maintaining the structure of the resulting protoplasts is a crucial step to ensure isotropic expansion. For this reason, specific experimental strategies are needed, and only a few protocols are currently available. We have developed a modified ExM protocol for S. cerevisiae, with 4x expansion factor, which allows the visualisation of the ultrastructure of the cells. Here, we describe the experimental procedure in detail, focusing on the most critical steps required to achieve isotropic expansion for ExM of S. cerevisiae.

","acknowledgements":"","authors":[{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["investigation","visualization","writing_originalDraft"],"email":"akorovesi@igc.gulbenkian.pt","firstName":"Artemis G.","lastName":"Korovesi","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":"","orcid":"https://orcid.org/0000-0003-1410-6757"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["investigation","visualization","writing_originalDraft"],"email":"lmorgado@igc.gulbenkian.pt","firstName":"Leonor","lastName":"Morgado","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":true,"WBId":"","orcid":"https://orcid.org/0000-0003-4510-8456"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["conceptualization","writing_reviewEditing"],"email":"mfumasoni@igc.gulbenkian.pt","firstName":"Marco","lastName":"Fumasoni","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":null,"WBId":"","orcid":"https://orcid.org/0000-0002-4507-7824"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal","MRC-Laboratory for Molecular Cell Biology. University College London, London, United Kingdom"],"departments":["",""],"credit":["conceptualization","writing_reviewEditing"],"email":"rjhenriques@igc.gulbenkian.pt","firstName":"Ricardo","lastName":"Henriques","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":null,"WBId":"","orcid":"https://orcid.org/0000-0002-2043-5234"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["supervision","dataCuration","writing_reviewEditing","conceptualization"],"email":"hsheil@igc.gulbenkian.pt","firstName":"Hannah S.","lastName":"Heil","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"https://orcid.org/0000-0003-4279-7022"},{"affiliations":["Instituto Gulbenkian de Ciência, Oeiras, Portugal"],"departments":[""],"credit":["supervision","dataCuration","writing_reviewEditing","conceptualization"],"email":"mrosario@igc.gulbenkian.pt","firstName":"Mario","lastName":"Del Rosario","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"https://orcid.org/0000-0002-0430-1463"}],"awards":null,"conflictsOfInterest":null,"dataTable":null,"extendedData":[{"description":"Supplementary figures of custom tools and troubleshooting table.","doi":"10.22002/D1.20159","resourceType":"Text","name":"ExM_yeast_Supp.docx","url":"https://portal.micropublication.org/uploads/a512f2d5c8a9017f881bc69a9ab1c9d3.docx"},{"description":"Supplementary Video 1. The video contains footage and instructions showing how to proceed throughout the protocol, focusing on the most critical steps. ","doi":"10.22002/D1.20160","resourceType":"Audiovisual","name":"YeastExM_SuppVideo.mp4","url":"https://portal.micropublication.org/uploads/f9a5a37aca86fa241279a8a1e6fa2b82.mp4"}],"funding":"This work was supported by the Gulbenkian Foundation (AGK, LM, RH, MF, HSH, MDR) and received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. [101001332] to RH), the European Molecular Biology Organization (EMBO) (Installation Grant EMBO-2020-IG-4734 to RH and the postdoctoral fellowship ALTF 499-2021 to HSH) and the Wellcome Trust (203276/Z/16/Z to RH). MF is supported by The European Horizon 2020 Framework Programme, H2020-MSCA-IF-2020 - 101030203/ GENMAINEVO. LM is kindly supported by a joint-collaboration between Abbelight and the Instituto Gulbenkian de Ciência. AGK would like to acknowledge support from the Integrative Biology and Biomedicine (IBB) PhD programme from Instituto Gulbenkian de Ciência.","image":{"url":"https://portal.micropublication.org/uploads/6d92a01310d0edeee28f7d2b9bff5b16.png"},"imageCaption":"

a) Scheme of expansion microscopy protocol workflow. b) Maximum intensity projection of DIC/DAPI composite image of yeast, acquired with a Nikon HCS using a 100x/1.45 oil objective. c) Maximum intensity projection of fluorescence image of post-expanded yeast, acquired with a Zeiss LSM 980 using a 40x/1.1 water objective and airyscan detection. Signals of DAPI and DIC/NHS-Ester are represented in magenta and grey, respectively. Scale bars: 5 µm. Samples were assessed by manual segmentation and measurement of the area and circularity between expanded (n=13) and non-expanded (n=27) specimens. The average expansion factor across all experiments was 3.52 ± 0.42 (mean ± standard deviation) while the circularity was retained within the level 20% of the population’s standard deviation.

