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      Advances in Two-Photon Imaging in Plants

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          Abstract

          Live and deep imaging play a significant role in the physiological and biological study of organisms. Two-photon excitation microscopy (2PEM), also known as multiphoton excitation microscopy, is a fluorescent imaging technique that allows deep imaging of living tissues. Two-photon lasers use near-infrared (NIR) pulse lasers that are less invasive and permit deep tissue penetration. In this review, recent advances in two-photon imaging and their applications in plant studies are discussed. Compared to confocal microscopy, NIR 2PEM exhibits reduced plant-specific autofluorescence, thereby achieving greater depth and high-resolution imaging in plant tissues. Fluorescent proteins with long emission wavelengths, such as orange–red fluorescent proteins, are particularly suitable for two-photon live imaging in plants. Furthermore, deep- and high-resolution imaging was achieved using plant-specific clearing methods. In addition to imaging, optical cell manipulations can be performed using femtosecond pulsed lasers at the single cell or organelle level. Optical surgery and manipulation can reveal cellular communication during development. Advances in in vivo imaging using 2PEM will greatly benefit biological studies in plant sciences.

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          Two-photon laser scanning fluorescence microscopy.

          Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. The excitation of fluorophores having single-photon absorption in the ultraviolet with a stream of strongly focused subpicosecond pulses of red laser light has made possible fluorescence images of living cells and other microscopic objects. The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation. This technique also provides unprecedented capabilities for three-dimensional, spatially resolved photochemistry, particularly photolytic release of caged effector molecules.
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            Über Elementarakte mit zwei Quantensprüngen

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                Author and article information

                Contributors
                Journal
                Plant Cell Physiol
                Plant Cell Physiol
                pcp
                Plant and Cell Physiology
                Oxford University Press (UK )
                0032-0781
                1471-9053
                August 2021
                26 May 2021
                26 May 2021
                : 62
                : 8 , Special Issue Advances in Plant Imaging Technologies
                : 1224-1230
                Affiliations
                departmentInstitute for Advanced Research (IAR), Nagoya University , Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan
                departmentInstitute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University , Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan
                Author notes
                *Corresponding author: E-mail, mizuta.yoko@ 123456f.mbox.nagoya-u.ac.jp .
                Author information
                https://orcid.org/0000-0002-8086-2297
                Article
                pcab062
                10.1093/pcp/pcab062
                8579158
                34019083
                994e7aa8-8b15-4536-9e54-6ec3e4214e28
                © The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License ( https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

                History
                : 06 January 2021
                : 16 March 2021
                : 20 May 2021
                : 08 April 2021
                : 13 July 2021
                Page count
                Pages: 7
                Funding
                Funded by: Japan Society for the Promotion of Science, DOI 10.13039/501100001691;
                Award ID: 20H05778, 20H05779
                Funded by: Japan Society for the Promotion of Science, DOI 10.13039/501100001691;
                Award ID: JP16H06464, JP16K21727
                Funded by: Foundation of Kinoshita Memorial Enterprise;
                Award ID: 2020 Grant-in-Aid for Academic research
                Categories
                Special Issue - Review
                AcademicSubjects/SCI01210
                AcademicSubjects/SCI01180

                Plant science & Botany
                deep imaging,fluorophore,laser ablation,live imaging,plant clearing,two-photon microscopy

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