Holographic second harmonic generation (SHG) microscopy

The coherent nature of second harmonic generation (SHG) makes possible retrieval of both its amplitude and its phase, by use of a proper phase-sensitive imaging technique such as digital holographic SHG microscopy. In addition to retrieving both amplitude and phase of second harmonic signals the computer-based hologram reconstruction process also makes possible to bring in focus different sections of a specimen separated by distances exceeding the depth of field of the imaging system. Similarly, its numerical focusing feature is especially useful to compensate drifts of focus over long term experiments or in unstable environment. Holographic SHG microscopy, based on off-axis digital holography, is a nonscanning, single-shot image acquisition technique. As such, it is in principle limited in speed only by the camera frame rate and the available second harmonic signal, which depends in a non-saturating manner on the peak power density of the laser source. In other words, holographic SHG imaging is especially suited for real-time imaging, and has the potential to truly exploit the instantaneous response time of second harmonic generation. Finally, holographic SHG imaging has already found applications for determining the polarization component responsible for second harmonic generation [1], for revealing phase matching conditions in biological specimens [2], and for nanometric 3D tracking of SHG scatterers [3]. Here, we propose to make an overview of holographic SHG microscopy.

Holographic Second Harmonic Generation Microscopy

Etienne Shaffer

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.

EPFL2011

Digital holographic microscopy investigation of second harmonic generated at a glass/air interface

Christian Depeursinge, Jonas Kühn, Nicolas Pavillon, Etienne Shaffer

Optical second harmonic generation, thanks to its coherent nature, is a suitable signal for interferometric measurements, such as digital holography: a well-established imaging technique that allows recovering complex diffraction wavefields from which it is possible to extract both amplitude- contrast and quantitative phase images. Here, we report on a new, multi-functional form of microscopy: second harmonic generation digital holographic microscopy. As a proof of concept, we have investigated the second harmonic signal generated at the glass/air interface of a microscope slide under focused femtosecond laser illumination and propose for the first time a representation and interpretation of the recovered phase. In this simple, yet very educative case study, we observe that the second harmonic is generated by the axial component of the incident field polarization

2009

Digital holography for second harmonic microscopy

Christian Depeursinge, Etienne Shaffer

Quantitative phase images make digital holographic microscopy (DHM) an excellent instrument for metrological, but also for biological applications, where it can reveal deformations and morphological details at ultrahigh resolution in the order of a few nanometers only, while also precisely determining the refractive index across a sample (e.g. cell or neuron). On the other hand, non-linear light-matter interactions have also proved very useful in microscopy. For instance, second harmonic generation (SHG) allows marker-free identification of cell structures, tubulin or membranes and, because of its coherent nature, SHG is very sensitive to the local sample structure and to the direction of the laser polarization. In addition, since SHG does not result from light absorption and subsequent re-emission, in opposition to fluorescence, photo-bleaching of the studied material can be avoided by a judicious selection of the laser wavelength. These characteristics make SHG very interesting for biomedical imaging. We have designed and built a microscope that combines the fast and precise DHM imaging with tagging capabilities of non-linear light-matter interactions. Here, we present the technique and look into its possible applications to biological and life sciences. Among promising applications is the 3D tracking of colloidal gold nanoparticles.

SPIE2010

Holographic Second Harmonic Generation Microscopy

Etienne Shaffer

EPFL2011