Parallel Illumination Research Articles

Scanless two-photon voltage imaging

Two-photon voltage imaging has long been heralded as a transformative approach capable of answering many long-standing questions in modern neuroscience. However, exploiting its full potential requires the development of novel imaging approaches well suited to the photophysical properties of genetically encoded voltage indicators. We demonstrate that parallel excitation approaches developed for scanless two-photon photostimulation enable high-SNR two-photon voltage imaging. We use whole-cell patch-clamp electrophysiology to perform a thorough characterization of scanless two-photon voltage imaging using three parallel illumination approaches and lasers with different repetition rates and wavelengths. We demonstrate voltage recordings of high-frequency spike trains and sub-threshold depolarizations from neurons expressing the soma-targeted genetically encoded voltage indicator JEDI-2P-Kv. Using a low repetition-rate laser, we perform multi-cell recordings from up to fifteen targets simultaneously. We co-express JEDI-2P-Kv and the channelrhodopsin ChroME-ST and capitalize on their overlapping two-photon absorption spectra to simultaneously evoke and image action potentials using a single laser source. We also demonstrate in vivo scanless two-photon imaging of multiple cells simultaneously up to 250 µm deep in the barrel cortex of head-fixed, anaesthetised mice.

Parallel illumination for depletion microscopy through acousto-optic spatial light modulation

Several types of super-resolution microscopy techniques, such as Stimulated Emission Depletion (STED), Reversible Saturable Optical Fluorescence Transitions (RESOLFT) or Switching Laser Mode (SLAM) microscopies, employ Laguerre-Gaussian beams (also called vortex or doughnut beams) to obtain fluorescence information within a sub-wavelength region of the specimen under observation, thus breaking the diffraction limit and producing images of greatly improved quality. However, in general, these techniques operate on a point-by-point basis, so scanning the sample in order to build a full, meaningful image, takes time. Parallelization of the illumination is the only way to make these microscopy techniques more suitable for real-time live cell imaging applications. Here, we demonstrate the parallel production of arbitrary arrays of Gaussian and Laguerre-Gaussian lasers foci suitable for super-resolution microscopy, together with the possibility to fast scan through the sample, by means of acousto-optic spatial light modulation, a technique that we have pioneered in the past in several other fields. Parallelized illumination with both Gaussian and doughnut beams will be then used to acquire super-resolution images.

Conjugated Polythiophene Frameworks as a Hole‐Selective Layer on Ta3N5 Photoanode for High‐Performance Solar Water Oxidation

AbstractDiscovering a competent charge transport layer promoting charge separation in photoelectrodes is a perpetual pursuit in photoelectrochemical (PEC) water splitting to achieve high solar‐to‐hydrogen (STH) conversion efficiency. Here, a conjugated polythiophene framework (CPF‐TTB) on Ta3N5 is elaborately electropolymerized, substantiating the hole transport behavior in their heterojunction. Tailored band structures of the CPF‐TTB/Ta3N5 reinforce the separation of photogenerated carriers, elevating a fill factor of the photoanode modified with a cocatalyst. The enhanced hole extraction enables the NiFeOx/CPF‐TTB/Ta3N5/TiN photoanode to generate a remarkable water oxidation photocurrent density of 9.12 mA cm−2 at 1.23 V versus the reversible hydrogen electrode. A tandem device combining the photoanode with a perovskite/Si solar cell implements an unbiased solar water splitting with a STH conversion efficiency of 6.26% under parallel illumination mode. This study provides novel strategies in interface engineering for metal nitride‐based photoelectrodes, suggesting a promise of the organic–inorganic hybrid photoelectrode for high‐efficiency PEC water splitting.

MTIE-Net: Multi-technology fusion of low-light image enhancement network.

