Zhengyi Yang

A compact Airy beam light sheet microscope with a tilted cylindrical lens

Light-sheet imaging is rapidly gaining importance for imaging intact biological specimens. Many of the latest innovations rely on the propagation-invariant Bessel or Airy beams to form an extended light sheet to provide high resolution across a large field of view. Shaping light to realize propagation-invariant beams often relies on complex programming of spatial light modulators or specialized, custom made, optical elements. Here we present a straightforward and low-cost modification to the traditional light-sheet setup, based on the open-access light-sheet microscope OpenSPIM, to achieve Airy light-sheet illumination. This brings wide field single-photon light-sheet imaging to a broader range of endusers. Fluorescent microspheres embedded in agarose and a zebrafish larva were imaged to demonstrate how such a microscope can have a minimal footprint and cost without compromising on imaging quality.

Light Sheet Tomography (LST) for in situ imaging of plant roots

The production of crops capable of efficient nutrient use is essential for addressing the problem of global food security. The ability of a plants root system to interact with the soil micro-environment determines how effectively it can extract water and nutrients. In order to assess this ability and develop the fast and cost effective phenotyping techniques which are needed to establish efficient root systems, in situ imaging in soil is required. To date this has not been possible due to the high density of scatterers and absorbers in soil or because other growth substrates do not sufficiently model the heterogeneity of a soils microenvironment. We present here a new form of light sheet imaging with novel transparent soil containing refractive index matched particles. This imaging method does not rely on fluorescence, but relies solely on scattering from root material. We term this form of imaging Light Sheet Tomography (LST). We have tested LST on a range of materials and plant roots in transparent soil and gel. Due to the low density of root structures, i.e. relatively large spaces between adjacent roots, long-term monitoring of lettuce root development in situ with subsequent quantitative analysis was achieved.

A compact light-sheet microscope for the study of the mammalian central nervous system.

Investigation of the transient processes integral to neuronal function demands rapid and high-resolution imaging techniques over a large field of view, which cannot be achieved with conventional scanning microscopes. Here we describe a compact light sheet fluorescence microscope, featuring a 45° inverted geometry and an integrated photolysis laser, that is optimized for applications in neuroscience, in particular fast imaging of sub-neuronal structures in mammalian brain slices. We demonstrate the utility of this design for three-dimensional morphological reconstruction, activation of a single synapse with localized photolysis, and fast imaging of neuronal Ca2+ signalling across a large field of view. The developed system opens up a host of novel applications for the neuroscience community.

Macro-optical trapping for sample confinement in light sheet microscopy

Light sheet microscopy is a powerful approach to construct three-dimensional images of large specimens with minimal photo-damage and photo-bleaching. To date, the specimens are usually mounted in agents such as agarose, potentially restricting the development of live samples, and also highly mobile specimens need to be anaesthetized before imaging. To overcome these problems, here we demonstrate an integrated light sheet microscope which solely uses optical forces to trap and hold the sample using a counter-propagating laser beam geometry. Specifically, tobacco plant cells and living Spirobranchus lamarcki larvae were successfully trapped and sectional images acquired. This novel approach has the potential to significantly expand the range of applications for light sheet imaging.

A sonic screwdriver: Acoustic angular momentum transfer for ultrasonic manipulation

Dexterous manipulation of objects in travelling wave beams can be achieved by modulating the phase profile of a coherent source to generate reconfigurable and complex interference patterns that can impart momentum on objects in the field. In this paper we use a commercially available matrix array operating at 550 kHz to generate helical ultrasonic beams to demonstrate the complexity of beam shapes that can be achieved from a system with approximately 1000 elements. Helical ultrasonic beams carry orbital angular momentum, due to the inclination of the wavefront, that increases with vorticity, or number of intertwined helices. The beams are used to both levitate and rotate a 100 mm diameter disk of acoustic absorber material in a water-filled chamber above the matrix array. The observed levitation of the absorber decreases with increasing vorticity because of the inclination of the wavefront away from the beam axis. The angular momentum transferred to the absorber increases with both the vorticity and the power of the ultrasonic beam. There was no evidence of aliasing of phase pattern applied to the array even with vorticity greater than 10, demonstrating the capability of a matrix array system for dexterous manipulation of objects with ultrasonic beams.

