BACKGROUND: The visualization of internal 3D-structure of tissues at micron resolutions without staining by contrast reagents is desirable in plant researches, and it can be achieved by an X-ray computed tomography (CT) with a phase-retrieval technique. Recently, a laboratory-based X-ray microscope adopting the phase contrast CT was developed as a powerful tool for the observation of weakly absorbing biological samples. Here we report the observation of unstained pansy seeds using the laboratory-based X-ray phase-contrast CT. RESULTS: A live pansy seed within 2 mm in size was simply mounted inside a plastic tube and irradiated by in-house X-rays to collect projection images using a laboratory-based X-ray microscope. The phase-retrieval technique was applied to enhance contrasts in the projection images. In addition to a dry seed, wet seeds on germination with the poorer contrasts were tried. The phase-retrieved tomograms from both the dry and the wet seeds revealed a cellular level of spatial resolutions that were enough to resolve cells in the seeds, and provided enough contrasts to delineate the boundary of embryos manually. The manual segmentation allowed a 3D rendering of embryos at three different stages in the germination, which visualized an overall morphological change of the embryo upon germination as well as a spatial arrangement of cells inside the embryo. CONCLUSIONS: Our results confirmed an availability of the laboratory-based X-ray phase-contrast CT for a 3D-structural study on the development of small seeds. The present method may provide a unique way to observe live plant tissues at micron resolutions without structural perturbations due to the sample preparation.

10 Ray Research Laboratory Rigaku Corporation 3 9 12 Matsubara cho Akishima Tokyo 196 8666 Japan

CEITEC Central European Institute of Technology Brno University of Technology Purkynova 123 612 00 Brno Czech Republic

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Fischer RS, Wu Y, Kanchanawong P, Shroff H, Waterman CM. Microscopy in 3D: a biologist’s toolbox. Trends Cell Biol. 2011;21(12):682–691. doi: 10.1016/j.tcb.2011.09.008. PubMed DOI PMC

Denk W, Strickler JH, Webb WW. Two-photon laser scanning fluorescence microscopy. Science. 1990;248(4951):73–76. doi: 10.1126/science.2321027. PubMed DOI

Gustafsson MGL. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc. 2000;198(2):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(11):780–782. doi: 10.1364/OL.19.000780. 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(5793):1642–1645. doi: 10.1126/science.1127344. PubMed DOI

Komis G, Novák D, Ovečka M, Šamajová O, Šamaj J. Advances in imaging plant cell dynamics. Plant Physiol. 2018;176(1):80–93. doi: 10.1104/pp.17.00962. PubMed DOI PMC

Cotte Y, Toy F, Jourdain P, Pavillon N, Boss D, Magistretti P, Marquet P, Depeursinge C. Marker-free phase nanoscopy. Nat Photonics. 2013;7(2):113–117. doi: 10.1038/nphoton.2012.329. DOI

Knott G, Genoud C. Is EM dead? J Cell Sci. 2013;126(20):4545–4552. doi: 10.1242/jcs.124123. PubMed DOI

Marion J, Le Bars R, Satiat-Jeunemaitre B, Boulogne C. Optimizing CLEM protocols for plants cells: GMA embedding and cryosections as alternatives for preservation of GFP fluorescence in Arabidopsis roots. J Struct Biol. 2017;198(3):196–202. doi: 10.1016/j.jsb.2017.03.008. PubMed DOI

Denk W, Horstmann H. Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol. 2004;2(11):e329. doi: 10.1371/journal.pbio.0020329. PubMed DOI PMC

Płachno BJ, Świątek P, Jobson RW, Małota K, Brutkowski W. Serial block face SEM visualization of unusual plant nuclear tubular extensions in a carnivorous plant (Utricularia, Lentibulariaceae) Ann Bot. 2017;120(5):673–680. doi: 10.1093/aob/mcx042. PubMed DOI PMC

Bronnikov AV. Reconstruction formulas in phase-contrast tomography. Opt Commun. 1999;171:239–244. doi: 10.1016/S0030-4018(99)00575-1. DOI

Paganin D, Mayo SC, Gureyev TE, Miller PR, Wilkins SW. Simultaneous phase and amplitude extraction from a single defocused image of a homogeneous object. J Microsc. 2002;206(1):33–40. doi: 10.1046/j.1365-2818.2002.01010.x. PubMed DOI

Mayo SC, Davis TJ, Gureyev TE, Miller PR, Paganin D, Pogany A, Stevenson AW, Wilkins SW. X-ray phase-contrast microscopy and microtomography. Opt Express. 2003;11(19):2289–2302. doi: 10.1364/OE.11.002289. PubMed DOI

Myers GR, Mayo SC, Gureyev TE, Paganin DM, Wilkins SW. Polychromatic cone-beam phase-contrast tomography. Phys Rev A. 2007;76(4):045804. doi: 10.1103/PhysRevA.76.045804. DOI

De Witte Y, Boone M, Vlassenbroeck J, Dierick M, Van Hoorebeke L. Bronnikov-aided correction for x-ray computed tomography. J Opt Soc Am A. 2009;26(4):890–894. doi: 10.1364/JOSAA.26.000890. PubMed DOI

