Iterative tomography with digital adaptive optics permits hour-long intravital observation of 3D subcellular dynamics at millisecond scale.

Long-term subcellular intravital imaging in mammals is vital to study diverse intercellular behaviors and organelle functions during native physiological processes. However, optical heterogeneity, tissue opacity, and phototoxicity pose great challenges. Here, we propose a computational imaging framework, termed digital adaptive optics scanning light-field mutual iterative tomography (DAOSLIMIT), featuring high-speed, high-resolution 3D imaging, tiled wavefront correction, and low phototoxicity with a compact system. By tomographic imaging of the entire volume simultaneously, we obtained volumetric imaging across 225 × 225 × 16 µm3, with a resolution of up to 220 nm laterally and 400 nm axially, at the millisecond scale, over hundreds of thousands of time points. To establish the capabilities, we investigated large-scale cell migration and neural activities in different species and observed various subcellular dynamics in mammals during neutrophil migration and tumor cell circulation.

Visualizing Intracellular Organelle and Cytoskeletal Interactions at Nanoscale Resolution on Millisecond Timescales.

In eukaryotic cells, organelles and the cytoskeleton undergo highly dynamic yet organized interactions capable of orchestrating complex cellular functions. Visualizing these interactions requires noninvasive, long-duration imaging of the intracellular environment at high spatiotemporal resolution and low background. To achieve these normally opposing goals, we developed grazing incidence structured illumination microscopy (GI-SIM) that is capable of imaging dynamic events near the basal cell cortex at 97-nm resolution and 266 frames/s over thousands of time points. We employed multi-color GI-SIM to characterize the fast dynamic interactions of diverse organelles and the cytoskeleton, shedding new light on the complex behaviors of these structures. Precise measurements of microtubule growth or shrinkage events helped distinguish among models of microtubule dynamic instability. Analysis of endoplasmic reticulum (ER) interactions with other organelles or microtubules uncovered new ER remodeling mechanisms, such as hitchhiking of the ER on motile organelles. Finally, ER-mitochondria contact sites were found to promote both mitochondrial fission and fusion.

Real-time single-molecule observation of incipient collagen fibrillogenesis and remodeling.

The hierarchic assembly of fibrillar collagen into an extensive and ordered supramolecular protein fibril is critical for extracellular matrix function and tissue mechanics. Despite decades of study, we still know very little about the complex process of fibrillogenesis, particularly at the earliest stages where observation of rapidly forming, nanoscale intermediates challenges the spatial and temporal resolution of most existing microscopy methods. Using video rate scanning atomic force microscopy (VRS-AFM), we can observe details of the first few minutes of collagen fibril formation and growth on a mica surface in solution. A defining feature of fibrillar collagens is a 67-nm periodic banding along the fibril driven by the organized assembly of individual monomers over multiple length scales. VRS-AFM videos show the concurrent growth and maturation of small fibrils from an initial uniform height to structures that display the canonical banding within seconds. Fibrils grow in a primarily unidirectional manner, with frayed ends of the growing tip latching onto adjacent fibrils. We find that, even at extremely early time points, remodeling of growing fibrils proceeds through bird-caging intermediates and propose that these dynamics may provide a pathway to mature hierarchic assembly. VRS-AFM provides a unique glimpse into the early emergence of banding and pathways for remodeling of the supramolecular assembly of collagen during the inception of fibrillogenesis.

Indirect detector for ultra-high-speed X-ray micro-imaging with increased sensitivity to near-ultraviolet scintillator emission.

