Variations of imaging depth and chloroplast emission spectrum of Arabidopsis thaliana with excitation wavelength in two-photon microscopy

이광자현미경 여기 광 파장에 따른 Arabidopsis thaliana 촬영 깊이 및 엽록체 형광 스펙트럼의 변화

  • 주용준 (포항공과대학교 융합생명공학부) ;
  • 손시형 (포항공과대학교 기계공학과) ;
  • 김기현 (포항공과대학교 융합생명공학부)
  • Received : 2014.12.03
  • Accepted : 2014.12.24
  • Published : 2014.12.31


Two-photon microscopy (TPM) has been used in plant research as a high-resolution high-depth 3D imaging modality. However, TPM is known to induce photo-damage to the plant in case of long time exposure, and optimal excitation wavelength for plant imaging has not been investigated. Longer excitation wavelength may be appropriate for in vivo two-photon imaging of Arabidopsis thaliana leaves, and effects of longer excitation wavelength were investigated in terms of imaging depth, emission spectrum. Changes of emission spectrum as a function of exposure time at longer excitation wavelength were measured for in vivo longitudinal imaging. Imaging depth was not changed much probably because photon scattering at the cell wall was a limiting factor. Chloroplast emission spectrum showed its intensity peak shift by 20 nm with transition of excitation wavelength from 849 nm or below to 850 nm or higher. Emission spectrum showed different change patterns with excitation wavelengths in longitudinal imaging. Longer excitation wavelengths appeared to interact with chloroplasts differently in comparison with 780 nm excitation wavelength, and may be good for in vivo imaging.


  1. Kate Maxwell, Giles N. Johnson, 2000, "Chlorophyll fluorescence - a practical guide", Journal of Experimental botany, Vol.51, No. 345, pp.659-668.
  2. Zuzana Benediktyová, Ladishlave NEdbal, 2009, "Imaging of multi-color fluorescene emission from leaf tissues", Photosynth Res, Vol. 102, pp.169-175.
  3. Winfried Denk, James H. Strickler, Watt W. Webb, 1990, "Two-photon Laser Scanning Fluorescence microscopy", Science, Vol.248, pp.73-76.
  4. Peter T.C. So, Chen Y. Dong, Barry R. Masters, Keith M. Berland, 2000, "Two-photon Excitaion Fluorescence Microscopy", Annu. Rev. Biomed. Eng 02, pp.339-429.
  5. Warren R Zipfel, Rebecca M Williams, Watt W Webb, 2003, "Nonlinear magic: multiphoton microscopy in the biosciences", Nature Biotechnology Volume 21, Number 11, pp.1369-1377.
  6. Goeppert-Mayer, M., 1931, "Uber Elementarakte mit zwei Quantensprungen", Annals of Physics 9 pp.273-294.
  7. S.P. Schilders and M. Gu, 2000, "Limiting Factors on Image Quality in Imaging through Turbid Media under Single-photon and Two-photon excitation", Microscopy and Microanalysis 6, pp.156-160.
  8. Peter TC So, 2000, "Two-photon Fluorescence Light Microscopy", Encyclopedia of Life Science, pp.1-5.
  9. Ping-Chin Chen, 2006, "Interaction of Light with Botanical Specimens", Handbook of Biological Confocal Microscopy, Third Edition, pp.414-441.
  10. Ping-chin Cheng, Bai-Ling Lin, Fu-jen Kao, Min Gu, Ming-Gun Xu, XiaosangGan, Mao-Kuo Huang, Yung-Shun Wang, 2001, "Multi-photon fluorescence microscopy - the response of plant cells to high intensity illumination", Micron 32, pp.661-669.
  11. George R. Littlejohn, Jessica C. Mansfield, Jacqueline T. Christmas, Eleanor Witterick, Mark D. Fricker, Murray R. Grant, Nicholas Smirnoff, Richard M. Everson, Julian Moger and John Love, 2014, "An update: improvements in imaging perfluorocarbon-mounted plant leaves with implications for studies of plant pathology, physiology, development and cell biology", frontiers in PLANT SCIENCE, Vol. 5, Article 140.
  12. Little john G.. R., Gouveia J. D., Edner C., Smirnoff N., and Love J., 2010, "Perfluo- rodecalin enhances in vivo confocal microscopy resolution of Arabidopsis thaliana mesophyll", NewPhytol. 186, 1018-1025.
  13. Littlejohn G. R., and Love J., 2012, "A simple method for confocal imaging of Arabidopsis leaves with perfluorodecalin as infiltrative imaging medium". J. Vis.Exp. 16.
  14. Fu-Jen Kao, Yi-Ming Wang, Jian-Cheng Chen, Ping-Chin Cheng, Rung-Wi Chen, Bai-Ling Lin, 2002, "Micro-spectroscopy of chloroplasts in protoplasts from Arabidopsis thaliana under single and multi photon excitations", Journal of Luminescence 98, pp.107-114.
  15. Fu-Jen Kao, Yi-Min Wang, Jian-Cheng Chen, Ping-Chin Chen, Rung-Wu Chen, Bai-Ling Lin, 2002, "Photobleaching under single photon and multi-photon excitation: chloroplasts in protoplasts from Arabidopsis thaliana", Optics Communications 201, pp.85-91.
  16. Peter J. Walla, Jenny Yom, Brent P. Krueger, and Graham R. Fleming, 2000, "Two-Photon Excitation Spectrum of Light-Harvesting Complex II and Fluorescence Upconversion after One- and Two-Photon Excitation of the Carotenoids", J. Phys. Chem. B, Vol.104, pp.4799-4806.
  17. Roberto Pedros, Ismael Moya, Yves Goulas, Stephane Jacquemoud, 2008, "Chlorophyll fluorescence emission spectrum inside a leaf", Photochemical & Photobiological Sciences, Vol. 7, pp.498-502..