","imageTitle":"

Yeast expansion microscopy overview

","methods":"

Preparation of stock solutions:

Sorbitol buffer:

● 1.2 M sorbitol solution in 0.1 M KH2PO4.

Linking solution:

● 0.1 mg/mL acryloyl X-SE in PBS. Store in aliquots at -20 °C.

Monomer solution:

Stocks:

● 38% sodium acrylate (w/w, diluted with ddH2O): 25 g in 65.79 mL, store at -20 °C.

● 40% acrylamide stock: 20 g in 50 mL ddH2O, store at -20°C.

● 2% N,N’-Methylenebisacrylamide : 0.2 g in 10 mL ddH2O, store at -20 °C.

Reagent

Final concentration

PBS

1x

Sodium acrylate*

19 g/100 mL

Acrylamide

10 g/100 mL

N,N’-Methylenebisacrylamide

0.1 g/100 mL

Store in 493 µL aliquots at -20 °C.

*Sodium acrylate is provided with variable quality levels. One should test it before using it: the reagent should be fully dissolved and not show impurities in the solution, in the form of precipitates or discolouring.

Denaturation buffer:

Reagent

Final concentration

ddH2O

-

SDS

200 mM

NaCl

200 mM

Tris*

50 mM

Store in falcon tubes at -20 °C.

*Adjust pH to 9.

YPD Media:

1. Dissolve 5 g of yeast extract and 10 g of peptone in 375 mL of ddH2O by manually stirring the flask and autoclave the solution.

2. Add 100 mL of ddH2O, 20 mL of autoclaved 50% Glucose (Dextrose) and 5 mL 1% Adenine + 1% Tryptophan.

Coverslip cleaning

Notes: i) This procedure should be performed under a fume hood, and one should use safety gloves when handling chloroform. ii) The chloroform and NaOH solutions can be reused. iii) The preparation of NaOH is an exothermic reaction, thus the NaOH stock solution should be prepared on an ice bath. iv) Other coverslip cleaning protocols are available, such as a chloroform-free protocol (Pereira et al. 2015).

  1. Arrange single coverslips in a coverslip rack.

  2. Place a glass container in an ultrasonic bath and fill the bath with water.

  3. Place the rack in the glass container.

  4. Fill the glass container with chloroform and sonicate for 1 hour.

  5. Remove the rack with a tweezers/forceps and place it in an empty glass container. The coverslips will dry in a few minutes as the chloroform evaporates quickly.

  6. Pour the remaining chloroform back into the bottle through a funnel.

  7. Reinstall the rack in the glass container in the ultrasonic bath.

  8. Fill the glass container with a 5 M NaOH solution and sonicate for 1 hour.

  9. Pass the coverslips into a beaker with ddH2O and transfer the NaOH solution back into the bottle.

  10. Wash the coverslips three times in a volume of 500 mL fresh ddH2O.

  11. Pre-dry the coverslips with a nitrogen or filtered air gun and then place them in a drying cabinet until they are completely dried.

  12. Store in 100 % ethanol in a glass Petri dish.

Coverslip poly-L-lysine coating

  1. Dry the previously cleaned coverslips.

  2. Place the coverslips in a clean plastic plate, for example, in a 6-well plate.

  3. Add 300 µL of 0.1 mg/mL poly-L-lysine on each coverslip and incubate at room temperature (RT) for 10 minutes.

  4. Add water to each well until the coverslip is covered and incubate at RT for 5 minutes.

  5. Dry the coverslips.

  6. Store coverslips on a rack at 4 °C until needed for cell/gel mounting.

Day 1

Yeast cells cultivation and fixation

The Saccharomyces cerevisiae BY4741 strain (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was used in this protocol.

  1. Culture yeast cells in the conditions required to assess the relevant biological question. (In our case, we incubate yeast cells in fresh YPD media supplemented with adenine and tryptophan at 30 °C and 220 rpm of agitation until the culture growth reaches the exponential phase).

  2. Count yeast cells with a hemocytometer and calculate the volume required to collect 6x107 cells.

  3. Centrifuge cells at 4,000 rpm for 2 minutes in a Falcon tube.

  4. Discard the supernatant and resuspend in 3 mL of PBS 1X.

  5. Centrifuge cells at 4,000 rpm for 2 minutes in a Falcon tube.

  6. Resuspend the cells in 3 mL of 4% paraformaldehyde (PFA), and fix them at 4 °C for 30 minutes in a multi-rotator.

  7. Centrifuge the fixed yeast cells at 4,000 rpm for 2 minutes.

  8. Discard the supernatant and resuspend in 3 mL of PBS 1X.

  9. Centrifuge the fixed yeast cells at 4,000 rpm for 2 minutes.

  10. Discard the supernatant and resuspend in 6 mL of sorbitol buffer.

Cell wall digestion

  1. Add 0.3 μL of zymolyase (5 U/μL) to 200 μL of fixed cells solution (2x106 cells) for a final concentration of 0.0075 U/µL and incubate the reaction at 30 °C for 3 minutes*.

