High-resolution 3D optical microscopy inside the beating zebrafish heart using prospective optical gating

3D fluorescence imaging is a fundamental tool in the study of functional and developmental biology, but effective imaging is particularly difficult in moving structures such as the beating heart. We have developed a non-invasive real-time optical gating system that is able to exploit the periodic nature of the motion to acquire high resolution 3D images of the normally-beating zebrafish heart without any unnecessary exposure of the sample to harmful excitation light. In order for the image stack to be artefact-free, it is essential to use a synchronization source that is invariant as the sample is scanned in 3D. We therefore describe a scheme whereby fluorescence image slices are scanned through the sample while a brightfield camera sharing the same objective lens is maintained at a fixed focus, with correction of sample drift also included. This enables us to maintain, throughout an extended 3D volume, the same standard of synchronization we have previously demonstrated in and near a single 2D plane. Thus we are able image the complete beating zebrafish heart exactly as if the heart had been artificially stopped, but sidestepping this undesirable interference with the heart and instead allowing the heart to beat as normal

Adaptive Optimisation of Illumination Beam Profiles in Fluorescence Microscopy

Wide-field fluorescence microscope techniques such as single/selective plane illumination microscope (SPIM) are typically configured to image large regions of a sample at once. Here the illumination beam provides uniform excitation of several biological features across the region, `sliced' to a thickness of between 5-10 microns. In this paper we propose a simple alteration to the optical configuration of a SPIM by switching the light-sheet- forming cylindrical lens with a spatial light modulator. This has the potential to adaptively reconfigure the light sheet geometry to improve the optical sectioning of specific biological features, rather than the thicker sectioning of several features at once across a larger observation field-of-view. We present a prototype version of such a system, referred to as an Adaptive-SPIM (A-SPIM) system. We then suggest that the direct recording of illumination beam shapes within the working microscope system can better facilitate the analysis and subsequent re-configuration of the illumination beam to a specific geometry, and summarise the design and operation of a device that we have developed specifically for this purpose. We finally present reconstructed quantitative three dimensional flux maps of illumination beams from three microscope configurations taken using this miniature high-dynamic range beam profiling device, comparing the beam geometry of a regular SPIM system with our prototype A-SPIM system, and suggesting future improvements

Speeding up liquid crystal SLMs using overdrive with phase change reduction

Nematic liquid crystal spatial light modulators (SLMs) with fast switching times and high diffraction efficiency are important to various applications ranging from optical beam steering and adaptive optics to optical tweezers. Here we demonstrate the great benefits that can be derived in terms of speed enhancement without loss of diffraction efficiency from two mutually compatible approaches. The first technique involves the idea of overdrive, that is the calculation of intermediate patterns to speed up the transition to the target phase pattern. The second concerns optimization of the target pattern to reduce the required phase change applied to each pixel, which in addition leads to a substantial reduction of variations in the intensity of the diffracted light during the transition. When these methods are applied together, we observe transition times for the diffracted light fields of about 1 ms, which represents up to a tenfold improvement over current approaches. We experimentally demonstrate the improvements of the approach for applications such as holographic image projection, beam steering and switching, and real-time control loops

Refractive elements for the measurement of the orbital angular momentum of a single photon

We have developed a mode transformer comprising two custom refractive optical elements which convert orbital angular momentum states into transverse momentum states. This transformation allows for an efficient measurement of the orbital angular momentum content of an input light beam. We characterise the channel capacity of the system for 50 input modes, giving a maximum value of 3.46 bits per photon. Using an electron multiplying CCD (EMCCD) camera with a laser source attenuated such that on average there is less than one photon present within the system per measurement period, we demonstrate that the elements are efficient for the use in single photon experiments

Traceable interferometry using binary reconfigurable holograms

We describe the characterization of a ferroelectric-liquid-crystal-on-silicon (FLCOS) spatial light modulator (SLM) in the production of holograms for use in interferometric metrology. It has already been shown that such a device can be used in producing small-amplitude arbitrary reference surfaces with small but appreciable errors due to the contaminating effect of higher-order structures being propagated through the spatial filter. Here we further quantify the size of these residuals for increasingly large aberrations up to nine waves rms Zernike astigmatism, showing a Zernike-corrected rms wavefront error of roughly 0.06 waves with high vibrational stability. We also present measurements of a vacuum window element using the FLCOS device to drastically reduce interferometric fringe density, showing a residual wavefront error of 0.046 waves rms with dominant components originating from test piece structure rather than holographic errors

Emergent properties in optically bound matter

Sub-micron particles have been observed to spontaneously form regular two-dimensional structures in counterpropagating evanescent laser fields. We show that collective properties of large numbers of optically-trapped particles can be qualitatively different to the properties of small numbers. This is demonstrated both with a computer model and with experimental results. As the number of particles in the structure is increased, optical binding forces can be sufficiently large to overcome the optical landscape imposed by the interference fringes of the laser beams and impose a different, competing structure

Light sheet adaptive optics microscope for 3D live imaging

We report on the incorporation of adaptive optics (AO) into the imaging arm of a selective plane illumination microscope (SPIM). SPIM has recently emerged as an important tool for life science research due to its ability to deliver high-speed, optically sectioned, time-lapse microscope images from deep within in vivo selected samples. SPIM provides a very interesting system for the incorporation of AO as the illumination and imaging paths are decoupled and AO may be useful in both paths. In this paper, we will report the use of AO applied to the imaging path of a SPIM, demonstrating significant improvement in image quality of a live GFP-labeled transgenic zebrafish embryo heart using a modal, wavefront sensorless approach and a heart synchronization method. These experimental results are linked to a computational model showing that significant aberrations are produced by the tube holding the sample in addition to the aberration from the biological sample itself

A Shack-Hartmann wavefront sensor projected on to the sky with reduced focal anisoplanatism

A method for producing a laser guide star wavefront sensor for adaptive optics with reduced focal anisoplanatism is presented. A theoretical analysis and numerical simulations have been carried out and the results are presented. The technique, named Sky-Projected Laser Array Shack–Hartmann (SPLASH), is shown to suffer considerably less from focal anisoplanatism than a conventional laser guide star system. The method is potentially suitable for large telescope apertures (8 m), and possibly for extremely large telescopes

Adaptive optimisation of illumination beam profiles in fluorescence microscopy

Wide-field fluorescence microscope techniques such as single/selective plane illumination microscope (SPIM) are typically configured to image large regions of a sample at once. Here the illumination beam provides uniform excitation of several biological features across the region, `sliced' to a thickness of between 5-10 microns. In this paper we propose a simple alteration to the optical configuration of a SPIM by switching the light-sheet- forming cylindrical lens with a spatial light modulator. This has the potential to adaptively reconfigure the light sheet geometry to improve the optical sectioning of specific biological features, rather than the thicker sectioning of several features at once across a larger observation field-of-view. We present a prototype version of such a system, referred to as an Adaptive-SPIM (A-SPIM) system. We then suggest that the direct recording of illumination beam shapes within the working microscope system can better facilitate the analysis and subsequent re-configuration of the illumination beam to a specific geometry, and summarise the design and operation of a device that we have developed specifically for this purpose. We finally present reconstructed quantitative three dimensional flux maps of illumination beams from three microscope configurations taken using this miniature high-dynamic range beam profiling device, comparing the beam geometry of a regular SPIM system with our prototype A-SPIM system, and suggesting future improvements