Ultra-high-speed synchrotron-based hard X-ray (i.e. above 10 keV) imaging is gaining a growing interest in a number of scientific domains for tracking non-repeatable dynamic phenomena at spatio-temporal microscales. This work describes an optimized indirect X-ray imaging microscope designed to achieve high performance at micrometre pixel size and megahertz acquisition speed. The entire detector optical arrangement has an improved sensitivity within the near-ultraviolet (NUV) part of the emitted spectrum (i.e. 310-430 nm wavelength). When combined with a single-crystal fast-decay scintillator, such as LYSO:Ce (Lu2-xYxSiO5:Ce), it exploits the potential of the NUV light-emitting scintillators. The indirect arrangement of the detector makes it suitable for high-dose applications that require high-energy illumination. This allows for synchrotron single-bunch hard X-ray imaging to be performed with improved true spatial resolution, as herein exemplified through pulsed wire explosion and superheated near-nozzle gasoline injection experiments at a pixel size of 3.2 µm, acquisition rates up to 1.4 MHz and effective exposure time down to 60 ps.

Advancements in fluorescence lifetime imaging microscopy Instrumentation: Towards high speed and 3D.

Fluorescence lifetime imaging microscopy (FLIM) is a powerful imaging tool offering molecular specific insights into samples through the measurement of fluorescence decay time, with promising applications in diverse research fields. However, to acquire two-dimensional lifetime images, conventional FLIM relies on extensive scanning in both the spatial and temporal domain, resulting in much slower acquisition rates compared to intensity-based approaches. This problem is further magnified in three-dimensional imaging, as it necessitates additional scanning along the depth axis. Recent advancements have aimed to enhance the speed and three-dimensional imaging capabilities of FLIM. This review explores the progress made in addressing these challenges and discusses potential directions for future developments in FLIM instrumentation.

Ultra-fast Digital DPC Yielding High Spatio-temporal Resolution for Low-Dose Phase Characterization.

In the scanning transmission electron microscope, both phase imaging of beam-sensitive materials and characterization of a material's functional properties using in situ experiments are becoming more widely available. As the practicable scan speed of 4D-STEM detectors improves, so too does the temporal resolution achievable for both differential phase contrast (DPC) and ptychography. However, the read-out burden of pixelated detectors, and the size of the gigabyte to terabyte sized data sets, remain a challenge for both temporal resolution and their practical adoption. In this work, we combine ultra-fast scan coils and detector signal digitization to show that a high-fidelity DPC phase reconstruction can be achieved from an annular segmented detector. Unlike conventional analog data phase reconstructions from digitized DPC-segment images yield reliable data, even at the fastest scan speeds. Finally, dose fractionation by fast scanning and multi-framing allows for postprocess binning of frame streams to balance signal-to-noise ratio and temporal resolution for low-dose phase imaging for in situ experiments.

Quantifying Agglomerate-to-Wall Impaction in Dry Powder Inhalers.

PURPOSE: The probability of agglomerate-to-wall collision was quantified using a unique image processing technique applied to high-speed microscopic images. The study aimed to investigate the effects of flow rate and particle size on the percentage of colliding agglomerates detected within an in-house powder dispersion device. METHOD: The device consists of a swirl chamber and two tangential inlets in various configurations, designed to emulate the geometric features of commercial devices such as the Aerolizer® and Osmohaler®. The test cases were conducted with constant flow rates of 30 SLPM and 60 SLPM. Four powder samples were tested, including carrier Respitose® SV010 (median volume diameter 104 µm, span 1.7) and mannitol of three constituent primary particle sizes (3 µm, 5 µm and 7 µm; span 1.6 - 1.9). RESULTS: At the lower flow rate of 30 SLPM, collision frequencies were significantly different between powders of different constituent particle sizes, but the effects of powder properties diminished on increasing the flow rate to 60 SLPM. At the higher flow rate, all powders experienced a significant increase in the proportion of colliding particles. CONCLUSION: Analysis of collision events showed that the probability of collision for each agglomerate increased with agglomerate diameter and velocity. Experimental data of agglomerate-to-wall collision were utilised to develop a logistic regression model that can accurately predict collisions with various powders and flow rates.

How Fast is Your Detector? The Effect of Temporal Response on Image Quality.