  2. Place the samples on ice to block the enzymatic reaction.

* The length of the reaction provided is optimised for a population of exponentially growing cells. Samples collected in other conditions may require an adjusted treatment to optimise the cell wall digestion.

Mounting and fixating the cells on the coverslip

  1. Place the poly-L-lysine coated coverslips on a 6-well plate.

  2. Add 200 µL of treated cells to each coverslip and incubate them at RT for 10 minutes to allow the sample to sediment.

  3. Under a fume hood, add enough of the 4% PFA/1% Glutaraldehyde (GA) fixative to cover the sample and incubate at RT for 10 minutes.

  4. Wash 3x with PBS.

  5. Inspect the sample under a microscope to confirm that the cells are attached (i.e., cells are immobile and not floating) and if the majority of cells present no cell wall.

  6. If needed to calculate the cell expansion factor, image the cells at this time point to measure dimensions such as area or diameter of cells before expansion.

Linking

  1. Remove PBS, add 1 mL of 0.1 mg/mL acryloyl X-SE solution in PBS to each well and incubate for 12 hours (overnight) at RT.

Day 2

Gelation

Note: Proceed quickly through steps 3 to 5, as after the gelation solution is prepared, it will polymerise in minutes. For this reason, it is recommended to proceed with these steps with a maximum of four samples each time. Reagents and slides are maintained on ice to prevent premature polymerisation.

  1. Cool the reagents (monomer solution, TEMED, and APS) and the gelation chamber slides (see supplementary figure 1) on ice for at least 10 minutes.

  2. Remove the excess solution from the coverslip with a lint-free wipe and place it next to the slide on which it will be mounted (Note: the coverslip side with the sample should be facing up).

  3. Prepare the gelation solution: Add first the TEMED to the monomer solution, then APS and vortex. (Note: it is important to follow this order, as when APS is added the polymerisation reaction starts).

Volume

Reagents

Stock concentration

Final concentration

493 µL

Monomer solution

-

-

5 µL

APS

50%

0.5%

2.5 µL

TEMED

99%

0.5%

500 µL

Final Volume

-

-

  1. Add 120 µL of gelation solution in each gelation chamber, place the coverslip on top, with the side with the sample facing the solution, and gently press it. Carefully remove excess with a lint-free wipe.

  2. Keep the slides for 5 more minutes on ice.

  3. Incubate for 1 hour at 37 °C in a humidified chamber.

Denaturation

  1. Preheat a heating block to 95 °C.

  2. Thaw the denaturation buffer in a warm water bath and proceed only when the solution is clear.

  3. Carefully remove the coverslip from the slide and transfer the polymerised gel to a new 6-well plate (Note: sometimes the coverslip comes attached to the gel, and it can be transferred along).

  4. Measure the width and length of the polymerised gel before expansion.

  5. Add 2 mL of denaturation buffer to each well and incubate for 15 minutes at RT.

  6. Prepare an 1.5 mL Eppendorf tube for each sample and add 1 mL of fresh denaturation buffer in each of them.

  7. Transfer the gel into the Eppendorf tube with a metal spatula and incubate at 95 °C for 90 minutes.

Expansion

  1. Carefully pour the gel from the Eppendorf tube to a big clean plastic plate (e.g., Petri dish with a diameter of at least 60 mm) and remove the excess of denaturation buffer solution.

  2. Add ddH2O to Petri dish until the gel is completely submerged.

  3. Change ddH2O 2x after 30 minutes.

  4. Expand in ddH2O overnight at RT.

Day 3

NHS and DAPI staining

  1. Remove ddH2O.

  2. Measure the width and length of the gel after expansion. Divide these values by the ones measured before expansion to calculate the macro-expansion factor, which should be approximately 4x.

  3. Cut gel in small pieces using the custom cutter shape (see supplementary figure 2) and a razor blade.

  4. Select the pieces to be stained, cut the correct corner for reference of the gel orientation (see supplementary figure 1), and transfer them to a new 6-well plate with a metal spatula.

