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Proceedings Article

In-vivo third-harmonic generation microscopy at 1550nm three-dimensional long-term time-lapse studies in living C. elegans embryos

[+] Author Affiliations
Rodrigo Aviles-Espinosa, Susana I. C. O. Santos, Cesar Alonso-Ortega, Pablo Loza-Alvarez

Instituto de Ciencias Fotónicas (Spain)

Andreas Brodschelm, Wilhelm G. Kaenders

TOPTICA Photonics AG (Germany)

David Artigas

Instituto de Ciencias Fotónicas (Spain) and Univ. Politècnica de Catalunya (Spain)

Proc. SPIE 7904, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XVIII, 79041G (February 10, 2011); doi:10.1117/12.874931
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From Conference Volume 7904

  • Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XVIII
  • Jose-Angel Conchello; Carol J. Cogswell; Tony Wilson; Thomas G. Brown
  • San Francisco, California, USA | January 22, 2011

abstract

In-vivo microscopic long term time-lapse studies require controlled imaging conditions to preserve sample viability. Therefore it is crucial to meet specific exposure conditions as these may limit the applicability of established techniques. In this work we demonstrate the use of third harmonic generation (THG) microscopy for long term time-lapse three-dimensional studies (4D) in living Caenorhabditis elegans embryos employing a 1550 nm femtosecond fiber laser. We take advantage of the fact that THG only requires the existence of interfaces to generate signal or a change in the refractive index or in the χ3 nonlinear coefficient, therefore no markers are required. In addition, by using this wavelength the emitted THG signal is generated at visible wavelengths (516 nm) enabling the use of standard collection optics and detectors operating near their maximum efficiency. This enables the reduction of the incident light intensity at the sample plane allowing to image the sample for several hours. THG signal is obtained through all embryo development stages, providing different tissue/structure information. By means of control samples, we demonstrate that the expected water absorption at this wavelength does not severely compromise sample viability. Certainly, this technique reduces the complexity of sample preparation (i.e. genetic modification) required by established linear and nonlinear fluorescence based techniques. We demonstrate the non-invasiveness, reduced specimen interference, and strong potential of this particular wavelength to be used to perform long-term 4D recordings.

© (2011) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
Citation

Rodrigo Aviles-Espinosa ; Susana I. C. O. Santos ; Andreas Brodschelm ; Wilhelm G. Kaenders ; Cesar Alonso-Ortega, et al.
"In-vivo third-harmonic generation microscopy at 1550nm three-dimensional long-term time-lapse studies in living C. elegans embryos", Proc. SPIE 7904, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XVIII, 79041G (February 10, 2011); doi:10.1117/12.874931; http://dx.doi.org/10.1117/12.874931


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