With increasing interest in high-speed imaging, there should be an increased interest in the response times of our scanning transmission electron microscope detectors. Previous works have highlighted and contrasted the performance of various detectors for quantitative compositional or structural studies, but here, we shift the focus to detector temporal response, and the effect this has on captured images. The rise and decay times of eight detectors' single-electron response are reported, as well as measurements of their flatness, roundness, smoothness, and ellipticity. We develop and apply a methodology for incorporating the temporal detector response into simulations, showing that a loss of resolution is apparent in both the images and their Fourier transforms. We conclude that the solid-state detector outperforms the photomultiplier tube-based detectors in all areas bar a slightly less elliptical central hole and is likely the best detector to use for the majority of applications. However, using the tools introduced here, we encourage users to effectively evaluate which detector is most suitable for their experimental needs.

A Review on Methods for Measurement of Free Water Surface.

Turbulent free-surface flows are encountered in several engineering applications and are typically characterized by the entrainment of air bubbles due to intense mixing and surface deformation. The resulting complex multiphase structure of the air-water interface presents a challenge in precise and reliable measurements of the free-water-surface topography. Conventional methods by manometers, wave probes, point gauges or electromagnetic/ultrasonic devices are proven and reliable, but also time-consuming, with limited accuracy and are mostly intrusive. Accurate spatial and temporal measurements of complex three-dimensional free-surface flows in natural and man-made hydraulic structures are only viable by high-resolution non-contact methods, namely, LIDAR-based laser scanning, photogrammetric reconstruction from cameras with overlapping field of view, or laser triangulation that combines laser ranging with high-speed imaging data. In the absence of seeding particles and optical calibration targets, sufficient flow aeration is essential for the operation of both laser- and photogrammetry-based methods, with local aeration properties significantly affecting the measurement uncertainty of laser-based methods.

Shedding Light on Gas-Dynamic Effects in Laser Beam Fusion Cutting: The Potential of Background-Oriented Schlieren Imaging (BOS).

In laser beam fusion cutting of metals, the interaction of the gas jet with the melt determines the dynamics of the melt extrusion and the quality of the resulting cutting kerf. The gas-dynamic phenomena occurring during laser beam cutting are not fully known, especially regarding temporal fluctuations in the gas jet. The observation of gas and melt dynamics is difficult because the gas flow is not directly visible in video recordings and access to the process zone for observation is limited. In this study, the problem of imaging the gas jet from the cutting nozzle is addressed in a novel way by utilizing the striation pattern formed at the cutting kerf as a background pattern for background-oriented Schlieren imaging (BOS). In this first feasibility study, jets of different gas nozzles were observed in front of a solidified cutting kerf, which served as a background pattern for imaging. The results show that imaging of the characteristic shock diamonds of cutting nozzles is possible. Furthermore, the resulting shock fronts from an interaction of the gas jet with a model of a cutting front can be observed. The possibility of high-speed BOS with the proposed method is shown, which could be suitable to extend the knowledge of gas-dynamic phenomena in laser beam fusion cutting.

Irrigant flow in the root canal during ultrasonic activation: A numerical fluid-structure interaction model and its validation.

AIM: The aim of the study was (a) to develop a three-dimensional numerical model combining the oscillation of a tapered ultrasonic file and the induced irrigant flow along with their two-way interaction in the confinement of a root canal. (b) To validate this model through comparison with experiments and theoretical (analytical) solutions of the flow. METHODOLOGY: Two partial numerical models, one for the oscillation of the ultrasonic file and another one for the irrigant flow inside the root canal around the file, were created and coupled in order to take into account the two-way coupled fluid-structure interaction. Simulations were carried out for ultrasonic K-files and for smooth wires driven at four different amplitudes in air or inside an irrigant-filled straight root canal. The oscillation pattern of the K-files was determined experimentally by Scanning Laser Vibrometry, and the flow pattern inside an artificial root canal was analysed using high-speed imaging together with Particle Image Velocimetry. Analytical solutions were obtained from an earlier study. Numerical, experimental and analytical results were compared to assess the validity of the model. RESULTS: The comparison of the oscillation amplitude and node location of the ultrasonic files and of the irrigant flow field showed a close agreement between the simulations, experiments and theoretical solutions. CONCLUSIONS: The model is able to predict reliably the file oscillation and irrigant flow inside root canals during ultrasonic activation under similar conditions.

