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A food dye suppresses light scattering in biological tissues to enable deep in vivo imaging.
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Colorantes , Dispersión de Radiación , Animales , Luz , Imagen Óptica/métodos , RatonesRESUMEN
Motivation: Quantifying lateral resolution as a function of depth is important in the design of 3D microscopy experiments. However, for many specimens, resolution is non-uniform within the same optical plane because of factors such as tissue variability and differential light scattering. This precludes application of a simple resolution metric to the image as a whole. In such cases, it can be desirable to analyse resolution only within specific, well-defined features. Results: An algorithm and software are presented to characterize resolution as a function of depth in features of arbitrary shape in 3D samples. The tool can be used to achieve an objective comparison between different preparation methods, imaging parameters, and optical systems. It can also inform the design of experiments requiring resolution of structures at a specific scale. The method is demonstrated by quantifying the improvement in resolution of two-photon microscopy over confocal in the central brain of Drosophila melanogaster. Measurement of image quality increases by tuning a single parameter, laser power, is also shown. An ImageJ plugin implementation is provided for ease of use via a simple Graphical User Interface, with outputs in table, graph, and colourmap formats. Availability and implementation: Software and source code are available at https://www.imperial.ac.uk/rowlands-lab/resources/.
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A targeted imaging system has been developed for applications requiring recording from stationary samples at high spatiotemporal resolutions. It works by illuminating regions of interest in rapid sequence, and recording the signal from the whole field of view onto a single photodetector. It can be implemented at low cost on an existing microscope without compromising existing functionality. The system is characterized in terms of speed, spatial resolution, and tissue penetration depth, before being used to record individual action potentials from ASAP-3 expressing neurons in an ex vivo mouse brain slice preparation.
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OBJECTIVE: Super-resolution ultrasound (SRUS) imaging through localising and tracking sparse microbubbles has been shown to reveal microvascular structure and flow beyond the wave diffraction limit. Most SRUS studies use standard delay and sum (DAS) beamforming, where high side lobes and broad main lobes make isolation and localisation of densely distributed bubbles challenging, particularly in 3D due to the typically small aperture of matrix array probes. METHOD: This study aimed to improve 3D SRUS by implementing a new fast 3D coherence beamformer based on channel signal variance. Two additional fast coherence beamformers, that have been implemented in 2D were implemented in 3D for the first time as comparison: a nonlinear beamformer with p-th root compression and a coherence factor beamformer. The 3D coherence beamformers, together with DAS, were compared in computer simulation, on a microflow phantom and in vivo. RESULTS: Simulation results demonstrated that all three adaptive weight-based beamformers can narrow the main lobe, suppress the side lobes, while maintaining the weaker scatter signals. Improved 3D SRUS images of microflow phantom and a rabbit kidney within a 3-second acquisition were obtained using the adaptive weight-based beamformers, when compared with DAS. CONCLUSION: The adaptive weight-based 3D beamformers can improve the SRUS and the proposed variance-based beamformer performs best in simulations and experiments. SIGNIFICANCE: Fast 3D SRUS would significantly enhance the potential utility of this emerging imaging modality in a broad range of biomedical applications.
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Procesamiento de Imagen Asistido por Computador , Procesamiento de Señales Asistido por Computador , Conejos , Animales , Procesamiento de Imagen Asistido por Computador/métodos , Simulación por Computador , Algoritmos , Imagenología Tridimensional , Ultrasonografía/métodos , Fantasmas de ImagenRESUMEN
A new method of generating potentially arbitrary photoacoustic wavefronts with optical holograms is presented. This method uses nanosecond laser pulses at 1064 nm that are split into four time-delayed components by means of a configurable multipass optical delay apparatus, which serves to map the pulses onto phase-delayed regions of a given acoustic wavefront. A single spatial light modulator generates separate holograms for each component, which are imaged onto a photoacoustic transducer comprised of a thermoelastic polymer. As a proof of concept of the broader arbitrary wavefront construction technique, the spatially- and temporally-modulated holograms in this study produce a phased array effect that enables beam steering of the resulting acoustic pulse. For a first experimental demonstration of the method, as verified by simulation, the acoustic beam is steered in four directions by around 5 degrees.
