Abstract
Serial sectioning transmission electron microscopy (ssTEM) is a classical method of 3D reconstruction using serial sections obtained with an ultramicrotome. However, producing a long ribbon with homogeneity is difficult. Here, ultramicrotome movement was suspended after producing a ribbon of 15–30 serial sections (cutting intervals, 100 nm), and then, the ribbon was mounted on an individual one-slot grid. However, as this ssTEM method may include influencing factors such as incorrect intervals of section thickness and distortion of sections, which is produced by cutting sections using a diamond knife and beam interaction under TEM observation, qualitative and quantitative data on rice mesophyll cells and chloroplasts were compared with those obtained from a focused ion beam scanning electron microscopy (FIB-SEM) (cutting intervals, 50 nm). No structural distortion in 3D models was observed. In addition, no significant differences in the volume and surface area were observed between the two methods. The surface to volume ratio was significantly affected by the increase in section thickness, but not the difference of methodologies. Our method was useful for observing large volumes of plant cells and organelles, leading to the identification of various sizes and types of chloroplasts. The formation of a chloroplast pocket, which is a structure surrounding other intracellular compartments, was confirmed in rice leaves grown under moderate growth conditions using the ssTEM method. As only four out of 90 chloroplasts formed pocket structures, the formation was considered to be rare under the applied moderate growth conditions.
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References
Bang BG, Bang B (1957) Graphic reconstruction of the third dimension from serial electron microphotographs. J Ultrastruct Res 1:138–139. https://doi.org/10.1016/S0022-5320(57)80002-1
Birch-Andersen A (1955) Reconstruction of the nuclear sites of Salmonella typhimurium from electron micrographs of serial sections. J Gen Microbiol 13:327–329. https://doi.org/10.1099/00221287-13-2-327
Blumer MJF, Gahleitner P, Narzt T, Handl C, Ruthensteiner B (2002) Ribbons of semithin sections: an advanced method with a new type of diamond knife. J Neurosci Methods 120:11–16. https://doi.org/10.1016/S0165-0270(02)00166-8
Fahrenbach WH (1984) Continuous serial thin sectioning for electron microscopy. J Electron Microsc Tech 1:387–398. https://doi.org/10.1002/jemt.1060010407
Guest E, Baldock R (1995) Automatic reconstruction of serial sections using the finite element method. Bioimaging 3:154–167. https://doi.org/10.1002/1361-6374(199512)3:4%3c154::AID-BIO2%3e3.0.CO;2-M
Harris KM, Perry E, Bourne J, Feinberg M, Ostroff L, Hurlburt J (2006) Uniform serial sectioning for transmission electron microscopy. J Neurosci 22:12101–12103. https://doi.org/10.1523/JNEUROSCI.3994-06.2006
Hayworth KJ, Morgan JL, Schalek R, Berger DR, Hildebrand DGC, Lichtman JW (2014) Imaging ATUM ultrathin section libraries with WaterMapper: a multi-scale approach to EM reconstruction of neural circuits. Front Neural Circuits 8:68. https://doi.org/10.3389/fncir.2014.00068
Horstmann H, Körber C, Sätzler K, Aydin D, Kuner T (2012) Serial section scanning electron microscopy (S3EM) on silicon wafers for ultra-structural volume imaging of cells and tissues. PLoS ONE 7:e35172. https://doi.org/10.1371/journal.pone.0035172
Hughes L, Hawes C, Monteith S, Vaughan S (2014) Serial block face scanning electron microscopy – the future of cell ultrastructure imaging. Protoplasma 251:395–401. https://doi.org/10.1007/s00709-013-0580-1
Islam MS, Niwa Y, Takagi S (2009) Light-dependent intracellular positioning of mitochondria in Arabidopsis thaliana mesophyll cells. Plant Cell Physiol 50:1032–1040. https://doi.org/10.1093/pcp/pcp054
Karnovsky MJ (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 27:137A (http://www.jstor.org/stable/1604673)
Kittelmann M, Hawes C, Hughes L (2016) Serial block face scanning electron microscopy and the reconstruction of plant cell membrane systems. J Microsc 263:200–211. https://doi.org/10.1111/jmi.12424
Kizilyaprak C, Daraspe J, Humbel BM (2014) Focused ion bean scanning electron microscopy in biology. J Microsc 254:109–114. https://doi.org/10.1111/jmi.12127
Micheva KD, Smith S (2007) Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron 55:25–36. https://doi.org/10.1016/j.neuron.2007.06.014
Miranda K, Girard-dias W, Attias M, De Souza W, Ramos I (2015) Three dimensional reconstruction by electron microscopy in the life sciences: an introduction for cell and tissue biologists. Mol Reprod Dev 82:530–547. https://doi.org/10.1002/mrd.22455
