Instantaneous thermometry imaging using two-photon laser-induced fluorescence.

Measuring temperature in complex two-phase flows is crucial for understanding the dynamics of heat and mass transfer. In this Letter, we introduce a novel, to the best of our knowledge, optical approach based on the combination of two-photon laser-induced fluorescence (2p-LIF) imaging and two-color laser-induced fluorescence (2CLIF) for instantaneous temperature mapping of complex liquid media. Using Kiton Red (KR) and Rhodamine 560 (R560), a temperature sensitivity of 1.54%/∘C has been achieved over a range of 17-60°C. The monitoring of two-dimensional transient temperature dynamics in the heating and degassing of water shows the efficiency of the 2p-2CLIF. This new approach contributes to the toolkit of optical temperature measurement techniques, providing a robust solution for studying transient scattering media and high-speed two-phase flows.

High-Speed Fluorescence Imaging Corroborates Biological Data on the Influence of Different Nozzle Types on Cell Spray Viability and Formation.

Treating severe dermal disruptions often presents significant challenges. Recent advancements have explored biological cell sprays as a promising treatment, but their success hinges on efficient cell delivery and complete wound coverage. This requires a good spray distribution with a small droplet size, high particle number, and ample surface coverage. The type of nozzle used with the spray device can impact these parameters. To evaluate the influence of different nozzles on spray characteristics, we compared air-assisted and unassisted nozzles. The unassisted nozzle displayed small particle size, high particle number, good overall coverage, high cell viability, preserved cell metabolic activity, and low cytotoxicity. Air-assisted nozzles did not perform well regarding cell viability and metabolic activity. Flow visualization analysis comparing two different unassisted nozzles using high-speed imaging (100 kHz frame rate) revealed a tulip-shaped spray pattern, indicating optimal spray distribution. High-speed imaging showed differences between the unassisted nozzles. One unassisted nozzle displayed a bi-modal distribution of the droplet diameter while the other unassisted nozzle displayed a mono-modal distribution. These findings demonstrate the critical role of nozzle selection in successful cell delivery. A high-quality, certified nozzle manufactured for human application omits the need for an air-assisted nozzle and provides a simple system to use with similar or better performance characteristics than those of an air-assisted system.

Droplet sizing in atomizing sprays using polarization ratio with structured laser illumination planar imaging.

Previous research has shown that the polarization ratio technique allows the characterization of the surface mean diameter, D21, of droplets forming dilute sprays. However, its application to optically dense sprays has posed significant challenges due to the presence of multiple light scattering. Additionally, errors in measurement can arise from the angular dependence of the signal. In this Letter, we present a novel, to the best of our knowledge, method that addresses these challenges. Our approach combines the use of a telecentric objective with structured laser illumination, to both optimize light detection and suppress the unwanted intensity from multiple scattering. This approach enables the utilization of the polarization ratio technique for measuring the droplet size of challenging atomizing sprays. The method offers a promising solution for accurate and comprehensive spray characterization. It is applied, here, to a hollow-cone water spray running at 30, 50, and 70 bar injection pressure, reaching an optical depth up to three.

High-speed videography of transparent media using illumination-based multiplexed schlieren.

Schlieren photography is widely used for visualizing phenomena within transparent media. The technique, which comes in a variety of configurations, is based on detecting or extracting the degree to which light is deflected whilst propagating through a sample. To date, high-speed schlieren videography can only be achieved using high-speed cameras, thus limiting the frame rate of such configurations to the capabilities of the camera. Here we demonstrate, for the first time, optically multiplexed schlieren videography, a concept that allows such hardware limitations to be bypassed, opening up for, in principle, an unlimited frame rate. By illuminating the sample with a rapid burst of uniquely spatially modulated light pulses, a temporally resolved sequence can be captured in a single photograph. The refractive index variations are thereafter measured by quantifying the local phase shift of the superimposed intensity modulations. The presented results demonstrate the ability to acquire a series of images of flame structures at frame rates up to 1 Mfps using a standard 50 fps sCMOS camera.