Explaining the working mechanism of laser-activated irrigation and its action on microbial biofilms: A high-speed imaging study.

AIM: Laser-activated irrigation (LAI) using pulsed erbium lasers has been studied with regard to canal cleaning, but its working mechanism remains poorly understood. This study sought to unravel the method of action of LAI and to assess its effect on bacterial biofilms in a root canal model, by means of high-speed imaging. METHODOLOGY: A root canal model consisting of dentine and glass walls was used. Visualization of the canal space during activation was achieved with a high-speed camera, capturing 20-s activation series at 50 000 frames per second. Recordings were made of canal models filled with water, models filled with water containing glass microspheres, and models with a biofilm (an undefined biofilm originating from oral samples, a 1-week-old Enterococcus faecalis biofilm or a 11-day-old multispecies biofilm) grown on the dentine walls. LAI parameters were 2940 nm, 15 Hz, 50 µs, 20 mJ and 400 µm conical tip held at orifice level. Quantitative (measurement of size, life time and timing of cavitation bubbles; velocity and amplitude of root canal content movement) and qualitative (descriptive) analysis of the intracanal events was performed using imaging software. RESULTS: During the implosion of the primary bubble, smaller cavitation bubbles emerged throughout the entire canal. This process began in the coronal canal part and continued in the apical direction. Expansion of these bubbles was followed by an implosion, and this volumetric change over a time span of a few 100 µs resulted in a very rapid vertical movement of the canal content with a mean amplitude of 900 µm. The succession of these movements with every pulse, resulted in biofilm detachment from the root canal walls and the gradual displacement of fragments coronally, until their complete removal. The pattern of the biofilm removal was the same for all groups. LAI was able to remove biofilm from the root canal models. CONCLUSIONS: The hydrodynamic effect of LAI is based on the generation of small cavitation bubbles throughout the entire canal, far from the primary bubble. Their volumetric oscillation results in a small yet very fast vertical movement of the root canal content and local liquid streaming on each pulse, resulting in biofilm detachment and coronal displacement.

Comparison of High-Speed Polarization Imaging Methods for Biological Tissues.

We applied a polarization filter array and high-speed camera to the imaging of biological tissues during large, dynamic deformations at 7000 frames per second. The results are compared to previous measurements of similar specimens using a rotating polarizer imaging system. The polarization filter eliminates motion blur and temporal bias from the reconstructed collagen fiber alignment angle and retardation images. The polarization imaging configuration dose pose additional challenges due to the need for calibration of the polarization filter array for a given sample in the same lighting conditions as during the measurement.

A Dual-Mode 303-Megaframes-per-Second Charge-Domain Time-Compressive Computational CMOS Image Sensor.

An ultra-high-speed computational CMOS image sensor with a burst frame rate of 303 megaframes per second, which is the fastest among the solid-state image sensors, to our knowledge, is demonstrated. This image sensor is compatible with ordinary single-aperture lenses and can operate in dual modes, such as single-event filming mode or multi-exposure imaging mode, by reconfiguring the number of exposure cycles. To realize this frame rate, the charge modulator drivers were adequately designed to suppress the peak driving current taking advantage of the operational constraint of the multi-tap charge modulator. The pixel array is composed of macropixels with 2 × 2 4-tap subpixels. Because temporal compressive sensing is performed in the charge domain without any analog circuit, ultrafast frame rates, small pixel size, low noise, and low power consumption are achieved. In the experiments, single-event imaging of plasma emission in laser processing and multi-exposure transient imaging of light reflections to extend the depth range and to decompose multiple reflections for time-of-flight (TOF) depth imaging with a compression ratio of 8× were demonstrated. Time-resolved images similar to those obtained by the direct-type TOF were reproduced in a single shot, while the charge modulator for the indirect TOF was utilized.

Capturing the Influence of Jet Fluctuations on Particles in Plasma Spraying.

