44 results found
Ward EN, Hecker L, Christensen CN, et al., 2022, Machine learning assisted interferometric structured illumination microscopy for dynamic biological imaging., Nat Commun, Vol: 13
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.
Whiteley I, Song C, Howe GA, et al., 2022, DIRECT, a low-cost system for high-speed, low-noise imaging of fluorescent biological samples
A targeted imaging system has been developed for applications requiringrecording from stationary samples at high spatiotemporal resolutions. It worksby illuminating regions of interest in rapid sequence, and recording the signalfrom the whole field of view onto a single photodetector, and can beimplemented at low cost on an existing microscope without compromising existingfunctionality. The system is characterized in terms of speed, spatialresolution, and tissue penetration depth, before being used to recordindividual action potentials from ASAP-3 expressing neurons in an ex vivo mousebrain slice preparation.
Strohl F, Bruggeman E, Rowlands CJ, et al., 2022, Quantification of the NA dependent change of shape in the image formation of a z-polarized fluorescent molecule using vectorial diffraction simulations, Microscopy Research and Technique, Pages: 1-7, ISSN: 1059-910X
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.
Whiteley I, Rowlands CJ, 2022, Towards two- and three -dimensional, DMD-based holographic targeting of biological samples
Progress in the development of two- and three- dimensional binary amplitude holograms for use in digital micromirror device-based biological targeting experiments is shown. The intended application is high-speed photomodulation of biological processes like neural activity.
Sesen M, Rowlands C, 2021, Thermally-actuated microfluidic membrane valve for point-of-care applications, Microsystems and Nanoengineering, Vol: 7, Pages: 1-12, ISSN: 2055-7434
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 hours with 585 actuations. The presented valve is capable of actuating rectangular microfluidic channels of 500μm×50μm with an expected temperature increase of up to 5°C. The fastest actuation times achieved were 2seconds for valve closing (heating) and 9 seconds for valve opening (cooling).
Boualam A, Rowlands C, 2021, A method for assessing the spatiotemporal resolution of structured illumination microscopy (SIM), Biomedical Optics Express, Vol: 12, Pages: 790-801, ISSN: 2156-7085
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.
Whiteley I, Song C, Knopfel T, et al., 2021, Targeted imaging system for voltage indicator readout
A system for quasi-simultaneous direct recording from multiple neurons is presented. A digital micromirror device (DMD) is used to selectively illuminate specific neurons, switching between them at high speed to isolate voltage signals.
Bezer JH, Koruk H, Rowlands CJ, et al., 2020, Elastic deformation of soft tissue-mimicking materials using a single microbubble and acoustic radiation force, Ultrasound in Medicine and Biology, Vol: 46, Pages: 3327-3338, ISSN: 0301-5629
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.
Chazot CAC, Nagelberg S, Rowlands CJ, et al., 2020, Luminescent surfaces with tailored angular emission for compact dark-field imaging devices, Nature Photonics, Vol: 14, Pages: 310-315, ISSN: 1749-4885
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, as well as 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 that are compatible with simple and compact light microscopes.
Whiteley I, Song C, Knöpfel T, et al., 2020, Optical readout of voltage indicators using an improved targeted direct patterning concept.
An improved method for the optical readout of voltage indicators is presented, whereby fluorescence from the whole surface of a neuron is integrated onto a single detector, dramatically increasing the recording bandwidth over conventional techniques.
Rowlands CJ, Bruns O, Franke D, et al., 2019, Increasing the penetration depth of temporal focusing multiphoton microscopy for neurobiological applications., Journal of Physics D: Applied Physics, Vol: 52, ISSN: 0022-3727
The first ever demonstration of temporal focusing with Short Wave InfraRed (SWIR) excitation and emission is demonstrated, achieving a penetration depth of 500µm 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.
Xue Y, Berry KP, Boivin JR, et al., 2019, Scanless volumetric imaging by selective access multifocal multiphoton microscopy, Optica, Vol: 6, Pages: 76-83, ISSN: 2334-2536
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 Ca 2 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.
Rowlands CJ, Ströhl F, Vallejo Ramirez P, et al., 2018, Flat-field super-resolution localization microscopy with a low-cost refractive beam-shaping element, Scientific Reports, Vol: 8, ISSN: 2045-2322
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.