Images obtained in low-light scenes are often accompanied by problems such as low visibility, blurred details, and color distortion, enhancing them can effectively improve the visual effect and provide favorable conditions for advanced visual tasks. In this study, we propose a Multi-Technology Fusion of Low-light Image Enhancement Network (MTIE-Net) that modularizes the enhancement task. MTIE-Net consists of a residual dense decomposition network (RDD-Net) based on Retinex theory, an encoder-decoder denoising network (EDD-Net), and a parallel mixed attention-based self-calibrated illumination enhancement network (PCE-Net). The low-light image is first decomposed by RDD-Net into a lighting map and reflectance map; EDD-Net is used to process noise in the reflectance map; Finally, the lighting map is fused with the denoised reflectance map as an input to PCE-Net, using the Fourier transform for illumination enhancement and detail recovery in the frequency domain. Numerous experimental results show that MTIE-Net outperforms the comparison methods in terms of image visual quality enhancement improvement, denoising, and detail recovery. The application in nighttime face detection also fully demonstrates its promise as a pre-processing means in practical applications.

High-axial-resolution speckle-free holographic reconstruction via cylindrical quadratic phase method and temporal focusing.

Holographic techniques enable precise laser manipulation, but suffer from two considerable limitations: speckle and deterioration of axial distribution. Here, we propose a cylindrical quadratic phase (CQP) method with temporal focusing (TF) to generate speckle-free holographic illumination with high axial resolution. TF-CQP utilizes a superposed cylindrical phase as the initial guess to iteratively optimize phase hologram, realizing speckle-free holographic reconstruction on the target focal plane and eliminating secondary focus on the defocused planes. TF-CQP further disperses defocused beams symmetrically by a blazed grating, placed conjugate to the focal plane, which enhances axial confinement. Simulation and experimental results show that TF-CQP reconstructs speckle-free illumination with arbitrary shapes and <10 µm axial resolution. Compared to TF-GS (Gerchberg-Saxton algorithm), widely used in holographic optogenetics, TF-CQP shows increased uniformity of 200% and improved modulation efficiency of 32.33% for parallel holographic illumination, as well as a 10% increment in axial resolution.

Application of Benthic Microbial Fuel Cells in Systems of Year-Round Monitoring of Water Environment Parameters

The bioelectrogenic activity of sediments of natural microbial association of the Peter’s Bay of Japanese sea research was performed in a year-round experiment with parallel temperature, illumination and water electrical conductivity monitoring by means of benthic microbial fuel cell (MFC) and automatic online-monitoring. Several variants of underwater devices, including benthic microbial fuel cells, monitoring water environment sensor,information collection and transmission systems, have been developed. This device make electrical voltage up to 216 mV, specific power up to 239 mW/m2. Electrogenic activity of natural microflora depends on water temperature and reach maximum on summer with temperature about 20–25°C. The introduction of toxicants in form as hydrocarbons and cadmium into the sluge led to the suppression of microbial electrogenesis. However the introduction of inductor substances of microbial sulfidogenesis led to the stimulation of microbial electrogenesis. The possibility of functioning of the benthic MFC in the field of the Peter’s Great Bay in various climatic periods is shown. It is shown that such experimental devices serve as a basis for autonomous stations monitoring the state of the aquatic environment for a long time and in a wide range of conditions change. Thus, automatic registration of temperature, illumination and salinity of water with a frequency of 48 times a day was carried out for 13 months (11/28/2019–12/31/2020). The electrogenic activity of this microbiota upon MFC scaling can potentially become a new renewable energy source for low-power marine electronics, including those used in mariculture.

Optimizing experimental parameters for orbital mapping

A new material characterization technique is emerging for the transmission electron microscope (TEM). Using electron energy-loss spectroscopy, real space mappings of the underlying electronic transitions in the sample, so called orbital maps, can be produced. Thus, unprecedented insight into the electronic orbitals responsible for most of the electrical, magnetic and optical properties of bulk materials can be gained. However, the incredibly demanding requirements on spatial as well as spectral resolution paired with the low signal-to-noise ratio severely limits the day-to-day use of this new technique. With the use of simulations, we strive to alleviate these challenges as much as possible by identifying optimal experimental parameters. In this manner, we investigate representative examples of a transition metal oxide, a material consisting entirely of light elements, and an interface between two different materials to find and compare acceptable ranges for sample thickness, acceleration voltage and electron dose for a scanning probe as well as for parallel illumination.

Speckle suppression in arbitrary parallel holographic illumination by the spatial frequency regaining method.

