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A rapid and effective optical-clearing technique for deep tissue fluorescence imaging in trees

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Abstract

Key message

This non-invasive clearing technique allows for deep clear imaging of plant tissue and 3D structural analysis of leaf cells.

Abstract

Optical-clearing technology allows the reduction of light scattering and absorption in internal biological tissues. This technology allows microscopy techniques, such as confocal laser scanning microscopy, to obtain structural information deep within the internal tissue and also allows for three-dimensional (3D) tissue analysis. However, the transparent method in plants remains to be further elucidated. In this study, we first report on a non-invasive, rapid and effective clearing technique that increases the transmission of light through plants. Importantly, we found that the leaf terminal veins of four species had different characteristics based on fluorescence imaging observations. Moreover, through the combination of transparent technology and microscopic observation, we clearly observed the three-dimensional structure of plant leaf cells. Our simple and rapid clearing method is useful for fluorescence imaging the intact internal structure. Taken together, the results of this study will provide valuable information for further microscopic experimental investigations in trees.

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References

  1. Amitrano C, Arena C, Rouphael Y, Pascale SD, Micco VD (2019) Vapour pressure deficit: The hidden driver behind plant morphofunctional traits in controlled environments. Ann Appl Biol 175:313–325

  2. Adeniyi IM, Adejoba OR, Akinlabi FM, Alao OJ (2016) Vegetable oils as clearing agents. Ach Life Sci 10:1–4

  3. Alwahaibi N, Aljaradi S, Alazri H (2018) Alternative to xylene as a clearing agent in histopathology. J Lab Phys 10:189–193

  4. Bashkatov AN, Genina EA, Tuchin VV, Altshuler GB (2009) Skin optical clearing for improvement of laser tattoo removal. Laser Phys 19:1312–1322

  5. Choi B, Tsu L, Chen E, Ishak TS, Iskandar SM, Chess S, Nelson JS (2005) Determination of chemical agent optical clearing potential using in vitro human skin. Laser Surg Med 36:72–75

  6. Cicchi R, Sampson D, Massi D, Pavone F (2005) Contrast and depth enhancement in two-photon microscopy of human skin ex vivo by use of optical clearing agents. Opt Express 13:2337–2344

  7. Economo MN, Clack NG, Lavis LD, Gerfen CR, Svoboda K, Myers EW, Chandrashekar J (2016) A platform for brain-wide imaging and reconstruction of individual neurons. Elife 5:e10566

  8. Feijo JA, Moreno N (2004) Imaging plant cells by two-photon excitation. Protoplasma 223:1–32

  9. Feng W, Shi R, Zhang C, Zhu D (2017) In vivo skin optical clearing efficiency of sucrose and fructose. J Opt Soc Am B.https://doi.org/10.1364/PIBM.2017.W3A.22

  10. Gardner RO (1975) An overview of botanical clearing technique. Stain Tech 50:99–105

  11. Genina EA, Bashkatov AN, Kochubey VI, Tuchin VV (2005) Optical clearing of human dura mater. Opt Spectrosc 98:470–476

  12. Genina EA, Bashkatov AN, Sinichkin YP, Tuchin VV (2006) Optical clearing of the eye sclera in vivo caused by glucose. Quantum Electron 36:1119–1124

  13. Genina EA, Bashkatov AN, Korobko AA, Zubkova EA, Tuchin VV, Yaroslavsky I, Altshuler GB (2008) Optical clearing of human skin: comparative study of permeability and dehydration of intact and photothermally perforated skin. J Biomed Opt 13:1–8

  14. Grabe DF, Peters JA (1998) Lactic acid clearing of grass seeds in tetrazolium tests. Seed Sci Technol 20:106–108

  15. Hama H, Kurokawa H, Kawano H, Ando R, Shimogori T, Noda H, Fukami K, Sakaue-Sawano A, Miyawaki A (2011) Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat Neurosci 14:1481–1488

  16. Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2:932–940

  17. Hoyer H (1882) Beiträ ge zur histologischen Technik. Biol Centralbl 2:23–24

  18. Ito M, Shirakawa R, Arita R, Karasawa Y, Imaki J, Amano S, Kobayashi Y, Takeuchi M (2014) Observation of whole-mount meibomian glands from cadaveric eyelids using a fructose-based optical clearing agent. IVOS 55:4442

  19. Jahrling N, Becker K, Schonbauer C, Schnorrer F, Dodt HU (2010) Three-dimensional reconstruction and segmentation of intact Drosophila by ultramicroscopy. Front Neuroendocrin 4:1

  20. Ke MT, Fujimoto S, Imai T (2013) SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nat Neurosci 16:1154–1161

  21. Knapp E, Flores R, Scheiblin D, Scheiblin D, Modla S, Czymmek K, Czymmek K, Yusibov V (2012) A cryohistological protocol for preparation of large plant tissue sections for screening intracellular fluorescent protein expression. BioTechniques 52:31–37

  22. Kost B, Spielhofer P, Chua NH (1998) A GFP-mouse talin fusion protein labels plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen tubes. Plant J 16:393–401

  23. Kurihara D, Mizuta Y, Sato Y, Higashiyama T (2015) ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging. Development 142:4168–4179

