BACKGROUND: In the present work, we provide an account of structured illumination microscopy (SIM) imaging of fixed and immunolabeled plant probes. We take advantage of SIM, to superresolve intracellular structures at a considerable z-range and circumvent its low temporal resolution capacity during the study of living samples. Further, we validate the protocol for the imaging of fixed transgenic material expressing fluorescent protein-based markers of different subcellular structures. RESULTS: Focus is given on 3D imaging of bulky subcellular structures, such as mitotic and cytokinetic microtubule arrays as well as on the performance of SIM using multichannel imaging and the quantitative correlations that can be deduced. As a proof of concept, we provide a superresolution output on the organization of cortical microtubules in wild-type and mutant Arabidopsis cells, including aberrant preprophase microtubule bands and phragmoplasts in a cytoskeletal mutant devoid of the p60 subunit of the microtubule severing protein KATANIN and refined details of cytoskeletal aberrations in the mitogen activated protein kinase (MAPK) mutant mpk4. We further demonstrate, in a qualitative and quantitative manner, colocalizations between MPK6 and unknown dually phosphorylated and activated MAPK species and we follow the localization of the microtubule associated protein 65-3 (MAP65-3) in telophase and cytokinetic microtubular arrays. CONCLUSIONS: 3D SIM is a powerful, versatile and adaptable microscopy method for elucidating spatial relationships between subcellular compartments. Improved methods of sample preparation aiming to the compensation of refractive index mismatches, allow the use of 3D SIM in the documentation of complex plant cell structures, such as microtubule arrays and the elucidation of their interactions with microtubule associated proteins.

Department of Cell Biology Centre of the Region Haná for Biotechnological and Agricultural Research Faculty of Science Palacký University Olomouc Olomouc Czech Republic

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Antosch M, Schubert V, Holzinger P, Houben A, Grasser KD. Mitotic lifecycle of chromosomal 3xHMG-box proteins and the role of their N-terminal domain in the association with rDNA loci and proteolysis. New Phytol. 2015;208:1067–1077. doi: 10.1111/nph.13575. PubMed DOI

Arigovindan M, Sedat JW, Agard DA. Effect of depth dependent spherical aberrations in 3D structured illumination microscopy. Opt Express. 2012;20:6527–6541. doi: 10.1364/OE.20.006527. PubMed DOI

Banaei-Moghaddam AM, Schubert V, Kumke K, Weiß O, Klemme S, Nagaki K, Macas J, González-Sánchez M, Heredia V, Gómez-Revilla D, González-García M, Vega JM, Puertas MJ, Houben A. Nondisjunction in favor of a chromosome: the mechanism of rye B chromosome drive during pollen mitosis. Plant Cell. 2012;24:4124–4134. doi: 10.1105/tpc.112.105270. PubMed DOI PMC

Baroux C, Schubert V. Technical review: microscopy and image processing tools to analyze plant chromatin: practical considerations. In: Baroux MBC, editor. Plant chromatin dynamics: methods and protocols. New York: Humana Press; 2018. pp. 537–589. PubMed

Beck M, Komis G, Müller J, Menzel D, Samaj J. Arabidopsis homologs of nucleus- and phragmoplast-localized kinase 2 and 3 and mitogen-activated protein kinase 4 are essential for microtubule organization. Plant Cell. 2010;22:755–771. doi: 10.1105/tpc.109.071746. PubMed DOI PMC

Beck M, Komis G, Ziemann A, Menzel D, Samaj J. Mitogen-activated protein kinase 4 is involved in the regulation of mitotic and cytokinetic microtubule transitions in Arabidopsis thaliana. New Phytol. 2011;189:1069–1083. doi: 10.1111/j.1469-8137.2010.03565.x. PubMed DOI

Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF. Imaging intracellular fluorescent proteins at nanometer resolution. Science. 2006;313:1642–1645. doi: 10.1126/science.1127344. PubMed DOI

Cole RW, Jinadasa T, Brown CM. Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control. Nat Protoc. 2011;6:1929–1941. doi: 10.1038/nprot.2011.407. PubMed DOI

Combs CA. Fluorescence microscopy: a concise guide to current imaging methods. Curr Protoc Neurosci. 2010 doi: 10.1002/0471142301.ns0201s50. PubMed DOI PMC

Costes SV, Daelemans D, Cho EH, Dobbin Z, Pavlakis G, Lockett S. Automatic and quantitative measurement of protein-protein colocalization in live cells. Biophys J. 2004;86:3993–4003. doi: 10.1529/biophysj.103.038422. PubMed DOI PMC