This Letter proposes a spatial frequency regaining method for parallel holographic illumination (SFR-PHI) to suppress speckle noise in phase-only computer-generated holography (CGH). Based on the accurate calculation of the beam bandwidth, this method uses the bandwidth-limited quadratic initial phase and weighted constraint iteration to generate the optimized phase hologram, which can provide the accurate spatial frequency of multiple illumination patterns. The results show that SFR-PHI performs superiorly in speckle suppression for generating dozens of illumination patterns in parallel and with arbitrary shapes and numbers. Compared with other speckle-suppression methods, it exhibits significant advantages in terms of accuracy and modulation efficiency.

Parallel illumination for depletion microscopy through acousto-optic spatial light modulation

Several types of super-resolution microscopy, such as Stimulated Emission Depletion (STED), Reversible Saturable Optical Fluorescence Transitions (RESOLFT) or Switching Laser Mode (SLAM) microscopies, employ Laguerre-Gaussian beams (also called vortex or doughnut beams) to obtain fluorescence information within a sub-wavelength region of the specimen under observation, thus breaking the diffraction limit and producing images of greatly improved quality. However, in general, these techniques operate on a point-by-point basis, so we need to raster scan the sample in order to build a full, meaningful image, which takes time. Parallelization of the illumination is the only way to make these microscopies more suitable for live cell imaging applications. Here, we demonstrate the parallel production of arbitrary arrays of Gaussian and Laguerre-Gaussian lasers foci suitable for super-resolution microscopy, together with the possibility to fast scan through the sample, by means of acousto-optic spatial light modulation, a technique that we have pioneered in the past in several other fields.

Design and Research of Chromatic Confocal System for Parallel Non-Coaxial Illumination Based on Optical Fiber Bundle.

Conventional chromatic confocal systems are mostly single-point coaxial illumination systems with a low signal-to-noise ratio, light energy utility and measurement efficiency. To overcome the above shortcomings, we propose a parallel non-coaxial-illumination chromatic-confocal-measurement system based on an optical fiber bundle. Based on the existing single-point non-coaxial-illumination system, the optical fiber bundle is used as the optical beam splitter to achieve parallel measurements. Thus, the system can yield measurements through line scanning, which greatly improves measurement efficiency. To verify the measurement performance of the system, based on the calibration experiment, the system realizes the measurement of the height of the step, the thickness of the transparent specimen and the reconstruction of the three-dimensional topography of the surface of the step and coin. The experimental results show that the measuring range of the system is 200 μm. The measurement accurcy can reach micron level, and the system can realize a good three-dimensional topography reconstruction effect.

Frequency-resolved frozen phonon multislice method and its application to vibrational electron energy loss spectroscopy using parallel illumination

We explore the capabilities of the frequency-resolved frozen phonon multislice method introduced in Phys. Rev. Lett. 124, 025501 (2020) to model inelastic vibrational scattering in transmission electron microscopy. We review the method in detail and discuss advantages of using a so called hotspot thermostat instead of the $\delta$-thermostat used in our first report. We apply the method to simulate vibrational electron energy loss spectra of hexagonal boron nitride under plane wave illumination. Simulated spectroscopic information well represents the theoretical phonon bandstructure of the studied material, both in terms of energies as well as polarization vectors of individual phonon modes.

Protein and Organic-Molecular Crystallography With 300kV Electrons on a Direct Electron Detector.

Electron 3D crystallography can reveal the atomic structure from undersized crystals of various samples owing to the strong scattering power of electrons. Here, a direct electron detector DE64 was tested for small and thin crystals of protein and an organic molecule using a JEOL CRYO ARM 300 electron microscope. The microscope is equipped with a cold-field emission gun operated at an accelerating voltage of 300 kV, quad condenser lenses for parallel illumination, an in-column energy filter, and a stable rotational goniometer stage. Rotational diffraction data were collected in an unsupervised manner from crystals of a heme-binding enzyme catalase and a representative organic semiconductor material Ph-BTBT-C10. The structures were determined by molecular replacement for catalase and by the direct method for Ph-BTBT-C10. The analyses demonstrate that the system works well for electron 3D crystallography of these molecules with less damaging, a smaller point spread, and less noise than using the conventional scintillator-coupled camera.

Setting Up Parallel Illumination on the Talos Arctica for High-Resolution Data Collection.