  24. Lersten NR (1986) Modified clearing method to show sieve tubes in minor veins of leaves. Stain Tech 61:231–234

  25. McNichols RJ, Fox MA, Gowda A, Tuya S, Bell B, Motamedi M (2005) Temporary dermal scatter reduction: quantitative assessment and implications for improved laser tattoo removal. Laser Surg Med 36:289–296

  26. Miyamichi K, Amat F, Moussavi F, Wang C, Wickersham I, Wall NR, Taniguchi H, Tasic B, Huang ZJ, He Z, Callaway EM, Horowitz MA, Luo L (2011) Cortical representations of olfactory input by trans-synaptic tracing. Nature 472:191–196

  27. Morley T (1968) Accelerated clearing of plant leaves by NaOH in association with oxygen. Stain Technol 433:15–19

  28. Paddock SW, Eliceiri KW (2014) Laser scanning confocal microscopy: history, applications, and related optical sectioning techniques. Methods Mol Biol 1075:9–47

  29. Palmer WM, Martin AP, Flynn JR, Reed SL, White RG, Furbank RT, Grof CPL (2015) PEA-CLARITY: 3D molecular imaging of whole plant organs. Sci Rep 5:13492

  30. Petra K, Matthias M (2019) Pretreatment: removing DNA contamination from ancient bones and teeth using sodium hypochlorite and phosphate. Ancient DNA.https://doi.org/10.1007/978-1-4939-9176-1_2

  31. Popham RE (1950) Cedarwood oil as a clearing agent with acetic alcohol fixatives. Biotech Histochem 25:112–113

  32. Sermadi W, Prabhu S, Acharya S, Javali SB (2014) Comparing the efficacy of coconut oil and xylene as a clearing agent in the histopathology laboratory. J Oral Maxillofac Pathol 18:S49–S53

  33. Sdobnov AY, Darvin ME, Geninab EA, Bashkatov AN, Lademann J, Tuchin VV (2018) Recent progress in tissue optical clearing for spectroscopic application. Spectrochim Acta A Mol Biomol Spectrosc 197:216–229

  34. Steward O, Zheng B, Tessier-Lavigne M (2003) False resurrections: distinguishing regenerated from spared axons in the injured central nervous system. J Comp Neurol 459:1–8

  35. Sudheendran N, Mohamed M, Ghosn MG, Tuchin VV, Larin KV (2010) Assessment of tissue optical clearing as a function of glucose concentration using optical coherence tomography. J Innov Opt Heal Sci 3:169–176

  36. Tuchina DK, Genin VD, Bashkatov AN, Genina EA, Tuchin VV (2016) Optical clearing of skin tissue ex vivo with polyethylene glycol. Opt Spectrosc+ 120:28–37

  37. Tuchin VV, Maksimova IL, Zimnyakov DA, Kon IL, Mavlyutov AH, Mishin AA (1997) Light propagation in tissues with controlled optical properties. J Biomed Opt 2:401–417

  38. Tuchin VV (2005) Optical clearing of tissues and blood using the immersion method. J Phys D Appl Phys 38:2497–2518

  39. Tuchin VV (2009) Hand book of optical sensing of glucose in biological fluids and tissues. CRC Press, Boca Raton, p 709

  40. Vargas G, Barton JK, Welch AJ (2008) Use of hyperosmotic chemical agent to improve the laser treatment of cutaneous vascular lesions. J Biomed Opt 13:021114

  41. Villani TS, Koroch AR, Simon JE (2013) An improved clearing and mounting solution to replace chloral hydrate in microscopic applications. Appl Plant Sci 1:1300016

  42. Warner CA, Biedrzycki ML, Jacobs SS, Wisser RJ, Caplan JL, Sherrier DJ (2014) An optical clearing technique for plant tissues allowing deep imaging and compatible with fluorescence microscopy. Plant Physiol 166:1684–1687

  43. Weatherburn MW (1967) Phenol-hypochlorite reaction for determination of ammonia. J Infect Dis 39:971–974

  44. Wen X, Tuchin VV, Luo QM, Zhu D (2009) Controling the scattering of Intralipid by using optical clearing agents. Phys Med Biol 54:6917–6930

  45. Zhu D, Larin KV, Luo Q, Tuchin VV (2013) Recent progress in tissue optical clearing. Laser Photon Rev 7:732–757

  46. Zingg B, Hintiryan H, Gou L, Song MY, Bay M, Bienkowski MS, Foster NN, Yamashita S, Bowman I, Toga AW, Dong HW (2014) Neural networks of the mouse neocortex. Cell 156:1096–1111

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (Nos. 31970182, 31670182, 31761133009 and 31401149), the State ‘13.5’ Key Research Program of China (No. 2016YFD0600102), and the Fundamental Research Funds for the Central Universities (No. 2019ZY29).

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Correspondence to Ruili Li.

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Communicated by V. De Micco.

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Cite this article

Lu, L., Cao, Y., He, Q. et al. A rapid and effective optical-clearing technique for deep tissue fluorescence imaging in trees. Trees (2020). https://doi.org/10.1007/s00468-020-01957-0

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Keywords

  • Tissue optical clearing
  • Deep fluorescence imaging
  • Three-dimensional structure
  • Terminal veins