High contrast, isotropic, and uniform 3D-imaging of centimeter-scale scattering samples using structured illumination light-sheet microscopy with axial sweeping.

Light-sheet fluorescent microscopy (LSFM) has, in recent years, allowed for rapid 3D-imaging of cleared biomedical samples at larger and larger scale. However, even in cleared samples, multiple light scattering often degrades the imaging contrast and widens the optical sectioning. Accumulation of scattering intensifies these negative effects as light propagates inside the tissue, which accentuates the issues when imaging large samples. With axially swept light-sheet microscopy (ASLM), centimeter-scale samples can be scanned with a uniform micrometric optical sectioning. But to fully utilize these benefits for 3D-imaging in biomedical tissue samples, suppression of scattered light is needed. Here, we address this by merging ASLM with light-sheet based structured illumination into Structured Illumination Light-sheet Microscopy with Axial Sweeping (SILMAS). The SILMAS method thus enables high-contrast imaging, isotropic micrometric resolution and uniform optical sectioning in centimeter-scale scattering samples, creating isotropic 3D-volumes of e.g., whole mouse brains without the need for any computation-heavy post-processing. We demonstrate the effectiveness of the approach in agarose gel phantoms with fluorescent beads, and in an PFF injected alpha-synuclein transgenic mouse model tagged with a green fluorescent protein (SynGFP). SILMAS imaging is compared to standard ASLM imaging on the same samples and using the same optical setup, and is shown to increase contrast by as much as 370% and reduce widening of optical sectioning by 74%. With these results, we show that SILMAS improves upon the performance of current state-of-the-art light-sheet microscopes for large and imperfectly cleared tissue samples and is a valuable addition to the LSFM family.

Transient Motion Classification Through Turbid Volumes via Parallelized Single-Photon Detection and Deep Contrastive Embedding.

Fast noninvasive probing of spatially varying decorrelating events, such as cerebral blood flow beneath the human skull, is an essential task in various scientific and clinical settings. One of the primary optical techniques used is diffuse correlation spectroscopy (DCS), whose classical implementation uses a single or few single-photon detectors, resulting in poor spatial localization accuracy and relatively low temporal resolution. Here, we propose a technique termed C lassifying R apid decorrelation E vents via P arallelized single photon d E tection (CREPE), a new form of DCS that can probe and classify different decorrelating movements hidden underneath turbid volume with high sensitivity using parallelized speckle detection from a 32 × 32 pixel SPAD array. We evaluate our setup by classifying different spatiotemporal-decorrelating patterns hidden beneath a 5 mm tissue-like phantom made with rapidly decorrelating dynamic scattering media. Twelve multi-mode fibers are used to collect scattered light from different positions on the surface of the tissue phantom. To validate our setup, we generate perturbed decorrelation patterns by both a digital micromirror device (DMD) modulated at multi-kilo-hertz rates, as well as a vessel phantom containing flowing fluid. Along with a deep contrastive learning algorithm that outperforms classic unsupervised learning methods, we demonstrate our approach can accurately detect and classify different transient decorrelation events (happening in 0.1-0.4 s) underneath turbid scattering media, without any data labeling. This has the potential to be applied to non-invasively monitor deep tissue motion patterns, for example identifying normal or abnormal cerebral blood flow events, at multi-Hertz rates within a compact and static detection probe.

Imaging Dynamics Beneath Turbid Media via Parallelized Single-Photon Detection.

Noninvasive optical imaging through dynamic scattering media has numerous important biomedical applications but still remains a challenging task. While standard diffuse imaging methods measure optical absorption or fluorescent emission, it is also well-established that the temporal correlation of scattered coherent light diffuses through tissue much like optical intensity. Few works to date, however, have aimed to experimentally measure and process such temporal correlation data to demonstrate deep-tissue video reconstruction of decorrelation dynamics. In this work, a single-photon avalanche diode array camera is utilized to simultaneously monitor the temporal dynamics of speckle fluctuations at the single-photon level from 12 different phantom tissue surface locations delivered via a customized fiber bundle array. Then a deep neural network is applied to convert the acquired single-photon measurements into video of scattering dynamics beneath rapidly decorrelating tissue phantoms. The ability to reconstruct images of transient (0.1-0.4 s) dynamic events occurring up to 8 mm beneath a decorrelating tissue phantom with millimeter-scale resolution is demonstrated, and it is highlighted how the model can flexibly extend to monitor flow speed within buried phantom vessels.

