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      Imaging plant cells and organs with light-sheet and super-resolution microscopy

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          Abstract

          The documentation of plant growth and development requires integrative and scalable approaches to investigate and spatiotemporally resolve various dynamic processes at different levels of plant body organization. The present update deals with vigorous developments in mesoscopy, microscopy and nanoscopy methods that have been translated to imaging of plant subcellular compartments, cells, tissues and organs over the past 3 years with the aim to report recent applications and reasonable expectations from current light-sheet fluorescence microscopy (LSFM) and super-resolution microscopy (SRM) modalities. Moreover, the shortcomings and limitations of existing LSFM and SRM are discussed, particularly for their ability to accommodate plant samples and regarding their documentation potential considering spherical aberrations or temporal restrictions prohibiting the dynamic recording of fast cellular processes at the three dimensions. For a more comprehensive description, advances in living or fixed sample preparation methods are also included, supported by an overview of developments in labeling strategies successfully applied in plants. These strategies are practically documented by current applications employing model plant Arabidopsis thaliana (L.) Heynh., but also robust crop species such as Medicago sativa L. and Hordeum vulgare L. Over the past few years, the trend towards designing of integrative microscopic modalities has become apparent and it is expected that in the near future LSFM and SRM will be bridged to achieve broader multiscale plant imaging with a single platform.

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          Most cited references109

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          Reactive oxygen species produced by NADPH oxidase regulate plant cell growth.

          Cell expansion is a central process in plant morphogenesis, and the elongation of roots and root hairs is essential for uptake of minerals and water from the soil. Ca2+ influx from the extracellular store is required for (and sets the rates of) cell elongation in roots. Arabidopsis thaliana rhd2 mutants are defective in Ca2+ uptake and consequently cell expansion is compromised--rhd2 mutants have short root hairs and stunted roots. To determine the regulation of Ca2+ acquisition in growing root cells we show here that RHD2 is an NADPH oxidase, a protein that transfers electrons from NADPH to an electron acceptor leading to the formation of reactive oxygen species (ROS). We show that ROS accumulate in growing wild-type (WT) root hairs but their levels are markedly decreased in rhd2 mutants. Blocking the activity of the NADPH oxidase with diphenylene iodonium (DPI) inhibits ROS formation and phenocopies Rhd2-. Treatment of rhd2 roots with ROS partly suppresses the mutant phenotype and stimulates the activity of plasma membrane hyperpolarization-activated Ca2+ channels, the predominant root Ca2+ acquisition system. This indicates that NADPH oxidases control development by making ROS that regulate plant cell expansion through the activation of Ca2+ channels.
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            Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution.

            Contrary to the well known diffraction limit, the fluorescence microscope is in principle capable of unlimited resolution. The necessary elements are spatially structured illumination light and a nonlinear dependence of the fluorescence emission rate on the illumination intensity. As an example of this concept, this article experimentally demonstrates saturated structured-illumination microscopy, a recently proposed method in which the nonlinearity arises from saturation of the excited state. This method can be used in a simple, wide-field (nonscanning) microscope, uses only a single, inexpensive laser, and requires no unusual photophysical properties of the fluorophore. The practical resolving power is determined by the signal-to-noise ratio, which in turn is limited by photobleaching. Experimental results show that a 2D point resolution of <50 nm is possible on sufficiently bright and photostable samples.
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              Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy.

              Lateral resolution that exceeds the classical diffraction limit by a factor of two is achieved by using spatially structured illumination in a wide-field fluorescence microscope. The sample is illuminated with a series of excitation light patterns, which cause normally inaccessible high-resolution information to be encoded into the observed image. The recorded images are linearly processed to extract the new information and produce a reconstruction with twice the normal resolution. Unlike confocal microscopy, the resolution improvement is achieved with no need to discard any of the emission light. The method produces images of strikingly increased clarity compared to both conventional and confocal microscopes.
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                Author and article information

                Journal
                Plant Physiol
                Plant Physiol
                plphys
                Plant Physiology
                Oxford University Press
                0032-0889
                1532-2548
                February 2022
                26 August 2021
                26 August 2021
                : 188
                : 2
                : 683-702
                Affiliations
                [1 ] Department of Cell Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University Olomouc , Šlechtitelů 27, 783 71 Olomouc, Czech Republic
                [2 ] Centre of the Region Haná for Biotechnological and Agricultural Research, Czech Advanced Technology and Research Institute, Palacký University Olomouc , Šlechtitelů 27, 783 71 Olomouc, Czech Republic
                Author notes
                Author for communication: jozef.samaj@ 123456upol.cz
                [†]

                Senior author.

                [‡]

                These authors contributed equally to this work.

                Author information
                https://orcid.org/0000-0002-1570-2174
                https://orcid.org/0000-0002-7673-9640
                https://orcid.org/0000-0001-8021-8082
                https://orcid.org/0000-0002-6672-2722
                https://orcid.org/0000-0001-5557-6152
                https://orcid.org/0000-0001-9007-2398
                https://orcid.org/0000-0003-4750-2123
                Article
                kiab349
                10.1093/plphys/kiab349
                8825356
                35235660
                8388dce4-d8c3-417c-9259-86ea904bc643
                © The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence ( https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

                History
                : 9 May 2021
                : 12 July 2021
                : 16 September 2021
                Page count
                Pages: 20
                Funding
                Funded by: Czech Science Foundation GAČR;
                Award ID: 19-18675S
                Funded by: ERDF project “Plants;
                Award ID: CZ.02.1.01/0.0/0.0/16_019/0000827
                Categories
                Focus Issue on the Plant Cell Atlas
                Update
                Cell Biology
                AcademicSubjects/SCI02286
                AcademicSubjects/SCI02287
                AcademicSubjects/SCI01270
                AcademicSubjects/SCI01280
                AcademicSubjects/SCI02288

                Plant science & Botany
                Plant science & Botany

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