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1.
Proc Natl Acad Sci U S A ; 121(43): e2404232121, 2024 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-39401353

RESUMO

Spontaneous Raman imaging has emerged as powerful label-free technique for investigating the molecular composition of medicines and biological specimens. Although Raman imaging can facilitate understanding of complex biological phenomena in vivo, current imaging modalities are limited in speed and sample compatibility. Here, we introduce a single-objective line-scanning light-sheet microscope, named [Formula: see text]-OPM, which records Raman images on a timescale of minutes to seconds. To demonstrate its function, we use [Formula: see text]-OPM to map and identify microplastic particles based on their Raman spectral characteristics. In live zebrafish embryos, we show that [Formula: see text]-OPM can capture wound dynamics at five-minute intervals, revealing rapid changes in cellular and extracellular matrix composition in the wounded region. Finally, we use [Formula: see text]-OPM to synchronize and average 36,800 individual frames to obtain hyperspectral videos of a zebrafish embryo's beating heart at an effective 28 frames per second, recording compositional changes throughout the cardiac cycle.


Assuntos
Embrião não Mamífero , Análise Espectral Raman , Peixe-Zebra , Animais , Peixe-Zebra/embriologia , Análise Espectral Raman/métodos , Microscopia/métodos , Coração/diagnóstico por imagem , Microplásticos , Imageamento Hiperespectral/métodos , Matriz Extracelular/metabolismo
2.
Proc Natl Acad Sci U S A ; 114(11): E2068-E2076, 2017 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-28242687

RESUMO

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 (∼1.6 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.


Assuntos
Técnicas Biossensoriais , Heme/metabolismo , Plasmodium/metabolismo , Animais , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Expressão Gênica , Genes Reporter , Heme/química , Heme/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Plasmodium/genética , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
3.
J Phys D Appl Phys ; 52(26): 264001, 2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-33191950

RESUMO

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.

4.
Proc Natl Acad Sci U S A ; 110(38): 15189-94, 2013 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-24003124

RESUMO

Tissue-conserving surgery is used increasingly in cancer treatment. However, one of the main challenges in this type of surgery is the detection of tumor margins. Histopathology based on tissue sectioning and staining has been the gold standard for cancer diagnosis for more than a century. However, its use during tissue-conserving surgery is limited by time-consuming tissue preparation steps (1-2 h) and the diagnostic variability inherent in subjective image interpretation. Here, we demonstrate an integrated optical technique based on tissue autofluorescence imaging (high sensitivity and high speed but low specificity) and Raman scattering (high sensitivity and high specificity but low speed) that can overcome these limitations. Automated segmentation of autofluorescence images was used to select and prioritize the sampling points for Raman spectroscopy, which then was used to establish the diagnosis based on a spectral classification model (100% sensitivity, 92% specificity per spectrum). This automated sampling strategy allowed objective diagnosis of basal cell carcinoma in skin tissue samples excised during Mohs micrographic surgery faster than frozen section histopathology, and one or two orders of magnitude faster than previous techniques based on infrared or Raman microscopy. We also show that this technique can diagnose the presence or absence of tumors in unsectioned tissue layers, thus eliminating the need for tissue sectioning. This study demonstrates the potential of this technique to provide a rapid and objective intraoperative method to spare healthy tissue and reduce unnecessary surgery by determining whether tumor cells have been removed.


Assuntos
Técnicas de Diagnóstico por Cirurgia , Microscopia/métodos , Neoplasias/diagnóstico , Imagem Óptica/métodos , Análise Espectral Raman/métodos , Técnicas Histológicas/métodos , Humanos , Neoplasias/patologia
5.
Science ; 385(6713): 1046-1047, 2024 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-39236198

RESUMO

A food dye suppresses light scattering in biological tissues to enable deep in vivo imaging.


