During this seminar, I will discuss recent advances in nonlinear microscopy aiming at increasing the number of available contrasts and the imaging speed. In particularly, I will present results obtained during my thesis in the Laboratory for Optics and Biosciences at Ecole Polytechnique.

First, I will introduce the use of synchronized pulse trains for multimodal nonlinear microscopy in a standard point scanning geometry and show that this configuration allows the simultaneous and effective two photon excitation of blue, green-yellow and red fluorescent proteins [1]. In collaboration with the group of J. Livet (Vision Institute, Paris), we applied this technique to image large volume of Brainbow-labelled tissues [2]. Potential applications include the mapping of neural networks and the analysis of cells interactions during the development of the central nervous system.

Eventually, I will introduce strategies to increase the imaging frame rate of nonlinear microscopy and show that the combination of two-photon light-sheet illumination [3] with synchronized pulse trains provides fast multicolour two-photon imaging.

[1] Mahou et al, Nature Methods 9, 815–818 (2012).

[2] Livet et al, Nature 450, 56–62 (2007).

[3] Truong et al, Nature Methods 8, 757–760 (2011).