Kalasová D, Zikmund T, Pína L, Takeda Y, Horváth M, Omote K, Kaiser J. Characterization of a laboratory-based X-ray computed nanotomography system for propagation-based method of phase contrast imaging. IEEE Trans Instrum Meas. 2019 doi: 10.1109/TIM.2019.2910338. DOI

Rousseau D, Widiez T, Di Tommaso S, Rositi H, Adrien J, Maire E, Langer M, Olivier C, Peyrin F, Rogowsky P. Fast virtual histology using X-ray in-line phase tomography: application to the 3D anatomy of maize developing seeds. Plant Methods. 2015;11:55. doi: 10.1186/s13007-015-0098-y. PubMed DOI PMC

Larsson DH, Vågberg W, Yaroshenko A, Yildirim AÖ, Hertz HM. High-resolution short-exposure small-animal laboratory x-ray phase-contrast tomography. Sci Rep. 2016;6:39074. doi: 10.1038/srep39074. PubMed DOI PMC

Töpperwien M, Krenkel M, Vincenz D, Stöber F, Oelschlegel AM, Goldschmidt J, Salditt T. Three-dimensional mouse brain cytoarchitecture revealed by laboratory-based x-ray phase-contrast tomography. Sci Rep. 2017;7:42847. doi: 10.1038/srep42847. PubMed DOI PMC

Müller M, de Sena Oliveria I, Allner S, Ferstl S, Bidola P, Mechlem K, Fehringer A, Hehn L, Dierolf M, Achterhold K, Gleich B, Hammel JU, Jahn H, Mayer G, Pfeiffer F. Myoanatomy of the velvet worm leg revealed by laboratory-based nanofocus X-ray source tomography. Proc Natl Acad Sci USA. 2017;114(47):12378–12383. doi: 10.1073/pnas.1710742114. PubMed DOI PMC

Töpperwien M, Markus A, Alves F, Salditt T. Contrast enhancement for visualizing neuronal cytoarchitecture by propagation-based x-ray phase-contrast tomography. Neuroimage. 2019;199:70–80. doi: 10.1016/j.neuroimage.2019.05.043. PubMed DOI

Bino RJ, Aartse JW, van der Burg WJ. Non-destructive X-ray analysis of Arabidopsis embryo mutants. Seed Sci Res. 1993;3(3):167–170. doi: 10.1017/S0960258500001744. DOI

Rousseau D, Chéné Y, Belin E, Semaan G, Trigui G, Boudehri K, Franconi F, Chapeau-Blondeau F. Multiscale imaging of plants: current approaches and challenges. Plant Methods. 2015;11:6. doi: 10.1186/s13007-015-0050-1. PubMed DOI PMC

Moran CJ, Pierret A, Stevenson AW. X-ray absorption and phase contrast imaging to study the interplay between plant roots and soil structure. Plant Soil. 2000;223(1–2):101–117. doi: 10.1023/A:1004835813094. DOI

Bidola P, Morgan K, Willner M, Fehringer A, Allner S, Prade F, Pfeiffer F, Achterhold K. Application of sensitive, high-resolution imaging at a commercial lab-based X-ray micro-CT system using propagation-based phase retrieval. J Microsc. 2017;266(2):211–220. doi: 10.1111/jmi.12530. PubMed DOI

Mathers AW, Hepworth C, Baillie AL, Sloan J, Jones H, Lundgren M, Fleming AJ, Mooney SJ, Sturrock CJ. Investigating the microstructure of plant leaves in 3D with lab-based X-ray computed tomography. Plant Methods. 2018;14:99. doi: 10.1186/s13007-018-0367-7. PubMed DOI PMC

Kass M, Witkin A, Terzopoulos D. Snakes: active contour models. Int J Comp Vision. 1988;1(4):321–331. doi: 10.1007/BF00133570. DOI

Box GEP, Wilson KB. On the experimental attainment of optimum conditions. J Royal Stat Soc B. 1951;13(1):1–38.

Rosenblatt M. Remarks on some nonparametric estimates of a density function. Ann Math Statist. 1956;27(3):832–837. doi: 10.1214/aoms/1177728190. DOI

Parzen E. On estimation of a probability density function and mode. Ann Math Statist. 1962;33(3):1065–1076. doi: 10.1214/aoms/1177704472. DOI

Carzaniga R, Domart MC, Collinson LM, Duke E. Cryo-soft X-ray tomography: a journey into the world of the native-state cell. Protoplasma. 2014;251(2):449–458. doi: 10.1007/s00709-013-0583-y. PubMed DOI PMC

Fogelqvist E, Kördel M, Carannante V, Önfelt B, Hertz HM. Laboratory cryo x-ray microscopy for 3D cell imaging. Sci Rep. 2017;7:13433. doi: 10.1038/s41598-017-13538-2. PubMed DOI PMC

Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671–675. doi: 10.1038/nmeth.2089. PubMed DOI PMC

van Aarle W, Palenstijn WJ, Cant J, Janssens E, Bleichrodt F, Dabravolski A, De Beenhouwer J, Joost Batenburg K, Sijbers J. Fast and flexible X-ray tomography using the ASTRA toolbox. Opt Express. 2016;24(22):25129–25147. doi: 10.1364/OE.24.025129. PubMed DOI

Limaye A. Drishti—volume exploration and presentation tool. Poster presentation In: IEEE Visualization 2006 (Vis 2006), Baltimore, Maryland, USA, 2006.