Illuminating a specimen with a parallel electron beam is critical for many experiments in transmission electron microscopy as deviations from this condition cause considerable deterioration of image quality. Carefully establishing parallel illumination is particularly important on two-condenser lens transmission electron microscopes (TEMs) as the parallel illumination condition is limited to a single beam intensity value on these instruments. It was recently shown that a Thermo Fisher Scientific Talos Arctica, a two-condenser lens TEM operating at 200kV, equipped with a Gatan K2 Summit direct electron detector is capable of resolving frozen-hydrated macromolecules of various sizes and internal symmetries to better than 3Å resolution using single particle methodologies. A critical aspect of the success of these findings was the careful alignment of the electron microscope to ensure the specimen was illuminated with a parallel electron beam. Here, this chapter describes how to establish parallel illumination conditions in a Talos Arctica TEM for high-resolution cryogenic data collection for structure determination.

Advances in cryo-EM and ED with a cold-field emission beam and energy filtration -Refinements of the CRYO ARM 300 system in RIKEN SPring-8 center.

We have designed and evaluated a cryo-electron microscopy (cryo-EM) system for higher-resolution single particle analysis and high-precision electron 3D crystallography. The system comprises a JEOL CRYO ARM 300 electron microscope-the first machine of this model-and a direct detection device camera, a scintillator-coupled camera, GPU clusters connected with a camera control computer and software for automated-data collection and efficient and accurate operation. The microscope provides parallel illumination of a highly coherent 300-kV electron beam to a sample from a cold-field emission gun and filters out energy-loss electrons through the sample with an in-column energy filter. The gun and filter are highly effective in improving imaging and diffraction, respectively, and have provided high quality data since July 2018. We here report on the characteristics of the cryo-EM system, updates, our progress and future plan for running such cryo-EM machines in RIKEN SPring-8 Center.

Parallel Polarization Illumination with a Multifocal Axicon Metalens for Improved Polarization Imaging.

Polarization imaging is an important branch of the microscopy technique that can provide additional information and enhanced contrast. The illumination system of a polarization microscope enables many different polarizations but makes the setup bulky, complicated, and slow. Here, we design and fabricate an ultrathin planar axicon metalens that also enables parallel illumination with different polarizations. Our results reveal a diffraction-limited size and high degree of linear polarization. To verify our approach, we accurately map the polarization angle of an aluminum grating, which is used as a polarizer. Furthermore, we demonstrate that elliptical polarization can be generated without additional design. A single metalens has the same capabilities as a conventional illumination module containing a polarizer, compensator, and rotation-stage/optical modulator. In addition, our device has the potential to enable rapid super-resolution polarization imaging. The new method could be useful in many applications and areas, including, e.g., materials research and biomedicine.

Wettable photothermal hollow fibers arrays for efficient solar-driven desalination under omnidirectional illumination without salt precipitation

Solar-driven evaporation based on photothermal materials is emerging as a sustainable fresh water generation technology for alleviating water scarcity. To realize the efficient and stable desalination under omnidirectional illumination, here, a “Pinscreen”-inspired photothermal hollow fibers arrays has been developed for high-efficiency and stable solar-driven evaporation under omnidirectional illumination. In the photothermal arrays design, driven by the capillary action, water was automatically taken up through the lumen side of hydrophilic alumina hollow fiber coated with polypyrrole (PPy), which also shows high light-harvesting. Thanks to the designed morphology of arrays, the evaporation amount of PPy-AHFs arrays is over 0.65 g h−1 even under parallel one-sun illumination. During a long-term desalination, photothermal hollow fibers arrays can realize controllable salt-precipitation attributed to ideal water transport. Our results of wettable photothermal hollow fibers arrays for efficient photothermal evaporation can be considered as a significant attempt to inspire the future development of novel photothermal materials in sustainable desalination.

