Holographic Second Harmonic Generation Microscopy

This dissertation is dedicated to the application of digital holography methods to second harmonic generation microscopy, and to the development of the resulting imaging technique: holographic second harmonic generation microscopy. Second harmonic generation microscopy was originally developed alongside of incoherent nonlinear microscopy (multiphoton excitation fluorescence), and largely contributed to the emergence of coherent nonlinear microscopy (e.g. higher harmonic generation or coherent anti-Stokes Raman scattering). One particularity of coherent nonlinear microscopy is that the detected signals originate from instantaneous interaction of incident electromagnetic radiation with the specimen, thus making ultrafast imaging possible. This is because these nonlinear interactions are of a scattering nature and, as such, do not involve absorption of light by the specimen. As a consequence, the light source for nonlinear microscopy does not need to be limited to a narrow absorption band, as it is the case in fluorescence, and can be, oppositely, selected to match a spectral window of transmission for the specimen, in order to avoiding photo-damage. Another advantage of second harmonic generation – and, more generally, of coherent nonlinear microscopy – lies in its coherent nature, which makes possible the retrieval of both its amplitude and its phase with a proper phase-sensitive imaging technique such as holographic interferometry. Here, we present our work on holographic second harmonic generation microscopy. At first, we explain how a holographic second harmonic generation setup can be implemented and second harmonic holograms be digitally recorded. Discussing of possible setup implementations, we insist on some key components, most especially the light source and the detector, to explain both why holographic second harmonic generation imaging has only recently been made possible and why it appears more and more appealing. Addressing the core of the technique, we describe the numerical reconstruction process of holograms that yield both amplitude and phase of the object wavefront, before commenting further on the types of image contrast accessible with holographic second harmonic generation imaging. We also propose a clever way for multiplexing on the digital sensor the second harmonic hologram with a bright field image facilitating its interpretation. Finally, we review a few fields of application of holographic second harmonic generation imaging. Notably, we discuss of how it is possible to deduce the polarization component of the fundamental excitation that is responsible for second harmonic generation. We also detail applications of holographic second harmonic generation microscopy to imaging of label-free biological tissues, and to real-time, nanometric 3D-tracking of nanoparticles.