Search

Light sheet fluorescence microscopy techniques have revolutionized biological microscopy enabling low-phototoxic long-term 3D imaging of living samples. Although there exist many light sheet microscopy (LSM) implementations relying on fluorescence, just a few works have paid attention to the laser elastic scattering source of contrast available in every light sheet microscope. Interestingly, elastic scattering can potentially disclose valuable information from the structure and composition of the sample at different spatial scales. However, when coherent scattered light is detected with a camera sensor, a speckled intensity is generated on top of the native imaged features, compromising their visibility. In this work, we propose a novel light sheet based optical setup which implements three strategies for dealing with speckles of elastic scattering images: (i) polarization filtering; (ii) reducing the temporal coherence of the excitation laser light; and, (iii) reducing the spatial coherence of the light sheet. Finally, we show how these strategies enable pristine light-sheet elastic-scattering imaging of structural features in challenging biological samples avoiding the deleterious effects of speckle, and without relying on, but complementing, fluorescent labelling.

We describe a computational method for accurate, quantitative tomographic reconstructions in Optical Projection Tomography, based on phase retrieval algorithms. Our method overcomes limitations imposed by light scattering in opaque tissue samples under the memory effect regime, as well as reduces artifacts due to mechanical movements, misalignments or vibrations. We make use of Gerchberg-Saxton algorithms, calculating first the autocorrelation of the object and then retrieving the associated phase under four numerically simulated measurement conditions. By approaching the task in such a way, we avoid the projection alignment procedure, exploiting the fact that the autocorrelation sinogram is always aligned and centered. We thus propose two new, projection-based, tomographic imaging flowcharts that allow registration-free imaging of opaque biological specimens and unlock three-dimensional tomographic imaging of hidden objects. Two main reconstruction approaches are discussed in the text, focusing on their efficiency in the tomographic retrieval and discussing their applicability under four different numerical experiments.

The combined use of a wavefront modulator and a scattering medium forms an "opaque lens" which forces the light to focus tightly. The adaptive focus has the same shape as the correlation function of the original speckle pattern and it can be generated at defined positions with resolution up to hundreds of nanometers. We have demonstrated that manipulating the speckle pattern spatial components can structure the shape of the focus. Exploiting selectively spatial-frequencies from the speckle components we realized opaque lenses able to produce sub-correlation foci and Bessel beams.

Recently great progress has been made in biological and biomedical imaging by combining non-invasive optical methods, novel adaptive light manipulation and computational techniques for intensity-based phase recovery and three dimensionalimage reconstruction. In particular and in relation to the work presented here, Optical Projection Tomography (OPT) is awell-established technique for imaging mostly transparent absorbing biological models such as C. Elegans and Danio Rerio. On the contrary, scattering layers like the cocoon surrounding the Drosophila during the pupae stage constitutes achallenge for three dimensional imaging through such a complex structure. However, recent studies enabled image reconstruction through scattering curtains up to few transport mean free paths via phase retrieval iterative algorithms allowing to uncover objects hidden behind complex layers. By combining these two techniques we explore the possibility to perform a three dimensional image reconstruction of fluorescent objects embedded between scattering layers without compromising its structural integrity. Dynamical cross correlation registration was implemented for the registration process due to translational and flipping ambiguity of the phase retrieval problem, in order to provide the correct aligned set of data to perform the back-projection reconstruction. We have thus managed to reconstruct a hidden complex object between static scattering curtains and compared with the effective reconstruction to fully understand the process before the in-vivo biological implementation. Keywords: Phase Retrieval , Scattering, Optical Projection Tomography, Three Dimensional Imaging, BiomedicalImaging, Image Reconstruction , Turbid Lens , Memory Effect.

Bessel beams are non-diffracting light structures, which maintain their spatial features after meters of propagation and are realized with simple optical elements such as axicon lenses, spatial filters, and lasers. In this paper, we demonstrate a method for generating non diffractive Bessel-like beams through a heavily scattering system, exploiting wavefronts shaped by a spatial light modulator. With the proposed method starting from amorphous speckle patterns, it is possible to produce at user defined positions configurable and non-diffracting light distributions which can improve depth-of-field in speckled illumination microscopy.

Adaptive optics can focus light through opaque media by compensating the random phase delay acquired while crossing a scattering curtain. The technique is commonly exploited in many fields, including astrophysics, microscopy, biomedicine and biology. A turbid lens has the capability of producing foci with a resolution higher than conventional optics, however it has a fundamental limit: to obtain a sharp focus one has to introduce a strongly scattering medium in the optical path. Indeed a tight focusing needs strong scattering and, as a consequence, high resolution focusing is obtained only for weakly transmitting samples. Here we describe a novel method allowing to obtain highly concentrated optical spots even by introducing a minimum amount of scattering in the beam path with semi-transparent materials. By filtering the pseudo-ballistic components of the transmitted beam we are able to experimentally overcome the limits of the adaptive focus resolution, gathering light on a spot with a diameter which is one third of the original speckle correlation function.

By compensating the random phase delay acquired while a light beam crosses a scattering curtain, it is possible to address the light at selected target position beyond the obstacle. An opaque lens can produce foci with a resolution higher than conventional optics if a strongly scattering medium is exploited. In practice, subwavelength resolution is obtained only for weakly transmitting samples. Herein we present a method which allows obtaining tiny bright optical spots even in presence of a minimum amount of scattering (semi-transparent media) in the beam path. Using a High-Pass spatial filter we block the pseudo-ballistic components of the transmitted beam, we are able to gather light on a spot with a diameter which is one third of the typical speckle grain in absence of the filter.

Strong scattering in the optical paths can be proactively exploited for determining light propagation and focusing through turbid media and ultimately improve optical imaging and light manipulation capabilities. The use of light shapers together with strongly scattering structures enables the production of foci confined in the nanoscale or on the other hand may provide significantly enhanced fields of view. Exploiting this concept, we introduce ad hoc engineered scattering structures, by direct femtosecond laser writing in the bulk of glasses, presenting isotropy in one dimension, and together with precisely phase-shaped illuminating light we produce submicrometer thin light sheets at user-defined positions. Our approach permits us to focus light of different wavelengths onto the same defined position without moving any optical element and correcting for chromatic aberrations. Furthermore, we demonstrate that natural biological tissues presenting isotropy in one dimension can also be used for generating light sheets directly in the biological sample.