Davis AM, Hall A, Millar AJ, Darrah C, Davis SJ. Protocol: streamlined sub-protocols for floral-dip transformation and selection of transformants in Arabidopsis thaliana. Plant Methods. 2009;5:1–7. doi: 10.1186/1746-4811-5-3. PubMed DOI PMC

Demmerle J, Innocent C, North AJ, Ball G, Müller M, Miron E, Matsuda A, Dobbie IM, Markaki Y, Schermelleh L. Strategic and practical guidelines for successful structured illumination microscopy. Nat Protoc. 2017;12:988–1010. doi: 10.1038/nprot.2017.019. PubMed DOI

Fišerová J, Efenberková M, Sieger T, Maninová M, Uhlířová J, Hozák P. Chromatin organization at the nuclear periphery as revealed by image analysis of structured illumination microscopy data. J Cell Sci. 2017;130:2066–2077. doi: 10.1242/jcs.198424. PubMed DOI

Fitzgibbon J, Bell K, King E, Oparka K. Super-resolution imaging of plasmodesmata using three-dimensional structured illumination microscopy. Plant Physiol. 2010;153:1453–1463. doi: 10.1104/pp.110.157941. PubMed DOI PMC

Gustafsson MG. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc. 2000;198:82–87. doi: 10.1046/j.1365-2818.2000.00710.x. PubMed DOI

Hell SW, Wichmann J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett. 1994;19:780–782. doi: 10.1364/OL.19.000780. PubMed DOI

Ho CM, Hotta T, Guo F, Roberson RW, Lee YR, Liu B. Interaction of antiparallel microtubules in the phragmoplast is mediated by the microtubule-associated protein MAP65-3 in Arabidopsis. Plant Cell. 2011;23:2909–2923. doi: 10.1105/tpc.110.078204. PubMed DOI PMC

Jonak C, Okrész L, Bögre L, Hirt H. Complexity, cross talk and integration of plant MAP kinase signalling. Curr Opin Plant Biol. 2002;5:415–424. doi: 10.1016/S1369-5266(02)00285-6. PubMed DOI

Jonkman J, Brown CM. Any way you slice it—a comparison of confocal microscopy techniques. J Biomol Tech. 2015;26:54–65. PubMed PMC

Kartasalo K, Pölönen RP, Ojala M, Rasku J, Lekkala J, Aalto-Setälä K, Kallio P. CytoSpectre: a tool for spectral analysis of oriented structures on cellular and subcellular levels. BMC Bioinform. 2015;16:344. doi: 10.1186/s12859-015-0782-y. PubMed DOI PMC

Komis G, Luptovčiak I, Ovečka M, Samakovli D, Šamajová O, Šamaj J. Katanin effects on dynamics of cortical microtubules and mitotic arrays in Arabidopsis thaliana revealed by advanced live-cell imaging. Front Plant Sci. 2017;8:866. doi: 10.3389/fpls.2017.00866. PubMed DOI PMC

Komis G, Mistrik M, Samajová O, Doskočilová A, Ovečka M, Illés P, Bartek J, Samaj J. Dynamics and organization of cortical microtubules as revealed by superresolution structured illumination microscopy. Plant Physiol. 2014;165:129–148. doi: 10.1104/pp.114.238477. PubMed DOI PMC

Komis G, Mistrik M, Šamajová O, Ovečka M, Bartek J, Šamaj J. Superresolution live imaging of plant cells using structured illumination microscopy. Nat Protoc. 2015;10:1248–1263. doi: 10.1038/nprot.2015.083. PubMed DOI

Komis G, Novák D, Ovečka M, Šamajová O, Šamaj J. Advances in imaging plant cell dynamics. Plant Physiol. 2018;176:80–93. doi: 10.1104/pp.17.00962. PubMed DOI PMC

Komis G, Šamajová O, Ovečka M, Šamaj J. Super-resolution microscopy in plant cell imaging. Trends Plant Sci. 2015;20:834–843. doi: 10.1016/j.tplants.2015.08.013. PubMed DOI

Kraus F, Miron E, Demmerle J, Chitiashvili T, Budco A, Alle Q, Matsuda A, Leonhardt H, Schermelleh L, Markaki Y. Quantitative 3D structured illumination microscopy of nuclear structures. Nat Protoc. 2017;12:1011–1028. doi: 10.1038/nprot.2017.020. PubMed DOI

Lin F, Krishnamoorthy P, Schubert V, Hause G, Heilmann M, Heilmann I. A dual role for cell plate-associated PI4Kbeta in endocytosis and phragmoplast dynamics during plant somatic cytokinesis. EMBO J. 2019 doi: 10.15252/embj.2018100303. PubMed DOI PMC

