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

Rodrigo Aviles-Espinosa; Susana I. C. O. Santos; Andreas Brodschelm; Wilhelm G. Kaenders; Cesar Alonso-Ortega; David Artigas; Pablo Loza-Alvarez

doi:10.1117/12.874931

11 February 2011 In-vivo third-harmonic generation microscopy at 1550nm three-dimensional long-term time-lapse studies in living C. elegans embryos

Rodrigo Aviles-Espinosa, Susana I. C. O. Santos, Andreas Brodschelm, Wilhelm G. Kaenders, Cesar Alonso-Ortega, David Artigas, Pablo Loza-Alvarez

Author Affiliations +

Proceedings Volume 7904, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XVIII; 79041G (2011) https://doi.org/10.1117/12.874931
Event: SPIE BiOS, 2011, San Francisco, California, United States

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.

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Rodrigo Aviles-Espinosa, Susana I. C. O. Santos, Andreas Brodschelm, Wilhelm G. Kaenders, Cesar Alonso-Ortega, David Artigas, and Pablo Loza-Alvarez "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 (11 February 2011); https://doi.org/10.1117/12.874931

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KEYWORDS

Tissues

Microscopy

Image processing

Fiber lasers

In vivo imaging

Microscopes

Time lapse microscopy

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