  5. Wash 2x for 15 minutes with PBS. (Note: the gels will shrink by 50%.)

  6. Prepare 10 µg/mL NHS Ester and 300 µM DAPI in PBS (Note: confirm that the NHS and DAPI solution is completely homogenised).

  7. Add 2 mL to each well, wrap it in aluminium foil and incubate for 90 minutes at RT on a rocking platform.

  8. Wash 3x for 15 minutes with PBS+Tween-20 0.1%.

  9. Add ddH2O to each well until the gel is completely submerged.

  10. Change ddH2O 2x after 30 minutes.

  11. Expand in ddH2O overnight at RT.

Imaging day

Immobilisation of the gel

  1. Place a poly-L-Lysine coated coverslip in an Attofluor™ Cell Chamber (or another comparable mounting device).

  2. Carefully dry a piece of gel on a spatula using lint-free wipes and transfer it to the coverslip, with the sample facing the coverslip (use cut corner as reference of gel orientation).

  3. Incubate at RT for 7 minutes. Confirm if the gel is immobilised on the coverslip.

  4. Add ddH2O until the gel is completely submerged.

","reagents":"

Resources

Supplier

Article number

Acrylamide

Sigma

A9099

Acryloyl-X SE

Invitrogen

A20770

Adenine

Sigma

A9126

APS - Ammonium Persulfate

Roth

9592.3

DAPI

Invitrogen

D1306

DMAA - N,N’-Methylenebisacrylamide

Sigma

M7279-25G

GA - Glutaraldehyde

Sigma

G5882-10X1ML

Glucose (Dextrose)

Sigma

G7021

KH2PO4 - Potassium dihydrogen phosphate

Sangon Biotech

A100781

NaCl

Roth

HN00.2

NHS-Ester

Thermo Fisher Scientific

D2184

Peptone

Thermo Fisher Scientific

211820

PFA – Paraformaldehyde

Thermo Fisher Scientific

43368

Poly-L-Lysine

Sigma

P8920

SDS – Sodium Dodecyl Sulfate

Sigma

L3771

Sodium acrylate

Sigma

408220

Sorbitol solution

Sangon Biotech

A100691

TEMED – N,N,N′,N′-Tetramethylethylenediamine

Sigma

T9281

Tris

Carl Roth

5429.1

Tryptophan

Sigma

T0254

Tween-20

Roth

9127.1

Yeast extract

Thermo Fisher Scientific

288620

Zymolyase enzyme

Zymo research

E1004

Attofluor™ Cell Chamber

Thermo Fisher Scientific

A7816

Coverslip (24 mm round #1.5)

Marienfeld

117640

Coverslip rack

Diversified Biotech

WSDR-1000

Razor blade

Carl Roth

CK08.1

","patternDescription":"

The unicellular eukaryote S. cerevisiae represents an invaluable resource for the study of basic eukaryotic cellular and molecular processes. The combination of a high genetic amenability, numerous genetic tools, and vast genomic resources makes it one of the most versatile model organisms, employed in a wide range of basic research disciplines. However, its small size compared to other eukaryotic organisms has limited its use for the study of sub-cellular structures. The diameter of unbudded yeast cells ranges approximately between 4 μm in haploids and 6 μm in diploids (Milo and Phillips 2015), complicating conventional diffraction-limited light microscopy approaches.

The last decade witnessed the rise of super-resolution (SR) techniques that enable sub-diffraction resolution fluorescence imaging of cellular structures. Expansion microscopy (ExM), for instance, allows for increasing imaging resolution by physically enlarging a fixed sample that is embedded and cross-linked to a swellable gel and is then expanded isotropically in water (Wassie et al. 2019). By doing so, fluorescently-labelled structures that previously would be too small or too close together to be distinguished can be resolved in 3 dimensions. The increased z-resolution can be exploited by z-sectioning, using confocal or light-sheet microscopy. In addition, ExM can also be used in combination with other super-resolution methods, such as airyscan confocal microscopy and Structured Illumination Microscopy (SIM), further enhancing resolution. ExM holds great potential to study the cellular structures of yeast, especially when paired with pan-labelling techniques to visualise total protein content inside cells. ExM protocols present a robust tool to study cellular organelles, obtaining super-resolution results with the advantage of employing inexpensive and straightforward equipment compared to other techniques that require more complex and expensive setups, such as optical SR approaches and electron microscopy.

