Ben Leshem

Two-photon excitation in scattering media by spatiotemporally shaped beams and their application in optogenetic stimulation

The use of wavefront shaping to generate extended optical excitation patterns which are confined to a predetermined volume has become commonplace on various microscopy applications. For multiphoton excitation, three-dimensional confinement can be achieved by combining the technique of temporal focusing of ultra-short pulses with different approaches for lateral light shaping, including computer generated holography or generalized phase contrast. Here we present a theoretical and experimental study on the effect of scattering on the propagation of holographic beams with and without temporal focusing. Results from fixed and acute cortical slices show that temporally focused spatial patterns are extremely robust against the effects of scattering and this permits their three-dimensionally confined excitation for depths more than 500 µm. Finally we prove the efficiency of using temporally focused holographic beams in two-photon stimulation of neurons expressing the red-shifted optogenetic channel C1V1.

Structural Basis for the Brilliant Colors of the Sapphirinid Copepods

Males of sapphirinid copepods use regularly alternating layers of hexagonal-shaped guanine crystals and cytoplasm to produce spectacular structural colors. In order to understand the mechanism by which the different colors are produced, we measured the reflectance of live individuals and then characterized the organization of the crystals and the cytoplasm layers in the same individuals using cryo-SEM. On the basis of these measurements, we calculated the expected reflectance spectra and found that they are strikingly similar to the measured ones. We show that variations in the cytoplasm layer thickness are mainly responsible for the different reflected colors and also that the copepod color strongly depends on the angular orientation relative to the incident light, which can account for its appearance and disappearance during spiral swimming in the natural habitat.

The Structural Basis for Enhanced Silver Reflectance in Koi Fish Scale and Skin

Fish have evolved biogenic multilayer reflectors composed of stacks of intracellular anhydrous guanine crystals separated by cytoplasm, to produce the silvery luster of their skin and scales. Here we compare two different variants of the Japanese Koi fish; one of them with enhanced reflectivity. Our aim is to determine how biology modulates reflectivity, and from this to obtain a mechanistic understanding of the structure and properties governing the intensity of silver reflectance. We measured the reflectance of individual scales with a custom-made microscope, and then for each individual scale we characterized the structure of the guanine crystal/cytoplasm layers using high-resolution cryo-SEM. The measured reflectance and the structural-geometrical parameters were used to calculate the reflectance of each scale, and the results were compared to the experimental measurements. We show that enhanced reflectivity is obtained with the same basic guanine crystal/cytoplasm stacks, but the structural arrangement between the stack, inside the stacks, and relative to the scale surface is varied when reflectivity is enhanced. Finally, we propose a model that incorporates the basic building block parameters, the crystal orientation inside the tissue, and the resulting reflectance and explains the mechanistic basis for reflectance enhancement.

When can temporally focused excitation be axially shifted by dispersion

Temporal focusing (TF) allows for axially confined wide-field multi-photon excitation at the temporal focal plane. For temporally focused Gaussian beams, it was shown both theoretically and experimentally that the temporal focus plane can be shifted by applying a quadratic spectral phase to the incident beam. However, the case for more complex wave-fronts is quite different. Here we study the temporal focus plane shift (TFS) for a broader class of excitation profiles, with particular emphasis on the case of temporally focused computer generated holography (CGH) which allows for generation of arbitrary, yet speckled, 2D patterns. We present an analytical, numerical and experimental study of this phenomenon. The TFS is found to depend mainly on the autocorrelation of the CGH pattern in the direction of the beam dispersion after the grating in the TF setup. This provides a pathway for 3D control of multi-photon excitation patterns.

Direct phase retrieval in double blind Fourier holography

Phase measurement is a long-standing challenge in a wide range of applications, from X-ray imaging to astrophysics and spectroscopy. While in some scenarios the phase is resolved by an interferometric measurement, in others it is reconstructed via numerical optimization, based on some a-priori knowledge about the signal. The latter commonly use iterative algorithms, and thus have to deal with their convergence, stagnation, and robustness to noise. Here we combine these two approaches and present a new scheme, termed double blind Fourier holography, providing an efficient solution to the phase problem in two dimensions, by solving a system of linear equations. We present and experimentally demonstrate our approach for the case of lens-less imaging.

Direct single-shot phase retrieval for separated objects(Conference Presentation)

The phase retrieval problem arises in various fields ranging from physics and astronomy to biology and microscopy. Computational reconstruction of the Fourier phase from a single diffraction pattern is typically achieved using iterative alternating projections algorithms imposing a non-convex computational challenge. A different approach is holography, relying on a known reference field. Here we present a conceptually new approach for the reconstruction of two (or more) sufficiently separated objects. In our approach we combine the constraint the objects are finite as well as the information in the interference between them to construct an overdetermined set of linear equations. We show that this set of equations is guaranteed to yield the correct solution almost always and that it can be solved efficiently by standard numerical algebra tools. Essentially, our method combine commonly used constraint (that the object is finite) with a holographic approach (interference information). It differs from holographic methods in the fact that a known reference field is not required, instead the unknown objects serve as reference to one another (hence blind holography). Our method can be applied in a single-shot for two (or more) separated objects or with several measurements with a single object. It can benefit phase imaging techniques such as Fourier phytography microscopy, as well as coherent diffractive X-ray imaging in which the generation of a well-characterized, high resolution reference beam imposes a major challenge. We demonstrate our method experimentally both in the optical domain and in the X-ray domain using XFEL pulses.