Multi-scattering software part II: experimental validation for the light intensity distribution.

This article, Part II of an article series on GPU-accelerated Monte Carlo simulation of photon transport through turbid media, focuses on the validation of the online software Multi-Scattering. While Part I detailed the implementation of the computational model, simulated and experimental results are now compared for the distribution of the scattered light intensity. The scattering phantoms prepared here are aqueous dispersions of polystyrene microspheres of diameter D = 0.5, 2 and 5 µm and at various concentrations, resulting in optical depth ranging from OD = 1 to 17.5. The Lorenz-Mie scattering phase functions used in the simulations have been verified experimentally at low particle concentrations by analyzing the angular light intensity distribution at the Fourier plane of a collecting lens. The validation approach herein accounts for the specific light collection and image formation by the camera. The front and side surfaces of the medium are imaged and the corresponding light intensity distributions are compared qualitatively and quantitatively. It is concluded that the model enables reliable simulations over the tested parameters, offering predictive simulations of transmitted intensities with a mean relative error ≤~19% over the full range. The online software is available at: https://multi-scattering.com/.

Transition from saliva droplets to solid aerosols in the context of COVID-19 spreading.

To control the evolution of a pandemic such as COVID-19, knowing the conditions under which the pathogen is being transmitted represents a critical issue, especially when implementing protection strategies such as social distancing and wearing face masks. For viruses and bacteria that spread via airborne and/or droplet pathways, this requires understanding how saliva droplets evolve over time after their expulsion by speaking or coughing. Within this context, the transition from saliva droplets to solid residues, due to water evaporation, is studied here both experimentally, considering the saliva from 5 men and 5 women, and via numerical modeling to accurately predict the dynamics of this process. The model assumes saliva to be a binary water/salt mixture and is validated against experimental results using saliva droplets that are suspended in an ultrasound levitator. We demonstrate that droplets with an initial diameter smaller than 21 µm will produce a solid residue that would be considered an aerosol of <5 µm diameter in less than 2 s (for any relative humidity less than 80% and/or any temperature greater than 20°C). Finally, the model developed here accounts for the influence of the saliva composition, relative humidity and ambient temperature on droplet drying. Thus, the travel distance prior to becoming a solid residue can be deduced. We found that saliva droplets of initial size below 80 µm, which corresponds to the vast majority of speech and cough droplets, will become solid residues prior to touching the ground when expelled from a height of 160 cm.

Stereoscopic high-speed imaging of iron microexplosions and nanoparticle-release.

In this work, the combustion behavior of seeded iron particles (d50 = 70 µm) in a laminar diffusion flame was studied in a modified Mckenna flat-flame burner. Two high speed cameras in stereo configuration allowed 3D position and 3D velocity measurements of burning iron particles as well as 3D evaluation of particle microexplosions. Microexplosive processes are important since it can affect both combustion stability and formation of product components. The observed microexplosions happened before particle extinction resulting in change of trajectories, velocities, radiation intensities and fragmentation into smaller particles. It was observed for the first time that fragments of these microexplosions tend to produce planar structures. A frequent release phenomenon was observed during the iron particle combustion using magnified thermal radiation imaging and high-speed shadowgraphy. This release phenomenon was indirectly confirmed with scanning electron microscopy of combust products, revealing multiple cracked particle shells and hollow structures. Black body radiation characteristics was observed indicating the release being in condensed phase and emission spectroscopy identified FeO as intermediate species during combustion. The observed release is believed to mainly consist of iron-oxide nanoparticles formed in the homogenous reaction between vapor iron and oxidizers.

Multi-Scattering software: part I: online accelerated Monte Carlo simulation of light transport through scattering media.