ExM can be applied in various organisms and cellular models, offering outstanding outcomes with expansion factors ranging from 4x to 20x (Faulkner et al. 2020, Truckenbrodt et al. 2018). However, certain cellular organelles are resistant to these processes, such as the cell wall of fungi and Gram-positive bacteria. These organisms present a cell wall composed of peptidoglycans or glycoproteins that are highly resistant to denaturation and conventional digestion processes. The digestion of the cell wall and the maintenance of the structure of the resulting protoplasts are crucial steps to achieve isotropic expansion. For this reason, specific experimental strategies are needed, and only a few protocols are currently available (Götz et al. 2020). In particular, only one study describes an ExM protocol applied to S. cerevisiae, showing different yeast structures in high resolution (Chen et al. 2021).

Here we describe an adapted and extended ExM protocol for S. cerevisiae (Figure 1) with a final expansion factor of ~3.5x. The major difference regarding to the protocol of Chen et al., is the staining strategy. While the previously published protocol is based on immunolabeling to visualise and super-resolve specific target proteins, here, samples are stained with DAPI and the pan-labelling reagent NHS-Ester BODIPY FL. This allows to provide an overview of the ultrastructural context of the whole cells. However, both protocols complement each other and could be combined, in principle. Here, we have compiled detailed step-by-step instructions for ExM to visualise the ultrastructure of S. cerevisiae, supported by supplementary video documentation (Supplementary Video 1).

Briefly, the first day begins with the preparation and fixation of yeast cells, which includes the digestion of the cell wall with zymolyase. The generated protoplasts are mounted on the coverslip, and linking takes place overnight, during which linker molecules will attach to the sample’s proteins, acting as anchors for the next step. On the second day, the first step is gelation, where the sample is embedded in a monomer solution that polymerases and forms a matrix to which the anchor molecules will be connected. This is followed by the denaturation step, where the sample structures are disintegrated. The samples are then left expanding overnight in water, swelling the polymerised gel, which will push the anchored molecules apart in an isotropic manner. Finally, on the third day, staining with DAPI and NHS-Ester takes place. Samples are left again expanding overnight, and they can be imaged from the next day onwards.

Assessing the expansion factor and specimen cellular integrity following sample preparation is a critical step that requires consideration. During sample expansion, the denaturation/digestion steps facilitate isotropic expansion of the sample but different organelle structures might possess specific requirements that need to be identified during the experimental planning phase of the experiment. The simplest solution to assess specimen integrity and expansion factor is to acquire microscopy data of the same field of view and specimen before and after expansion. This process will allow the user to detect immediate changes in the sample but carries the complication of being difficult to execute since finding the same sample, particularly in yeast, can be difficult and time-consuming. Other options, such as the one included in this protocol, image multiple expanded and non-expanded cells and compare their features (area and circularity) as means to assess expansion (average circularity 0.892 ± 0.063 on expanded samples (mean ± standard deviation, n=13) and 0.906 ± 0.035 on non-expanded ones (n=27)). Individual organelle expansion should be obtained via specific labelling of the structure and determining before and after expansion sizes and compare it to the expansion factor of the whole cell.

","references":[{"reference":"

Chen L, Yao L, Zhang L, Fei Yn, Mi L, Ma J. 2021. Applications of Super Resolution Expansion Microscopy in Yeast. Frontiers in Physics. 9:650353.

","pubmedId":"","doi":"10.3389/fphy.2021.650353"},{"reference":"Faulkner EL, Thomas SG, Neely RK. 2020. An introduction to the methodology of expansion microscopy. Int J Biochem Cell Biol 124: 105764.","pubmedId":"32407880","doi":""},{"reference":"Götz R, Panzer S, Trinks N, Eilts J, Wagener J, Turrà D, Di Pietro A, Sauer M, Terpitz U. 2020. Expansion Microscopy for Cell Biology Analysis in Fungi. Front Microbiol 11: 574.","pubmedId":"32318047","doi":""},{"reference":"

Milo, R., & Phillips, R. 2015. Cell Biology by the numbers (1st ed.). Garland Science.

","pubmedId":"","doi":"10.1201/9780429258770"},{"reference":"Pereira PM, Almada P, Henriques R. 2015. High-content 3D multicolor super-resolution localization microscopy. Methods Cell Biol 125: 95-117.","pubmedId":"25640426","doi":""},{"reference":"Truckenbrodt S, Maidorn M, Crzan D, Wildhagen H, Kabatas S, Rizzoli SO. 2018. X10 expansion microscopy enables 25-nm resolution on conventional microscopes. EMBO Rep 19: .","pubmedId":"29987134","doi":""},{"reference":"

Wassie AT, Zhao Y, Boyden ES. 2019. Expansion microscopy: principles and uses in biological research. Nat Methods 16: 33-41.

","pubmedId":"30573813","doi":""}],"title":"

Expansion Microscopy on Saccharomyces cerevisiae

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