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Proceedings Paper

In-vivo third-harmonic generation microscopy at 1550nm three-dimensional long-term time-lapse studies in living C. elegans embryos
Author(s): Rodrigo Aviles-Espinosa; Susana I. C. O. Santos; Andreas Brodschelm; Wilhelm G. Kaenders; Cesar Alonso-Ortega; David Artigas; Pablo Loza-Alvarez
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Paper 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.

Paper Details

Date Published: 11 February 2011
PDF: 8 pages
Proc. SPIE 7904, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XVIII, 79041G (11 February 2011); doi: 10.1117/12.874931
Show Author Affiliations
Rodrigo Aviles-Espinosa, Instituto de Ciencias Fotónicas (Spain)
Susana I. C. O. Santos, Instituto de Ciencias Fotónicas (Spain)
Andreas Brodschelm, TOPTICA Photonics AG (Germany)
Wilhelm G. Kaenders, TOPTICA Photonics AG (Germany)
Cesar Alonso-Ortega, Instituto de Ciencias Fotónicas (Spain)
David Artigas, Instituto de Ciencias Fotónicas (Spain)
Univ. Politècnica de Catalunya (Spain)
Pablo Loza-Alvarez, Instituto de Ciencias Fotónicas (Spain)

Published in SPIE Proceedings Vol. 7904:
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XVIII
Jose-Angel Conchello; Carol J. Cogswell; Tony Wilson; Thomas G. Brown, Editor(s)

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