Light-sheet microscopy with attenuation-compensated propagation-invariant beams

Tailoring beams to overcome attenuation allows light-sheet microscopy to image at greater depth with enhanced contrast. Scattering and absorption limit the penetration of optical fields into tissue. We demonstrate a new approach for increased depth penetration in light-sheet microscopy: attenuation-compensation of the light field. This tailors an exponential intensity increase along the illuminating propagation-invariant field, enabling the redistribution of intensity strategically within a sample to maximize signal and minimize irradiation. A key attribute of this method is that only minimal knowledge of the specimen transmission properties is required. We numerically quantify the imaging capabilities of attenuation-compensated Airy and Bessel light sheets, showing that increased depth penetration is gained without compromising any other beam attributes. This powerful yet straightforward concept, combined with the self-healing properties of the propagation-invariant field, improves the contrast-to-noise ratio of light-sheet microscopy up to eightfold across the entire field of view in thick biological specimens. This improvement can significantly increase the imaging capabilities of light-sheet microscopy techniques using Airy, Bessel, and other propagation-invariant beam types, paving the way for widespread uptake by the biomedical community.

Fast volume-scanning light sheet microscopy reveals transient neuronal events

Light sheet fluorescence microscopy offers considerable potential to the cellular neuroscience community as it makes it possible to image extensive areas of neuronal structures, such as axons or dendrites, with a low light budget, thereby minimizing phototoxicity. However, the shallow depth of a light sheet, which is critical for achieving high contrast, well resolved images, adds a significant challenge if fast functional imaging is also required, as multiple images need to be collected across several image planes. Consequently, fast functional imaging of neurons is typically restricted to a small tissue volume where part of the neuronal structure lies within the plane of a single image. Here we describe a method by which fast functional imaging can be achieved across a much larger tissue volume; a custom-built light sheet microscope is presented that includes a synchronized galvo mirror and electrically tunable lens, enabling high speed acquisition of images across a configurable depth. We assess the utility of this technique by acquiring fast functional Ca2+ imaging data across a neuron’s dendritic arbour in mammalian brain tissue.

Three-photon light-sheet fluorescence microscopy

We present the first demonstration of three-photon excitation light-sheet fluorescence microscopy. Light-sheet fluorescence microscopy in single-and two-photon excitation modes has emerged as a powerful wide-field, low photo-damage technique for fast volumetric imaging of biological samples. We extend this imaging modality to the three-photon regime enhancing its penetration depth. Our present study uses a standard femtosecond pulsed laser at 1000 nm wavelength for the imaging of 450 μm diameter cellular spheroids. In addition, we show, through numerical simulations, the potential advantages in three-photon light-sheet microscopy of using propagation-invariant Bessel beams in preference to Gaussian beams. OCIS codes (110.0180) Microscopy; (190.4180) Multiphoton processes; (180.2520) Fluorescence microscopy; (180.4315) Nonlinear microscopy; (180.6900) Three-dimensional microscopy; (110.4100) Imaging through turbid media.

The dyslexia susceptibility KIAA0319 gene shows a highly specific expression pattern during zebrafish development supporting a role beyond neuronal migration.

Dyslexia is a common neurodevelopmental disorder that affects reading abilities and is predicted to be caused by a significant genetic component. Very few genetic susceptibility factors have been identified so far and amongst those, KIAA0319 is a key candidate. KIAA0319 is highly expressed during brain development but its function remains poorly understood. Initial RNA-interference studies in rats suggested a role in neuronal migration whereas subsequent work with double knock-out mouse models for both Kiaa0319 and its paralogue Kiaa0319-like reported effects in the auditory system but not in neuronal migration. To further understand the role of KIAA0319 during neurodevelopment, we carried out an expression study of the zebrafish orthologue at different embryonic stages. We report particularly high gene expression during the first few hours of development. At later stages, expression becomes localised in well-defined structures such as the eyes, the telencephalon and the notochord, supporting a role for kiaa0319 that is not restricted to brain development. Surprisingly, kiaa0319-like, which generally shows a similar expression pattern, was not expressed in the notochord suggesting a role specific to kiaa0319 in this structure. This study contributes to our understanding of KIAA0319 function during embryonic development which might involve additional roles in the visual system and in the notochord. Such a specific spatiotemporal expression pattern is likely to be under the controlled of tightly regulated sequences. Therefore, these data provide a framework to interpret the effects of the dyslexia-associated genetic variants that reside in KIAA0319 non-coding regulatory regions.

A few twists regarding the momentum of shaped beams

In the acoustic tweezers literature, it is sometimes stated that there would be no radiation pressure in the absence of nonlinearity. In fact, such a circumstance would allow violation of the Second Law of Thermodynamics, an issue we discuss in this talk. In the limit where the Second Law applies, all radiant forms of energy must carry an associated momentum. Shaped beams offer (along with shaped targets) opportunity for discussion of the respective advantages of conservative and non-conservative forces. Commonly in acoustic trapping, conservative, gradient-induced mechanisms (e.g., standing waves) are used to manipulate matter. Such situations are reasonably described in terms of potential energy landscapes, an approach also applied to optics, for applications such as cell sorting [MacDonald et al, Nature 426 (2003)]. No such description is possible for radiation pressure, which is non-conservative, a distinction that is sometimes muddled in the literature, although it was made clear even in early landmark papers.