Instabilities and fluctuations of the plasma jet in a thermal spray process can have a significant influence on the particle in-flight temperatures and velocities, affecting the properties of resulting plasma-sprayed coatings. Presented in this paper is a novel method for capturing the effects particles are exposed to in the plasma spraying process. High-speed camera images of a plasma jet generated by a cascaded three-cathode plasma generator (TriplexPro-210) were recorded for varying operating conditions. The images are processed using the inverse Abel transform. This transformation accounts for the fact that the images represent a 2D projection of the 3D jet and generates more accurate intensity values that the sprayed particles would experience. These images are then combined with particle tracks resulting from CFD simulations of the plasma jet to match the particles path with the recorded plasma jet. This new method allows a precise description of the plasma intensity experienced by individual particles with a high temporal resolution. The results show a high sensitivity of the method, even detecting the influence on the particles of the plasma jet originating from the cascaded triple arc plasma generator, which is considered as rather stable.

Jumping in lantern bugs (Hemiptera, Fulgoridae).

Lantern bugs are amongst the largest of the jumping hemipteran bugs, with body lengths reaching 44 mm and masses reaching 0.7 g. They are up to 600 times heavier than smaller hemipterans that jump powerfully using catapult mechanisms to store energy. Does a similar mechanism also propel jumping in these much larger insects? The jumping performance of two species of lantern bugs (Hemiptera, Auchenorrhyncha, family Fulgoridae) from India and Malaysia was therefore analysed from high-speed videos. The kinematics showed that jumps were propelled by rapid and synchronous movements of both hind legs, with their trochantera moving first. The hind legs were 20-40% longer than the front legs, which was attributable to longer tibiae. It took 5-6 ms to accelerate to take-off velocities reaching 4.65 m s-1 in the best jumps by female Kalidasa lanata. During these jumps, adults experienced an acceleration of 77 g, required an energy expenditure of 4800 µJ and a power output of 900 mW, and exerted a force of 400 mN. The required power output of the thoracic jumping muscles was 21,000 W kg-1, 40 times greater than the maximum active contractile limit of muscle. Such a jumping performance therefore required a power amplification mechanism with energy storage in advance of the movement, as in their smaller relatives. These large lantern bugs are near isometrically scaled-up versions of their smaller relatives, still achieve comparable, if not higher, take-off velocities, and outperform other large jumping insects such as grasshoppers.

Short Time Correlation Analysis of Melt Pool Behavior in Laser Metal Deposition Using Coaxial Optical Monitoring.

The development and improvement of monitoring and process control systems is one of the important ways of advancing laser metal deposition (LMD). The control of hydrodynamic, heat and mass transfer processes in LMD is extremely important, since these processes directly affect the crystallization of the melt and, accordingly, the microstructural properties and the overall quality of the synthesized part. In this article, the data of coaxial video monitoring of the LMD process were used to assess the features of melt dynamics. The obtained images were used to calculate the time dependences of the characteristics of the melt pool (MP) (temperature, width, length and area), which were further processed using the short-time correlation (STC) method. This approach made it possible to reveal local features of the joint behavior of the MP characteristics, and to analyze the nature of the melt dynamics. It was found that the behavior of the melt in the LMD is characterized by the presence of many time periods (patterns), during which it retains a certain ordered character. The features of behavior that are important from the point of view of process control systems design are noted. The approach used for the analysis of melt dynamics based on STC distributions of MP characteristics, as well as the method for determining the moments of pattern termination through the calculation of the correlation power, can be used in processing the results of online LMD diagnostics, as well as in process control systems.

Swimming kinematics and hydrodynamics of barnacle larvae throughout development.