Tunable and switchable soft adsorption of polymer-coated microparticles on a flat substrate

Soft adhesion to a horizontal flat substrate of micron-sized colloids coated by a controlled molar fraction f of PLL-g-PNIPAM is tuned and triggered by controlling the temperature of the sample. This mechanism is possible because PNIPAM is a thermo-responsive polymer, which undergoes a phase transition at a LCST (lower critical solution temperature) Tc=32±1°C. In order to capture the very final events before the immobilization of colloids the T-ramp protocol is designed: the particles suspension is injected in cell at room temperature, the temperature is increased at a constant rate up to 38°C>Tc, and kept constant until the end of the acquisition. Attraction between beads and the flat substrate is thereby triggered when crossing the critical temperature Tc. Ascending and descending ramp experiments are performed in order to access both adsorption and desorption kinetics. 3-D motion of beads is real-time tracked using slightly defocused microscopy in parallel illumination. We use track records to have access to pre-adsorption diffusion coefficients and to characterize adsorption and desorption dynamics. Present results corroborate that adsorption is controlled by PNIPAM doping, and indeed dominated by rolling and memory (aging) effect on the contact domain. On the contrary, the desorption kinetics is independent of doping. Moreover, the sharpness (in T) of the PNIPAM transition is quantified in a set of experiments at different ramp speeds. We show that the transition smearing can reach up to ±1°C for higher PNIPAM coverage, while it is not detectable for the lowest ones. The combination of all these observations paves the way to practical applications. As an example, we discuss a soft adhesion-based method to sort and separate colloids in microfluidic channels.

Photoacoustic tomography for imaging the prostate: a transurethral illumination probe design and application

In vivo imaging of prostate cancer with photoacoustic tomography is currently limited by the lack of sufficient local fluence for deep tissue penetration and the risk of over-irradiation near the laser-tissue contact surface. We propose the design of a transurethral illumination probe that addresses those limitations. A high energy of 50 mJ/pulse is coupled into a 1000-µm-core diameter multimode fiber. A 2 cm diffusing end is fabricated, which delivers light in radial illumination. The radial illumination is then reflected and reshaped by a parabolic cylindrical mirror to obtain nearly parallel side illumination with a doubled fluence. The fiber assembly is housed in a 25 Fr cystoscope sheath to provide protection of the fiber and maintain a minimal laser-tissue contact distance of 5 mm. A large laser-tissue contact surface area of 4 cm2 is obtained and the fluence on the tissue surface is kept below the maximum permissible exposure. By imaging a prostate mimicking phantom, a penetration depth of 3.5 cm at 10 mJ/cm2 fluence and 700 nm wavelength is demonstrated. The results indicate that photoacoustic tomography with the proposed transurethral probe has the potential to image the entire prostate while satisfying the fluence maximum permissible exposure and delivering a high power to the tissue.

A new cryo-EM system for single particle analysis

A new cryo-EM system has been investigated for single particle analysis of protein structures. The system provides parallel illumination of a highly-coherent 300 kV electron beam from a cold-field emission gun, and boosts image contrast with an in-column energy filter and a hole-free phase plate. It includes motorized cryo-sample loading and automated liquid-nitrogen filling for cooling multiple samples. In this study, we describe gun and electron beam characteristics, and demonstrate the suitability of this system for single particle reconstructions. The performance of the system is tested on two examples, a spherical virus and apoferritin. GUI programs have also been developed to control and monitor the system for correct illumination, imaging with less ellipticity and steady magnification, and timing of flashing and liquid-nitrogen filling. These programs are especially useful for efficient application of the system to single particle cryo-EM.

A new cryo-EM system for electron 3D crystallography by eEFD

A new cryo-EM system has been developed and investigated for use in protein electron 3D crystallography. The system provides parallel illumination of a coherent 300 kV electron beam to a sample, filters out energy-loss electrons through the sample with an in-column energy filter, and allows rotational data collection on a fast camera. It also possesses motorized cryo-sample loading and automated liquid-nitrogen filling for cooling of multiple samples. To facilitate its use, we developed GUI programs for efficient operation and accurate structure analysis. Here we report on the performance of the system and first results for thin 3D crystals of the protein complexes, catalase and membrane protein complex ExbBD. Data quality is remarkably improved with this approach, which we name eEFD (electron energy-filtered diffraction of 3D crystals), compared with those collected at 200 kV without energy filtration. Key advances include precise control of the microscope and recordings of lens fluctuations, which the programs process and respond to. We also discuss the merits of higher-energy electrons and filtration of energy-loss electrons in electron 3D crystallography.