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Structured Illumination Microscopy, SIM, is one of the most powerful optical imaging methods available to visualize biological environments at subcellular resolution. Its limitations stem from a difficulty of imaging in multiple color channels at once, which reduces imaging speed. Furthermore, there is substantial experimental complexity in setting up SIM systems, preventing a widespread adoption. Here, we present Machine-learning Assisted, Interferometric Structured Illumination Microscopy, MAI-SIM, as an easy-to-implement method for live cell super-resolution imaging at high speed and in multiple colors. The instrument is based on an interferometer design in which illumination patterns are generated, rotated, and stepped in phase through movement of a single galvanometric mirror element. The design is robust, flexible, and works for all wavelengths. We complement the unique properties of the microscope with an open source machine-learning toolbox that permits real-time reconstructions to be performed, providing instant visualization of super-resolved images from live biological samples.
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Iluminación , Aprendizaje Automático , Microscopía Fluorescente/métodos , InterferometríaRESUMEN
The point spread function of a fixed fluorophore with its dipole axis colinear to the optical axis appears donut-shaped when seen through a microscope, and its light distribution in the pupil plane is radially polarized. Yet other techniques, such as photolithography, report that this same light distribution in the pupil plane appears as a solid spot. How can this same distribution lead to a spot in one case but a donut in the other? Here, we show how the tube lens of the system plays a critical role in determining this shape. Using a vectorial treatment of image formation, we simulate the relative contributions of both longitudinal and radial components to the image of a dipole emitter and thus show how the donut (typically reported for z-polarized single molecule fluorescence microscopy) transforms into a solid spot (as commonly reported for photolithography) as the numerical aperture of the tube lens increases. We find that the transition point occurs around 0.7 NA, which is significantly higher than used for most microscopy systems and lower than for common photolithography systems, thus resolving the seeming paradox of dipole shape.
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Algoritmos , Lentes , Microscopía/métodosRESUMEN
Microfluidics has enabled low volume biochemistry reactions to be carried out at the point-of-care. A key component in microfluidics is the microfluidic valve. Microfluidic valves are not only useful for directing flow at intersections but also allow mixtures/dilutions to be tuned real-time and even provide peristaltic pumping capabilities. In the transition from chip-in-a-lab to lab-on-a-chip, it is essential to ensure that microfluidic valves are designed to require less peripheral equipment and that they are transportable. In this paper, a thermally-actuated microfluidic valve is presented. The valve itself is fabricated with off-the-shelf components without the need for sophisticated cleanroom techniques. It is shown that multiple valves can be controlled and operated via a power supply and an Arduino microcontroller; an important step towards transportable microfluidic devices capable of carrying out analytical assays at the point-of-care. It is been calculated that a single actuator costs less than $1, this highlights the potential of the presented valve for scaling out. The valve operation is demonstrated by adjusting the ratio of a water/dye mixture in a continuous flow microfluidic chip with Y-junction channel geometry. The power required to operate one microfluidic valve has been characterised both theoretically and experimentally. Cyclical operation of the valve has been demonstrated for 65 h with 585 actuations. The presented valve is capable of actuating rectangular microfluidic channels of 500 µm × 50 µm with an expected temperature increase of up to 5 °C. The fastest actuation times achieved were 2 s for valve closing (heating) and 9 s for valve opening (cooling).
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A method is proposed for assessing the temporal resolution of structured illumination microscopy (SIM), by tracking the amplitude of different spatial frequency components over time, and comparing them to a temporally-oscillating ground-truth. This method is used to gain insight into the performance limits of SIM, along with alternative reconstruction techniques (termed 'rolling SIM') that claim to improve temporal resolution. Results show that the temporal resolution of SIM varies considerably between low and high spatial frequencies, and that, despite being used in several high profile papers and commercial microscope software, rolling SIM provides no increase in temporal resolution over conventional SIM.