Changes in size strongly influence organisms' ecological performances. For aquatic organisms, they can transition from viscosity- to inertia-dominated fluid regimes as they grow. Such transitions are often associated with changes in morphology, swimming speed and kinematics. Barnacles do not fit into this norm as they have two morphologically distinct planktonic larval phases that swim differently but are of comparable sizes and operate in the same fluid regime (Reynolds number 100-101). We quantified the hydrodynamics of the rocky intertidal stalked barnacle Capitulum mitella from the nauplius II to cyprid stage and examined how kinematics and size increases affect its swimming performance. Cyprids beat their appendages in a metachronal wave to swim faster, more smoothly, and with less backwards slip per beat cycle than did all naupliar stages. Micro-particle image velocimetry showed that cyprids generated trailing viscous vortex rings that pushed water backwards for propulsion, contrary to the nauplii's forward suction current for particle capture. Our observations highlight that zooplankton swimming performance can shift via morphological and kinematic modifications without a significant size increase. The divergence in ecological functions through ontogeny in barnacles and the removal of feeding requirement likely contributed to the evolution of the specialized, taxonomically unique cyprid phase.

Effects of Flow-Induced Microfluidic Chip Wall Deformation on Imaging Flow Cytometry.

Imaging flow cytometry is a powerful tool by virtue of its capability for high-throughput cell analysis. The advent of high-speed optical imaging methods on a microfluidic platform has significantly improved cell throughput and brought many degrees of freedom to instrumentation and applications over the last decade, but it also poses a predicament on microfluidic chips. Specifically, as the throughput increases, the flow speed also increases (currently reaching 10 m/s): consequently, the increased hydrodynamic pressure on the microfluidic chip deforms the wall of the microchannel and produces detrimental effects lead to defocused and blur image. Here, we present a comprehensive study of the effects of flow-induced microfluidic chip wall deformation on imaging flow cytometry. We fabricated three types of microfluidic chips with the same geometry and different degrees of stiffness made of polydimethylsiloxane (PDMS) and glass to investigate material influence on image quality. First, we found the maximum deformation of a PDMS microchannel was >60 µm at a pressure of 0.6 MPa, while no appreciable deformation was identified in a glass microchannel at the same pressure. Second, we found the deviation of lag time that indicating velocity difference of migrating microbeads due to the deformation of the microchannel was 29.3 ms in a PDMS microchannel and 14.9 ms in a glass microchannel. Third, the glass microchannel focused cells into a slightly narrower stream in the X-Y plane and a significantly narrower stream in the Z-axis direction (focusing percentages were increased 30%, 32%, and 5.7% in the glass channel at flow velocities of 0.5, 1.5, and 3 m/s, respectively), and the glass microchannel showed stabler equilibrium positions of focused cells regardless of flow velocity. Finally, we achieved the world's fastest imaging flow cytometry by combining a glass microfluidic device with an optofluidic time-stretch microscopy imaging technique at a flow velocity of 25 m/s. © 2019 International Society for Advancement of Cytometry.

Do the enlarged hind legs of male thick-legged flower beetles contribute to take-off or mating?

The volume of the hind femora in the adult male flower beetle Oedemera nobilis is 38 times greater than in adult females. To determine what advantage limbs with swollen femora might provide, the behaviour of these insects was analysed with high-speed videography. First, because large hind legs are often associated with jumping and take-off, the performance of this behaviour by the two sexes was determined. Take-off was generated by a series of small-amplitude wing beats followed by larger ones, with the hind legs contributing little or no propulsion. The mean acceleration time to take-off was not significantly different in males (46.2 ms) and females (45.5 ms), but the mean take-off velocity of males was 10% higher than in females. Second, to determine if enlarged hind legs were critical in specifically male behaviour, interactions between males and females, and between males were videoed. The male mounted a female and then encircled her abdomen between the enlarged femora and tibiae of both his hind legs. The joint between these leg parts acted like a mole wrench (vice grip) so that when the tibia was fully flexed, a triangular space of 0.3 mm2 remained, in which a female abdomen (cross-sectional area 0.9 mm2) could be compressed and restrained firmly without inflicting damage. The flexor tibiae muscle in a male hind femur was 5.9 times larger than the extensor. In interactions between males, attempts to achieve a similar entrapment were frequently thwarted by the pursued male extending his hind legs vertically.