Assuntos
Corantes , Espalhamento de Radiação , Animais , Luz , Imagem Óptica/métodos , Camundongos
6.
Biomed Opt Express ; 15(9): 5009-5024, 2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-39296388

RESUMO

Biosynthesis in bioreactors plays a vital role in many applications, but tools for accurate in situ monitoring of the cells are still lacking. By engineering the cells such that their conditions are reported through fluorescence, it is possible to fill in the gap using fluorescence diffuse optical tomography (fDOT). However, the spatial accuracy of the reconstruction can still be limited, due to e.g. undersampling and inaccurate estimation of the optical properties. Utilizing controlled phantom studies, we use a two-step hybrid approach, where a preliminary fDOT result is first obtained using the classic model-based optimization, and then enhanced using a neural network. We show in this paper using both simulated and phantom experiments that the proposed method can lead to a 8-fold improvement (Intersection over Union) of fluorescence inclusion reconstruction in noisy conditions, at the same speed of conventional neural network-based methods. This is an important step towards our ultimate goal of fDOT monitoring of bioreactors.

7.
Bioinform Adv ; 3(1): vbad182, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38146539

RESUMO

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/.

8.
Biomed Opt Express ; 14(12): 6361-6380, 2023 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-38420325

RESUMO

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.

9.
Biomed Opt Express ; 14(6): 2565-2575, 2023 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-37342684

RESUMO

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.

10.
IEEE Trans Biomed Eng ; 70(9): 2752-2761, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37015124

RESUMO

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.


Assuntos
Processamento de Imagem Assistida por Computador , Processamento de Sinais Assistido por Computador , Coelhos , Animais , Processamento de Imagem Assistida por Computador/métodos , Simulação por Computador , Algoritmos , Imageamento Tridimensional , Ultrassonografia/métodos , Imagens de Fantasmas
11.
Analyst ; 137(18): 4119-22, 2012 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-22842554

RESUMO

A selective scanning method was used to measure spatially resolved Raman spectra of live Neospora caninum tachyzoites colonizing human brain microvascular-endothelial cells. The technique allowed the detection of nucleic acids, lipids and proteins linked to the parasites and their cellular micro-environment at ∼10× shorter acquisition time compared to raster scanning.


Assuntos
Encéfalo/parasitologia , Células Endoteliais/parasitologia , Neospora/química , Análise Espectral Raman , Interações Hospedeiro-Parasita , Humanos , Lipídeos/análise , Neospora/crescimento & desenvolvimento , Ácidos Nucleicos/análise , Proteínas de Protozoários/análise
12.
Microsc Res Tech ; 85(5): 2016-2022, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35045219

RESUMO

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.


Assuntos
Algoritmos , Lentes , Microscopia/métodos
13.
Nat Commun ; 13(1): 7836, 2022 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-36543776

RESUMO

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.


Assuntos
Iluminação , Aprendizado de Máquina , Microscopia de Fluorescência/métodos , Interferometria
15.
Biomed Opt Express ; 12(2): 790-801, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33680542

RESUMO

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.

16.
Microsyst Nanoeng ; 7: 48, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34567761

RESUMO

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).

17.
Chemphyschem ; 11(11): 2393-8, 2010 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-20623572

RESUMO

A method for fabricating infrared-transmitting waveguides that yields low optical losses and strong confinement of light is presented. The method minimises the number of fabrication steps by exploiting the photosensitivity of arsenic trisulfide glass, using it both as a photoresist and as a waveguiding material. Controlled annealing/remelting of the waveguides minimises scattering due to fluctuations in refractive index at the interface between the waveguide and the surrounding medium, allowing low losses to be realised. Bends and Y-splitter structures have been realised, as well as the longest As(2)S(3) serpentine planar waveguides yet reported.

18.
Ultrasound Med Biol ; 46(12): 3327-3338, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32919812

RESUMO

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.


Assuntos
Módulo de Elasticidade , Teste de Materiais , Microbolhas , Modelos Biológicos , Ultrassonografia , Acústica
19.
Nat Photonics ; 14(5): 310-315, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-33584848

RESUMO

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.

20.
Optica ; 6(1): 76-83, 2019 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-31984218

RESUMO

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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