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Mechanical effects of microbubbles on tissues are central to many emerging ultrasound applications. Here, we investigated the acoustic radiation force a microbubble exerts on tissue at clinically relevant therapeutic ultrasound parameters. Individual microbubbles administered into a wall-less hydrogel channel (diameter: 25-100 µm, Young's modulus: 2-8.7 kPa) were exposed to an acoustic pulse (centre frequency: 1 MHz, pulse length: 10 ms, peak-rarefactional pressures: 0.6-1.0 MPa). Using high-speed microscopy, each microbubble was tracked as it pushed against the hydrogel wall. We found that a single microbubble can transiently deform a soft tissue-mimicking material by several micrometres, producing tissue loading-unloading curves that were similar to those produced using other indentation-based methods. Indentation depths were linked to gel stiffness. Using a mathematical model fitted to the deformation curves, we estimated the radiation force on each bubble (typically tens of nanonewtons) and the viscosity of the gels. These results provide insight into the forces exerted on tissues during ultrasound therapy and indicate a potential source of bio-effects.
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Módulo de Elasticidad , Ensayo de Materiales , Microburbujas , Modelos Biológicos , Ultrasonografía , AcústicaRESUMEN
Dark-field microscopy is a standard imaging technique widely employed in biology that provides high image contrast for a broad range of unstained specimens1. Unlike bright-field microscopy, it accentuates high spatial frequencies and can therefore be used to emphasize and resolve small features. However, the use of dark-field microscopy for reliable analysis of blood cells, bacteria, algae, and other marine organisms often requires specialized, bulky microscope systems, and expensive additional components, such as dark-field-compatible objectives or condensers2,3. Here, we propose to simplify and downsize dark-field microscopy equipment by generating the high-angle illumination cone required for dark field microscopy directly within the sample substrate. We introduce a luminescent photonic substrate with a controlled angular emission profile and demonstrate its ability to generate high-contrast dark-field images of micrometre-sized living organisms using standard optical microscopy equipment. This new type of substrate forms the basis for miniaturized lab-on-chip dark-field imaging devices, compatible with simple and compact light microscopes.
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The first ever demonstration of temporal focusing with short wave infrared (SWIR) excitation and emission is demonstrated, achieving a penetration depth of 500 µm in brain tissue. This is substantially deeper than the highest previously-reported values for temporal focusing imaging in brain tissue, and demonstrates the value of these optimized wavelengths for neurobiological applications.
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Simultaneous, high-resolution imaging across a large number of synaptic and dendritic sites is critical for understanding how neurons receive and integrate signals. Yet, functional imaging that targets a large number of submicrometer-sized synaptic and dendritic locations poses significant technical challenges. We demonstrate a new parallelized approach to address such questions, increasing the signal-to-noise ratio by an order of magnitude compared to previous approaches. This selective access multifocal multiphoton microscopy uses a spatial light modulator to generate multifocal excitation in three dimensions (3D) and a Gaussian-Laguerre phase plate to simultaneously detect fluorescence from these spots throughout the volume. We test the performance of this system by simultaneously recording Ca2+ dynamics from cultured neurons at 98-118 locations distributed throughout a 3D volume. This is the first demonstration of 3D imaging in a "single shot" and permits synchronized monitoring of signal propagation across multiple different dendrites.
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Super-resolution single-molecule localization microscopy, often referred to as PALM/STORM, works by ensuring that fewer than one fluorophore in a diffraction-limited volume is emitting at any one time, allowing the observer to infer that the emitter is located at the center of the point-spread function. This requires careful control over the incident light intensity in order to control the rate at which fluorophores are switched on; if too many fluorophores are activated, their point-spread functions overlap, which impedes efficient localization. If too few are activated, the imaging time is impractically long. There is therefore considerable recent interest in constructing so-called 'top-hat' illumination profiles that provide a uniform illumination over the whole field of view. We present the use of a single commercially-available low-cost refractive beamshaping element that can be retrofitted to almost any existing microscope; the illumination profile created by this element demonstrates a marked improvement in the power efficiency of dSTORM microscopy, as well as a significant reduction in the propensity for reconstruction artifacts, compared to conventional Gaussian illumination.
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Three-photon wide-field depth-resolved excitation is used to overcome some of the limitations in conventional point-scanning two- and three-photon microscopy. Excitation of chromophores as diverse as channelrhodopsins and quantum dots is shown, and a penetration depth of more than 700 µm into fixed scattering brain tissue is achieved, approximately twice as deep as that achieved using two-photon wide-field excitation. Compatibility with live animal experiments is confirmed by imaging the cerebral vasculature of an anesthetized mouse; a complete focal stack was obtained without any evidence of photodamage. As an additional validation of the utility of wide-field three-photon excitation, functional excitation is demonstrated by performing three-photon optogenetic stimulation of cultured mouse hippocampal neurons expressing a channelrhodopsin; action potentials could reliably be excited without causing photodamage.