So PTC, Choi H, Yew E, et al., 2018, Principles and applications of temporal-focusing wide-field two-photon microscopy, Multiphoton Microscopy and Fluorescence Lifetime Imaging: Applications in Biology and Medicine, Pages: 103-140, ISBN: 9783110438987
Temporal focusing allows for rapid optically sectioned two-photon widefield microscopy. Depth sectioning is provided in a wide-field manner, without spatial focusing, by controlling the temporalwidth of femtosecond laser pulses near the focal plane. This spatial control of the temporal pulse width is achieved by diffracting the light off a grating resulting in spectral component separation and temporal broadening; these spectral components are only recombined at the focal plane to reproduce short, femtosecond pulses. Applications include (i) high speed functional imaging in the brain, (ii) fast FLIM and PLIM, (iii) cell-selective optogenetic excitation, and (iv) temporal focusing photodynamic therapy that may allow selective killing of cancer cells.
Rowlands CJ, Park D, Bruns OT, et al., 2017, Wide-field three-photon excitation in biological samples, Light: Science and Applications, Vol: 6, Pages: 1-9, ISSN: 2047-7538
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.
Bruns OT, Bischof TS, Harris DK, et al., 2017, Next-generation in vivo optical imaging with short-wave infrared quantum dots, NATURE BIOMEDICAL ENGINEERING, Vol: 1, ISSN: 2157-846X
Abshire JR, Rowlands CJ, Ganesan SM, et al., 2017, Quantification of labile heme in live malaria parasites using a genetically encoded biosensor, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 114, Pages: E2068-E2076, ISSN: 0027-8424
Park JK, Rowlands CJ, So PTC, 2017, Enhanced Axial Resolution of Wide-Field Two-Photon Excitation Microscopy by Line Scanning Using a Digital Micromirror Device, MICROMACHINES, Vol: 8, ISSN: 2072-666X
Xue Y, Berry KP, Rowlands CJ, et al., 2017, Monitoring neuronal signal integration by selective access multifoci multiphoton microscopy
Our project seeks to develop technology capable of monitoring how a single neuron integrate its inputs from over 104 synapses. This target requires optical imaging technology with high resolution and high speed for both structural and functional imaging. Parallel multifoci imaging is a significant method to improve the imaging speed while preserving high image signal-tonoise- ratio (SNR). Here we demonstrate a scanless method, selective access multifoci multiphoton microscopy (saMMM), for volumetric structure imaging and function imaging. The system is able to excite multifoci modulated by spatial light modulator, and more importantly, detect fluorescence from these multiple spots simultaneously using a Gaussian-Laguerre (GL) phase plate for phase modulation. The phase plate modulates a Gaussian point spread function to double-helix point spread function, which both extends the depth of focus and encodes axial position by rotation angle. We showed the multifoci advantage of the system by simultaneous recording calcium dynamics of cultured neuron from 149 locations across the whole field of view, and we showed the volumetric imaging advantage by simultaneously exciting and detecting multiple locations in three-dimension of a neuron and reconstruct the exact axial positions from a single plane image. Calcium dynamics recorded with and without modulation with a GL phase phate recorded and compared in terms of SNR. This “3D image by one shot“ strategy largely improved the signal to noise ratio of fluorescence images, therefore it is possible to accelerate the imaging speed. The selective access illumination further elevates the imaging speed by only recording labeled area, also avoid unnecessary photodamage to the specimen. The scanless design breakthrough the limit of mechanical scanning speed, ensured dynamic records from all the foci are strictly synchronized, and no perturbation to cell physiology during imaging. This saMMM system potentially can be appl
Uzel SGM, Platt RJ, Subramanian V, et al., 2016, Microfluidic device for the formation of optically excitable, three-dimensional, compartmentalized motor units, SCIENCE ADVANCES, Vol: 2, ISSN: 2375-2548
- Author Web Link
- Citations: 136
So PTC, Yew E, Rowlands C, 2016, Applications of Multiphoton Microscopy in Dermatology, IMAGING IN DERMATOLOGY, Editors: Hamblin, Avci, Gupta, Publisher: ACADEMIC PRESS LTD-ELSEVIER SCIENCE LTD, Pages: 241-268, ISBN: 978-0-12-802838-4
- Author Web Link
- Citations: 2
Berry KP, Xue Y, Rowlands CJ, et al., 2016, Next generation high-throughput random access imaging in vivo
Our team is developing a next-generation high-throughput random-access imaging system for real-time monitoring of sensory-driven synaptic activity. This monitoring takes place across all inputs to single living neurons in the context of the intact cerebral cortex, in order to better understand how these synaptic signals are integrated and processed. Our first target is to monitor calcium signals from approximately 10,000 locations corresponding to all excitatory synapses of a single neuron with 100 ms temporal resolution. While calcium imaging with GCaMP is well-established, the RCaMP excitation wavelength at about 1050 nm is more compatible with high power Yb-fiber based femtosecond laser sources.