In this article we present and describe an online freely accessible software called Multi-Scattering for the modeling of light propagation in scattering and absorbing media. Part II of this article series focuses on the validation of the model by rigorously comparing the simulated results with experimental data. The model is based on the use of the Monte Carlo method, where billions of photon packets are being tracked through simulated cubic volumes. Simulations are accelerated by the use of general-purpose computing on graphics processing units, reducing the computation time by a factor up to 200x in comparison with a single central processing unit thread. By using four graphic cards on a single computer, the simulation speed increases by a factor of 800x. For an anisotropy factor g = 0.86, this enables the transport path of one billion photons to be computed in 10 seconds for optical depth OD = 10 and in 20 minutes for OD = 500. Another feature of Multi-Scattering is the integration and implementation of the Lorenz-Mie theory in the software to generate the scattering phase functions from spherical particles. The simulations are run from a computer server at Lund University, allowing researchers to log in and use it freely without any prior need for programming skills or specific software/hardware installations. There are countless types of scattering media in which this model can be used to predict light transport, including medical tissues, blood samples, clouds, smoke, fog, turbid liquids, spray systems, etc. An example of simulation results is given here for photon propagation through a piece of human head. The software also includes features for modeling image formation by inserting a virtual collecting lens and a detection matrix which simulate a camera objective and a sensor array respectively. The user interface for setting-up simulations and for displaying the corresponding results is found at: https://multi-scattering.com/.

Beyond MHz image recordings using LEDs and the FRAME concept.

Many important scientific questions in physics, chemistry and biology rely on high-speed optical imaging techniques for their investigations. These techniques are either passive, relying on the rapid readout of photoactive elements, or active, relying on the illumination properties of specially designed pulse trains. Currently, MHz imaging speeds are difficult to realize; passive methods, being dictated by electronics, cause the unification of high spatial resolution with high frame rates to be very challenging, while active methods rely on expensive and complex hardware such as femto- and picosecond laser sources. Here we present an accessible temporally resolved imaging system for shadowgraphy based on multiplexed LED illumination that is capable of producing four images at MHz frame rates. Furthermore as the LEDs are independent of each other, any light burst configuration can be obtained, allowing for instance the simultaneous determination of low- and high speed events in parallel. To the best of the authors' knowledge, this is the fastest high speed imaging system that does not rely on pulsed lasers or fast detectors, in this case reaching up to 4.56 MHz.

Snapshot 3D reconstruction of liquid surfaces.

In contrast to static objects, liquid structures such as drops, blobs, as well as waves and ripples on water surfaces are challenging to image in 3D due to two main reasons: first, the transient nature of those phenomena requires snapshot imaging that is fast enough to freeze the motion of the liquid. Second, the transparency of liquids and the specular reflections from their surfaces induce complex image artefacts. In this article we present a novel imaging approach to reconstruct in 3D the surface of irregular liquid structures that only requires a single snapshot. The technique is named Fringe Projection - Laser Induced Fluorescence (FP-LIF) and uses a high concentration of fluorescent dye in the probed liquid. By exciting this dye with a fringe projection structured laser beam, fluorescence is generated primarily at the liquid surface and imaged at a backward angle. By analysing the deformation of the initial projected fringes using phase-demodulation image post-processing, the 3D coordinates of the liquid surface are deduced. In this article, the approach is first numerically tested by considering a simulated pending drop, in order to analyse its performance. Then, FP-LIF is applied for two experimental cases: a quasi-static pending drop as well as a transient liquid sheet. We demonstrate reconstruction RMS errors of 1.4% and 6.1% for the simulated and experimental cases respectively. The technique presented here demonstrates, for the first time, a fringe projection approach based on LIF detection to reconstruct liquid surfaces in 3D. FP-LIF is promising for the study of more complex liquid structures and is paving the way for high-speed 3D videography of liquid surfaces.

3D mapping of droplet Sauter mean diameter in sprays.

