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Nonlinear microscopy with broadband ultrashort pulses


Researchers :

Manuel Joffre
Emmanuel Beaurepaire

An important feature of any imaging method is to provide an information as complete and specific as possible. This can be achieved by combining complimentary images obtained using different techniques such as multiphoton fluorescence and harmonic generation, or by using different filters on the collected fluorescence. However, there is another degree of freedom associated with the control of the excitation frequency. Apart from the obvious method of frequency tuning a narrow-band exciting laser, which has some drawbacks due to the change in laser operating conditions, it is also possible to use a laser with a very broad spectral width (100 nm or more). The spectral resolution can then be recovered by using coherent control methods, as was previously demonstrated in the case of stimulated Raman microscopy (N. Dudovich et al., Nature 418, 512 (2002)).
In this context, the first challenge is to measure a spectrum (e.g. multiphoton excitation or Raman) with the spatial resolution of the nonlinear microscope. We have thus demonstrated that nonlinear Fourier-transform spectroscopy (a simplified 1D version of multidimensional spectroscopy) is a both simple and accurate method for measuring multiphoton [1] and Raman [2,4] spectra in a nonlinear microscope. For example, the figure below shows a 2D image (one frequency dimension and one spatial dimension) of a polystyrene bead immersed in a solvent. The image has been obtained through a Fourier transform of the data recorded as a function of both the time delay between two 20-fs pulses and the laser position scanned across the bead. The Raman spectra of both the solvent and polystyrene are clearly visible [2].

When the spectral information on the involved species is already known, it is extremely useful to be able to selectively excite a given species so as to record specific images using the nonlinear microscope. Such a result can be achieved by using coherent control of two-photon excitation. This approach consists of controlling interferences between the different excitation pathways occurring in the case of a broadband pulse. By shaping the spectral phase of the exciting pulse, it is indeed possible to induce constructive or destructive interferences depending on the actual excitation frequency. It is thus possible to shape the two-photon excitation spectrum by changing the pulse phase only, i.e. at constant pulse energy. This concept has been initially demonstrated in Caesium atoms (D. Meshulach, Y. Silberberg, Nature 396, 239 (1998)), and was later extended to molecules in solution (T. Brixner et al., Nature 414, 57 (2001); I. Pastirk et al., Opt. Express 11, 1695 (2003)). We have demonstrated that this approach could be also applied to imaging of biological objects, by using a scanning nonlinear microscope and an acousto-optic pulse shaper (Dazzler, Fastlite)[3], and more recently using a lab-designed SLM-based shaper switchable at kHz rates[5]. The figure below shows images obtained in vivo in Drosophila embryos with a spectral phase optimized to excite either a fluorescent protein present in the embryo periphery ("red pulse") or the endogenous fluorescence originating mostly from the embryo inner yolk ("blue pulse") [3,5]. These images clearly evidence the effect of selective excitation, despite an inevitable crosstalk which can be compensated for by using an appropriate linear combination between the two images, as shown in the image on the right which demonstrates the selective excitation of the two fluorescent species present in this Drosophila embryo.


Related publications :

"Dispersion-based pulse shaping for multiplexed two-photon fluorescence microscopy"
G. Labroille, R. S. Pillai, X. Solinas, C. Boudoux, N. Olivier, E. Beaurepaire & M. Joffre
Opt Lett 35(20), 3444 (2010).

Multiplexed two-photon microscopy of dynamic biological samples with shaped broadband pulses
R. S. Pillai, C. Boudoux, G. Labroille, N. Olivier, I. Veilleux, E. Farge, M. Joffre, E. Beaurepaire
Opt. Express 17, 12741-12752 (2009) PDF

Interferometric Fourier transform coherent anti-Stokes Raman scattering
M. Cui, M. Joffre, J. Skodack, J. P. Ogilvie
Opt. Express 14, 8448 (2006)

Use of coherent control for selective two-photon fluorescence microscopy in live organisms
J.P. Ogilvie, D. Débarre, X. Solinas, J.-L. Martin, E. Beaurepaire, M. Joffre
Opt. Express 14, 759 (2006) PDF

Fourier transform coherent anti-Stokes Raman scattering microscopy
J.P. Ogilvie, E. Beaurepaire, A. Alexandrou, M. Joffre
Opt. Lett. 31, 480 (2006) PDF

A. Stolow. NRC, Canada
N. Forget, D. Kaplan, T. Oksenhendler, P. Tournois, et al. Fastlite
J. P. Ogilvie. University of Michigan, Department of Physics

Guillaume Labroille (PhD std 2009-2011)
Rajesh S.Pillai (postdoc 2007-2009)
Nicolas Olivier (PhD std 2007-2009)
Caroline Boudoux (postdoc 2007-2008)
Antigoni Alexandrou (CNRS)
Delphine Débarre (PhD 2006)
Jennifer P. Ogilvie (postdoc)
Kevin J. Kubarych (postdoc)
Thomas Chastang (undergrad 2007)