Rowlands CJ, Bruns OT, Bawendi MG, et al., 2015, Objective, comparative assessment of the penetration depth of temporal-focusing microscopy for imaging various organs, JOURNAL OF BIOMEDICAL OPTICS, Vol: 20, ISSN: 1083-3668
- Author Web Link
- Citations: 7
Xue Y, Rowlands CJ, So PTC, 2015, Parallel and Flexible Imaging using Two-photon RESOLFT Microscopy with Spatial Light Modulator Control, Conference on Multiphoton Microscopy in the Biomedical Sciences XV, Publisher: SPIE-INT SOC OPTICAL ENGINEERING, ISSN: 0277-786X
- Author Web Link
- Citations: 3
Rowlands CJ, Wu J, Uzel SGM, et al., 2014, 3D-resolved targeting of photodynamic therapy using temporal focusing, Laser Physics Letters, Vol: 11, ISSN: 1612-2011
A method for selectively inducing apoptosis in tumor nodules is presented, with close-to-cellular level resolution, using 3D-resolved widefield temporal focusing illumination. Treatment times on the order of seconds were achieved using Verteporfin as the photosensitizer, with doses of 30 μg ml−1 and below. Results were achieved on both 2D and 3D cell cultures, demonstrating that treatment was possible through approximately one hundred microns of dense tumor nodules.
Kong K, Rowlands CJ, Varma S, et al., 2014, Increasing the speed of tumour diagnosis during surgery with selective scanning Raman microscopy, JOURNAL OF MOLECULAR STRUCTURE, Vol: 1073, Pages: 58-65, ISSN: 0022-2860
- Author Web Link
- Citations: 10
Yew E, Rowlands C, So PTC, 2014, Application of multiphoton microscopy in dermatological studies: A mini-review, JOURNAL OF INNOVATIVE OPTICAL HEALTH SCIENCES, Vol: 7, ISSN: 1793-5458
- Author Web Link
- Citations: 53
Rowlands CJ, Bruns OT, Franke D, et al., 2014, Near-infrared temporal focusing microscopy with quantum dot fluorophores
The tissue penetration depth of temporal focusing is increased by utilizing longer excitation wavelengths combined with quantum dots possessing extremely high two-photon crosssections. Cerebral vasculature is imaged in a mouse brain in vivo.
Marcu L, French PMW, Elson DS, et al., 2014, Preface, ISBN: 9781439861677
Wide-field time-gated fluorescence lifetime imaging (FLIM) essentially entails illuminating a sample with an ultrashort pulse of excitation radiation and sampling the resulting time varying fluorescence “image” following excitation by acquiring a series of gated fluorescence intensity images recorded at different relative delays with respect to the excitation pulse. This is represented schematically in Figure 8.1. In the simplest case, a map of the mean fluorescence decay times across the field of view is obtained. If the sampling of the fluorescence decay profiles is appropriately detailed, then the entire fluorescence decay profile for each image pixel can be acquired, and the resulting data set can be fitted to complex temporal decay models. For example, a double exponential decay model is frequently used to analyze data from Förster resonant energy transfer (FRET) experiments. The acquisition of time-gated fluorescence intensity images requires a 2-D detector, normally a charge-coupled device (CCD) camera, and some kind of fast “shutter” able to sample fluorescence decay profiles on subnanosecond timescales. Such a “shutter” function cannot be provided by mechanical means or yet by electronic circuitry and is typically provided by optical image intensifiers whose gain can be modulated by varying the applied voltage.
So PTC, Yew EYS, Rowlands C, 2013, High-Throughput Nonlinear Optical Microscopy, BIOPHYSICAL JOURNAL, Vol: 105, Pages: 2641-2654, ISSN: 0006-3495
- Author Web Link
- Citations: 29
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