Ohta K, Sadayama S, Togo A, Higashi R, Tanoue R, Nakamura K (2012) Beam deceleration for block-face scanning electron microscopy of embedded biological tissue. Micron 43:612–620. https://doi.org/10.1016/j.micron.2011.11.001
Oi T, Enomoto S, Nakao T, Arai S, Yamane K, Taniguchi M (2017) Three-dimensional of a whole rice mesophyll cell observed with FIB-SEM. Ann Bot 120:21–28. https://doi.org/10.1093/aob/mcx036
Oi T, Enomoto S, Nakao T, Arai S, Yamane K, Taniguchi M (2020) Three-dimensional ultrastructural change of chloroplasts in rice mesophyll cells responding to salt stress. Ann Bot 125:833–840. https://doi.org/10.1093/aob/mcz192
Ota S, Oshima K, Yamazaki T, Kim S, Yu Z, Yoshihara M, Takeda K, Takeshita T, Hirata A, Bišová K, Zachleder V, Hattori M, Kawano S (2016) Highly efficient lipid production in the green alga Parachlorella kessleri: draft genome and transcriptome endorsed by whole-cell 3D ultrastructure. Biotechnol Biofuels 9:13. https://doi.org/10.1186/s13068-016-0424-2
Ouk R, Oi T, Taniguchi M (2020) Three-dimensional anatomy of mesophyll cells in rice leaf tissue by serial section light microscopy. Plant Prod Sci 23:149–159. https://doi.org/10.1080/1343943X.2019.1702470
Reichelt M, Joubert L, Perrino J, Koh AL, Phanwar I, Arvin AM (2012) 3D reconstruction of VZV infected cell nuclei and PML nuclear cages by serial section array scanning electron microscopy and electron tomography. PLoS Pathog 8:e1002740. https://doi.org/10.1371/journal.ppat.1002740
Rieder CL (1981) Thick and thin serial sectioning for the three-dimensional reconstruction of biological ultrastructure. In: Turner JN (ed) Methods in cell biology: three-dimensional ultrastructure in biology. Academic Press INC, New York, pp 215–249
Saalfeld S, Fetter R, Cardona A, Tomancak P (2012) Elastic volume reconstruction from series of ultra-thin microscopy sections. Nat Methods 9:717e720. https://doi.org/10.1038/nmeth.2072
Sage TL, Sage RF (2009) The functional anatomy of rice leaves: implications for refixation of photorespiratory CO2 and efforts to engineer C4 photosynthesis into rice. Plant Cell Physiol 50:756–772. https://doi.org/10.1093/pcp/pcp033
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676e682. https://doi.org/10.1038/nmeth.2019
Stevens JK, Davis TL, Friedman N, Sterling PA (1980) A systematic approach to reconstructing microcircuitry by electron microscopy of serial sections. Braine Res Rev 2:265–293. https://doi.org/10.1016/0165-0173(80)90010-7
Uwizeye C, Decelle J, Jouneau P-H, Flori S, Gallet B, Keck J-B, Dal Bo D, Moriscot C, Seydoux C, Chevalier F, Schieber NL, Templin R, Allorent G, Courtois F, Curien G, Schwab Y, Schoehn G, Zeeman SC, Falconet D, Finazzi G (2021) Morphological bases of phytoplankton energy management and physiological responses unveiled by 3D subcellular imaging. Nature Commun. https://doi.org/10.1038/s41467-021-21314-0
Yamane K, Oi T, Enomoto S, Nakao T, Arai S, Miyake H, Taniguchi M (2018) Three-dimensional ultrastructure of chloroplast pockets formed under salinity stress. Plant Cell Environ 41:563–575. https://doi.org/10.1111/pce.13115
Yamane K, Oi T, Taniguchi M (2020) Three-dimensional analysis of chloroplast protrusion formed under osmotic stress by polyethylene glycol in rice leaves. Plant Prod Sci 23:160–171. https://doi.org/10.1080/1343943X.2019.1709513
Zellnig G, Zechmann B, Perktold A (2004) Morphological and quantitative data of plastids and mitochondria within drought-stressed spinach leaves. Protoplasma 223:221–227. https://doi.org/10.1007/s00709-003-0034-2
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This work was partially supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers JP18K05603 (to K. Y.), JP19K15823 (to T. O.), and JP20H02966 (to M. T.).
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Conceptualization: K. Y.; methodology: K. Y., and T. O.; software: K. Y. and T. O.; validation: T. O. and M. T.; formal analysis: K. Y.; investigation: K. Y.; Resources: K. Y., T. O., and M. T.; data curation, T. O. and M. T.; writing — original draft preparation:, K. Y.; writing — review and editing: K. Y., T. O., and M. T.; visualization: K. Y. and T. O; supervision: M. T.; funding acquisition: K. Y., T. O., and M. T.
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Yamane, K., Oi, T. & Taniguchi, M. Evaluation of the validity of large-scale serial sectioning TEM for three-dimensional reconstruction of rice mesophyll cells and chloroplasts. Protoplasma 259, 1219–1231 (2022). https://doi.org/10.1007/s00709-021-01728-9
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DOI: https://doi.org/10.1007/s00709-021-01728-9