In this study, we report on the three-dimensional (3D) characterization of a spray in terms of its droplet Sauter mean diameter (SMD) using the laser-induced fluorescence (LIF)/Mie ratio technique. The spray structure is analyzed for a multi-hole direct-injection spark ignition (DISI) injector. A calibration curve to convert the LIF/Mie ratio to droplet diameter is deduced using LIF/Mie imaging and analysis of single droplets generated by a droplet generator. The DISI spray investigated here is optically sectioned by means of two-phase structured laser illumination planar imaging to suppress the intensity of multiple light scattering from LIF and Mie images prior to their ratio. A series of calibrated LIF/Mie ratio images of spray is then recorded at several depths along the z direction following the light sheet scanning of the spray. The droplet SMD ranges from less than 5 µm up to a maximum of 50 µm in single-shot images. The averaged SMD results (1-30 µm) obtained by using the calibration curve from the droplet generator are compared with measurement results from phase-Doppler anemometry. Finally, a 3D map is reconstructed from the successive 2D layers generated from spray scanning. The resulting 3D representation of the droplet SMD shows a non-symmetric spray structure produced by the studied multi-hole injector, which cannot be resolved by analyzing only one central plane.

Crossed patterned structured illumination for the analysis and velocimetry of transient turbid media.

Imaging through turbid environments is experimentally challenging due to multiple light scattering. Structured laser illumination has proven to be effective to minimize errors arising from this phenomenon, allowing the interior of optically dense media to be observed. However, in order to preserve the image spatial resolution while suppressing the intensity contribution from multiple light scattering, the method relies on multiple acquisitions and thus sequential illumination. These requirements significantly limit the usefulness of structured illumination when imaging highly transient events. Here we present a method for achieving snapshot visualizations using structured illumination, where the spatial frequency domain is increased by a factor of two compared to past structured illumination snapshots. Our approach uses two crossed intensity-modulated patterns, allowing us to expand the spatial frequency response of the extracted data. The snapshot capability of this imaging approach allows tracking single particles and opens up for the extraction of velocity vectors by combining it with standard particle tracking/image velocimetry (PTV or PIV) equipment. In this paper we demonstrate the capabilities of this new method and, for the first time, use structured illumination to extract velocity vectors in 2D in a transient turbid medium, in this case an optically dense atomizing spray.

Light sheet fluorescence microscopic imaging for the primary breakup of diesel and gasoline sprays with real-world fuels.

This paper describes the adaptation of the laser-induced fluorescence measurement technique for the investigation of the primary breakup of modern diesel and gasoline direct injection sprays. To investigate the primary breakup, a microscopic technique is required, and with the help of special tracer dyes, a high fluorescence signal can be achieved in the visible range of the electromagnetic spectrum, resulting in good image quality with a nonintensified camera. Besides the optimization of the optical setup for the microscopic field of view, different tracer dyes are compared, and their solubility and fluorescence are tested in the desired surrogate and real-world fuels. As a tracer, the phenoxazine dye Nile Red was found to provide sufficient solubility in alkanes as well as suitable emission and excitation spectrum for the use of the second-harmonic frequency of a Nd:YAG laser (532 nm). The good quantum efficiency delivered by Nile Red also meant that single-shot images clearly showing spray structures in regions measuring up to 3 mm by 3 mm around the nozzle outlet could be recorded. Compared to relatively easy shadowgraph techniques and complex and costly x-ray synchrotron measurements, light sheet fluorescence microscopic imaging is not overly complex yet delivers excellent data on spray structures as well as qualitative fuel distribution.

Analysis of LIF and Mie signals from single micrometric droplets for instantaneous droplet sizing in sprays.

Planar droplet sizing (PDS) is a technique relying on the assumption that laser-induced fluorescence (LIF) and Mie scattering optical signals from spherical droplets depend on their volume and surface area, respectively. In this article, we verify the validity of this assumption by experimentally analyzing the light intensity of the LIF and Mie optical signals from micrometric droplets as a function of their diameter. The size of the droplets is controlled using a new flow-focusing monodisperse droplet generator capable of producing droplets of the desired size in the range of 21 µm to 60 µm. Ethanol droplets doped with eosin dye and excited at 532 nm are considered in this study, and the individual droplets were imaged simultaneously at microscopic and macroscopic scale. The effects of laser power, dye concentration, and temperature variation are systematically studied as a function of LIF/Mie ratio in the whole range of droplet sizes. Finally, a calibration curve at tracer concentration of 0.5 vol% is deduced and used to extract the droplet Sauter mean diameter (SMD) from instantaneous images of a transient ethanol spray. This droplet size mapping is done using structured laser illumination planar imaging (SLIPI), in order to suppress the artifacts induced by multiple light scattering.

