Multiphoton imaging with a nanosecond supercontinuum source

Claire Lefort; Rodney P. O'Connor; Véronique Blanquet; Fabienne Baraige; Vincent Tombelaine; Philippe Lévêque; Vincent Couderc; Philippe Leproux

doi:10.1117/12.2208134

14 March 2016 Multiphoton imaging with a nanosecond supercontinuum source

Claire Lefort, Rodney P. O'Connor, Véronique Blanquet, Fabienne Baraige, Vincent Tombelaine, Philippe Lévêque, Vincent Couderc, Philippe Leproux

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Proceedings Volume 9712, Multiphoton Microscopy in the Biomedical Sciences XVI; 97121D (2016) https://doi.org/10.1117/12.2208134
Event: SPIE BiOS, 2016, San Francisco, California, United States

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.

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Claire Lefort, Rodney P. O'Connor, Véronique Blanquet, Fabienne Baraige, Vincent Tombelaine, Philippe Lévêque, Vincent Couderc, and Philippe Leproux "Multiphoton imaging with a nanosecond supercontinuum source", Proc. SPIE 9712, Multiphoton Microscopy in the Biomedical Sciences XVI, 97121D (14 March 2016); https://doi.org/10.1117/12.2208134

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