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For in vivo imaging, the short-wavelength infrared region (SWIR; 1000-2000 nm) provides several advantages over the visible and near-infrared regions: general lack of autofluorescence, low light absorption by blood and tissue, and reduced scattering. However, the lack of versatile and functional SWIR emitters has prevented the general adoption of SWIR imaging by the biomedical research community. Here, we introduce a class of high-quality SWIR-emissive indium-arsenide-based quantum dots (QDs) that are readily modifiable for various functional imaging applications, and that exhibit narrow and size-tunable emission and a dramatically higher emission quantum yield than previously described SWIR probes. To demonstrate the unprecedented combination of deep penetration, high spatial resolution, multicolor imaging and fast-acquisition-speed afforded by the SWIR QDs, we quantified, in mice, the metabolic turnover rates of lipoproteins in several organs simultaneously and in real time as well as heartbeat and breathing rates in awake and unrestrained animals, and generated detailed three-dimensional quantitative flow maps of the mouse brain vasculature.
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Heme is ubiquitous, yet relatively little is known about the maintenance of labile pools of this cofactor, which likely ensures its timely bioavailability for proper cellular function. Quantitative analysis of labile heme is of fundamental importance to understanding how nature preserves access to the diverse chemistry heme enables, while minimizing cellular damage caused by its redox activity. Here, we have developed and characterized a protein-based sensor that undergoes fluorescence quenching upon heme binding. By genetically encoding this sensor in the human malarial parasite, Plasmodium falciparum, we have quantified cytosolic labile heme levels in intact, blood-stage parasites. Our findings indicate that a labile heme pool (â¼1.6 µM) is stably maintained throughout parasite development within red blood cells, even during a period coincident with extensive hemoglobin degradation by the parasite. We also find that the heme-binding antimalarial drug chloroquine specifically increases labile cytosolic heme, indicative of dysregulation of this homeostatic pool that may be a relevant component of the antimalarial activity of this compound class. We propose that use of this technology under various environmental perturbations in P. falciparum can yield quantitative insights into fundamental heme biology.
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Técnicas Biosensibles , Hemo/metabolismo , Plasmodium/metabolismo , Animales , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Expresión Génica , Genes Reporteros , Hemo/química , Hemo/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Plasmodium/genética , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Temporal focusing multiphoton microscopy is a technique for performing highly parallelized multiphoton microscopy while still maintaining depth discrimination. While the conventional wide-field configuration for temporal focusing suffers from sub-optimal axial resolution, line scanning temporal focusing, implemented here using a digital micromirror device (DMD), can provide substantial improvement. The DMD-based line scanning temporal focusing technique dynamically trades off the degree of parallelization, and hence imaging speed, for axial resolution, allowing performance parameters to be adapted to the experimental requirements. We demonstrate this new instrument in calibration specimens and in biological specimens, including a mouse kidney slice.
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Motor units are the fundamental elements responsible for muscle movement. They are formed by lower motor neurons and their muscle targets, synapsed via neuromuscular junctions (NMJs). The loss of NMJs in neurodegenerative disorders (such as amyotrophic lateral sclerosis or spinal muscle atrophy) or as a result of traumatic injuries affects millions of lives each year. Developing in vitro assays that closely recapitulate the physiology of neuromuscular tissues is crucial to understand the formation and maturation of NMJs, as well as to help unravel the mechanisms leading to their degeneration and repair. We present a microfluidic platform designed to coculture myoblast-derived muscle strips and motor neurons differentiated from mouse embryonic stem cells (ESCs) within a three-dimensional (3D) hydrogel. The device geometry mimics the spinal cord-limb physical separation by compartmentalizing the two cell types, which also facilitates the observation of 3D neurite outgrowth and remote muscle innervation. Moreover, the use of compliant pillars as anchors for muscle strips provides a quantitative functional readout of force generation. Finally, photosensitizing the ESC provides a pool of source cells that can be differentiated into optically excitable motor neurons, allowing for spatiodynamic, versatile, and noninvasive in vitro control of the motor units.