Manders EM, Stap J, Brakenhoff GJ, van Driel R, Aten JA. Dynamics of three-dimensional replication patterns during the S-phase, analysed by double labelling of DNA and confocal microscopy. J Cell Sci. 1992;103:857–862. PubMed

Marques A, Schubert V, Houben A, Pedrosa-Harand A. Restructuring of holocentric centromeres during meiosis in the plant Rhynchospora pubera. Genetics. 2016;204:555–568. doi: 10.1534/genetics.116.191213. PubMed DOI PMC

Müller S, Fuchs E, Ovecka M, Wysocka-Diller J, Benfey PN, Hauser MT. Two new loci, PLEIADE and HYADE, implicate organ-specific regulation of cytokinesis in Arabidopsis. Plant Physiol. 2002;130:312–324. doi: 10.1104/pp.004416. PubMed DOI PMC

Müller J, Beck M, Mettbach U, Komis G, Hause G, Menzel D, Samaj J. Arabidopsis MPK6 is involved in cell division plane control during early root development, and localizes to the pre-prophase band, phragmoplast, trans-Golgi network and plasma membrane. Plant J. 2010;61:234–248. doi: 10.1111/j.1365-313X.2009.04046.x. PubMed DOI

Musielak TJ, Slane D, Liebig C, Bayer M. A versatile optical clearing protocol for deep tissue imaging of fluorescent proteins in Arabidopsis thaliana. PLoS ONE. 2016;11(8):e0161107. doi: 10.1371/journal.pone.0161107. PubMed DOI PMC

Nakagawa T, Suzuki T, Murata S, Nakamura S, Hino T, Maeo K, et al. Improved gateway binary vectors: high-performance vectors for creation of fusion constructs in transgenic analysis of plants. Biosci Biotechnol Biochem. 2007;71:2095–2100. doi: 10.1271/bbb.70216. PubMed DOI

Nakamura M, Ehrhardt DW, Hashimoto T. Microtubule and katanin-dependent dynamics of microtubule nucleation complexes in the acentrosomal Arabidopsis cortical array. Nat Cell Biol. 2010;12:1064–1070. doi: 10.1038/ncb2110. PubMed DOI

Ovečka M, Takáč T, Komis G, Vadovič P, Bekešová S, Doskočilová A, Šamajová V, Luptovčiak I, Samajová O, Schweighofer A, Meskiene I, Jonak C, Křenek P, Lichtscheidl I, Škultéty L, Hirt H, Šamaj J. Salt-induced subcellular kinase relocation and seedling susceptibility caused by overexpression of Medicago SIMKK in Arabidopsis. J Exp Bot. 2014;65:2335–2350. doi: 10.1093/jxb/eru115. PubMed DOI PMC

Ovečka M, von Wangenheim D, Tomančák P, Šamajová O, Komis G, Šamaj J. Multiscale imaging of plant development by light-sheet fluorescence microscopy. Nat Plants. 2018;4:639–650. doi: 10.1038/s41477-018-0238-2. PubMed DOI

Palmer WM, Martin AP, Flynn JR, Reed SL, White RG, Furbank RT, Grof CP. PEA-CLARITY: 3D molecular imaging of whole plant organs. Sci Rep. 2015;5:13492. doi: 10.1038/srep13492. PubMed DOI PMC

Plotnikov A, Zehorai E, Procaccia S, Seger R. The MAPK cascades: signaling components, nuclear roles and mechanisms of nuclear translocation. Biochim Biophys Acta. 2011;1813:1619–1633. doi: 10.1016/j.bbamcr.2010.12.012. PubMed DOI

Ribeiro T, Marques A, Novák P, Schubert V, Vanzela AL, Macas J, Houben A, Pedrosa-Harand A. Centromeric and non-centromeric satellite DNA organisation differs in holocentric Rhynchospora species. Chromosoma. 2017;126:325–335. doi: 10.1007/s00412-016-0616-3. PubMed DOI

Rocchetti A, Hawes C, Kriechbaumer V. Fluorescent labelling of the actin cytoskeleton in plants using a cameloid antibody. Plant Methods. 2014;10:12. doi: 10.1186/1746-4811-10-12. PubMed DOI PMC

Rust MJ, Bates M, Zhuang X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) Nat Methods. 2006;3:793–795. doi: 10.1038/nmeth929. PubMed DOI PMC

Sahl SJ, Hell SW, Jakobs S. Fluorescence nanoscopy in cell biology. Nat Rev Mol Cell Biol. 2017;18:685–701. doi: 10.1038/nrm.2017.71. PubMed DOI

Samajová O, Komis G, Samaj J. Immunofluorescent localization of MAPKs and colocalization with microtubules in Arabidopsis seedling whole-mount probes. Methods Mol Biol. 2014;2014(1171):107–115. doi: 10.1007/978-1-4939-0922-3_9. PubMed DOI