Quantitative measurements of turbid liquids via structured laser illumination planar imaging where absorption spectrophotometry fails.

A comparison between the commonly used absorption spectrophotometry and a more recent approach known as structured laser illumination planar imaging (SLIPI) is presented for the characterization of scattering and absorbing liquids. Water solutions of milk and coffee are, respectively, investigated for 10 different levels of turbidity. For the milk solutions, scattering is the dominant process, while the coffee solutions have a high level of absorption. Measurements of the extinction coefficient are performed at both λ=450 nm and λ=638 nm and the ratio of their values has been extracted. We show that the turbidity limit of valid transmission measurements is reached at an optical depth of OD∼2.4, corresponding here to an extinction coefficient of µe=0.60 mm-1 when using a modern absorption spectrometer having a spatial Fourier filter prior to detection. Above this value, errors are induced due to the contribution of scattered and multiply scattered photons reaching the detector. On the contrary, the SLIPI measurements were found to be very reliable, even for an extinction coefficient three times as high, where µe=1.80 mm-1. This improvement is due to the capability of the technique in efficiently suppressing the contribution from multiple light scattering.

High dynamic spectroscopy using a digital micromirror device and periodic shadowing.

We present an optical solution called DMD-PS to boost the dynamic range of 2D imaging spectroscopic measurements up to 22 bits by incorporating a digital micromirror device (DMD) prior to detection in combination with the periodic shadowing (PS) approach. In contrast to high dynamic range (HDR), where the dynamic range is increased by recording several images at different exposure times, the current approach has the potential of improving the dynamic range from a single exposure and without saturation of the CCD sensor. In the procedure, the spectrum is imaged onto the DMD that selectively reduces the reflection from the intense spectral lines, allowing the signal from the weaker lines to be increased by a factor of 28 via longer exposure times, higher camera gains or increased laser power. This manipulation of the spectrum can either be based on a priori knowledge of the spectrum or by first performing a calibration measurement to sense the intensity distribution. The resulting benefits in detection sensitivity come, however, at the cost of strong generation of interfering stray light. To solve this issue the Periodic Shadowing technique, which is based on spatial light modulation, is also employed. In this proof-of-concept article we describe the full methodology of DMD-PS and demonstrate - using the calibration-based concept - an improvement in dynamic range by a factor of ~100 over conventional imaging spectroscopy. The dynamic range of the presented approach will directly benefit from future technological development of DMDs and camera sensors.

FRAME: femtosecond videography for atomic and molecular dynamics.

Many important scientific questions in physics, chemistry and biology require effective methodologies to spectroscopically probe ultrafast intra- and inter-atomic/molecular dynamics. However, current methods that extend into the femtosecond regime are capable of only point measurements or single-snapshot visualizations and thus lack the capability to perform ultrafast spectroscopic videography of dynamic single events. Here we present a laser-probe-based method that enables two-dimensional videography at ultrafast timescales (femtosecond and shorter) of single, non-repetitive events. The method is based on superimposing a structural code onto the illumination to encrypt a single event, which is then deciphered in a post-processing step. This coding strategy enables laser probing with arbitrary wavelengths/bandwidths to collect signals with indiscriminate spectral information, thus allowing for ultrafast videography with full spectroscopic capability. To demonstrate the high temporal resolution of our method, we present videography of light propagation with record high 200 femtosecond temporal resolution. The method is widely applicable for studying a multitude of dynamical processes in physics, chemistry and biology over a wide range of time scales. Because the minimum frame separation (temporal resolution) is dictated by only the laser pulse duration, attosecond-laser technology may further increase video rates by several orders of magnitude.