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Proceedings Paper

Multiphoton imaging with a nanosecond supercontinuum source
Author(s): Claire Lefort; Rodney P. O'Connor; Véronique Blanquet; Fabienne Baraige; Vincent Tombelaine; Philippe Lévêque; Vincent Couderc; Philippe Leproux
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Paper Abstract

Multiphoton microscopy is a well-established technique for biological imaging of several kinds of targets. It is classically based on multiphoton processes allowing two means of contrast simultaneously: two-photon fluorescence (TPF) and second harmonic generation (SHG). Today, the quasi exclusive laser technology used in that aim is femtosecond titanium sapphire (Ti: Sa) laser. We experimentally demonstrate that a nanosecond supercontinuum laser source (STM-250-VIS-IR-custom, Leukos, France; 1 ns, 600-2400 nm, 250 kHz, 1 W) allows to obtain the same kind of image quality in the case of both TPF and SHG, since it is properly filtered. The first set of images concerns the muscle of a mouse. It highlights the simultaneous detection of TPF and SHG. TPF is obtained thanks to the labelling of alpha-actinin with Alexa Fluor® 546 by immunochemistry. SHG is created from the non-centrosymmetric organization of myosin. As expected, discs of actin and myosin are superimposed alternatively. The resulting images are compared with those obtained from a standard femtosecond Ti: Sa source. The physical parameters of the supercontinuum are discussed. Finally, all the interest of using an ultra-broadband source is presented with images obtained in vivo on the brain of a mouse where tumor cells labeled with eGFP are grafted. Texas Red® conjugating Dextran is injected into the blood vessels network. Thus, two fluorophores having absorption wavelengths separated by 80 nm are imaged simultaneously with a single laser source.

Paper Details

Date Published: 14 March 2016
PDF: 5 pages
Proc. SPIE 9712, Multiphoton Microscopy in the Biomedical Sciences XVI, 97121D (14 March 2016); doi: 10.1117/12.2208134
Show Author Affiliations
Claire Lefort, Univ. de Limoges, Labex Sigma-Lim, INRA, CNRS (France)
Rodney P. O'Connor, Univ. de Limoges, Labex Sigma-Lim, INRA, CNRS (France)
Véronique Blanquet, Univ. de Limoges, Labex Sigma-Lim, INRA, CNRS (France)
Fabienne Baraige, Univ. de Limoges, Labex Sigma-Lim, INRA, CNRS (France)
Vincent Tombelaine, LEUKOS Innovative Optical Systems (France)
Philippe Lévêque, Univ. de Limoges, Labex Sigma-Lim, INRA, CNRS (France)
Vincent Couderc, Univ. de Limoges, Labex Sigma-Lim, INRA, CNRS (France)
Philippe Leproux, Univ. de Limoges, Labex Sigma-Lim, INRA, CNRS (France)
LEUKOS Innovative Optical Systems (France)


Published in SPIE Proceedings Vol. 9712:
Multiphoton Microscopy in the Biomedical Sciences XVI
Ammasi Periasamy; Peter T. C. So; Karsten König, Editor(s)

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