Šamaj J, Ovečka M, Hlavacka A, Lecourieux F, Meskiene I, Lichtscheidl I, Lenart P, Salaj J, Volkmann D, Bögre L, Baluska F, Hirt H. Involvement of the mitogen-activated protein kinase SIMK in regulation of root hair tip growth. EMBO J. 2002;21:3296–3306. doi: 10.1093/emboj/cdf349. PubMed DOI PMC

Sasabe M, Kosetsu K, Hidaka M, Murase A, Machida Y. Arabidopsis thaliana MAP65-1 and MAP65-2 function redundantly with MAP65-3/PLEIADE in cytokinesis downstream of MPK4. Plant Signal Behav. 2011;6:743–747. doi: 10.4161/psb.6.5.15146. PubMed DOI PMC

Sauer M, Paciorek T, Benková E, Friml J. Immunocytochemical techniques for whole-mount in situ protein localization in plants. Nat Protoc. 2006;1:98–103. doi: 10.1038/nprot.2006.15. PubMed DOI

Schmid VJ, Cremer M, Cremer T. Quantitative analyses of the 3D nuclear landscape recorded with super-resolved fluorescence microscopy. Methods. 2017;123:33–46. doi: 10.1016/j.ymeth.2017.03.013. PubMed DOI

Schubert V, Lermontova I, Schubert I. Loading of the centromeric histone H3 variant during meiosis—how does it differ from mitosis? Chromosoma. 2014;123:491–497. doi: 10.1007/s00412-014-0466-9. PubMed DOI

Schubert V, Lermontova I, Schubert I. The Arabidopsis CAP-D proteins are required for correct chromatin organisation, growth and fertility. Chromosoma. 2013;122:517–533. doi: 10.1007/s00412-013-0424-y. PubMed DOI

Schubert V. RNA polymerase II forms transcription networks in rye and Arabidopsis nuclei and its amount increases with endopolyploidy. Cytogenet Genome Res. 2014;143:69–77. doi: 10.1159/000365233. PubMed DOI

Schubert V. Super-resolution microscopy—applications in plant cell research. Front Plant Sci. 2017;8:531. doi: 10.3389/fpls.2017.00531. PubMed DOI PMC

Shaw SL, Ehrhardt DW. Smaller, faster, brighter: advances in optical imaging of living plant cells. Annu Rev Plant Biol. 2013;64:351–375. doi: 10.1146/annurev-arplant-042110-103843. PubMed DOI

Smékalová V, Luptovčiak I, Komis G, Šamajová O, Ovečka M, Doskočilová A, Takáč T, Vadovič P, Novák O, Pechan T, Ziemann A, Košútová P, Šamaj J. Involvement of YODA and mitogen activated protein kinase 6 in Arabidopsis post-embryogenic root development through auxin up-regulation and cell division plane orientation. New Phytol. 2014;203:1175–1193. doi: 10.1111/nph.12880. PubMed DOI PMC

Staudt T, Lang MC, Medda R, Engelhardt J, Hell SW. 2,2′-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy. Microsc Res Tech. 2007;70:1–9. doi: 10.1002/jemt.20396. PubMed DOI

Szczurek A, Contu F, Hoang A, Dobrucki J, Mai S. Aqueous mounting media increasing tissue translucence improve image quality in structured illumination microscopy of thick biological specimen. Sci Rep. 2018;8:13971. doi: 10.1038/s41598-018-32191-x. PubMed DOI PMC

Török P, Hewlett SJ, Varga P. The role of specimen-induced spherical aberration in confocal microscopy. J Microsc. 1997;1997(188):158–172. doi: 10.1046/j.1365-2818.1997.2440802.x. DOI

Vyplelová P, Ovečka M, Komis G, Šamaj J. Advanced microscopy methods for bioimaging of mitotic microtubules in plants. Methods Cell Biol. 2018;145:129–158. doi: 10.1016/bs.mcb.2018.03.019. PubMed DOI

Warner CA, Biedrzycki ML, Jacobs SS, Wisser RJ, Caplan JL, Sherrier DJ. An optical clearing technique for plant tissues allowing deep imaging and compatible with fluorescence microscopy. Plant Physiol. 2014;166:1684–1687. doi: 10.1104/pp.114.244673. PubMed DOI PMC

Werner S, Marillonnet S, Hause G, Klimyuk V, Gleba Y. Immunoabsorbent nanoparticles based on a tobamovirus displaying protein A. Proc Natl Acad Sci USA. 2006;103:17678–17683. doi: 10.1073/pnas.0608869103. PubMed DOI PMC

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