Single Particle Counting Research Articles

Determination of the response for the National Ignition Facility particle time of flight (PTOF) detector using single particle counting.

The Particle Time of Flight (PTOF) detector is a chemical vapor deposition diamond-based detector used to measure bang times in low-yield (≲ 1015 neutrons) experiments at the National Ignition Facility (NIF) at Lawrence Livermore National Laboratory (LLNL). Historically, the impulse response for PTOF diamond detectors has been obtained from x-ray timing shots on the NIF and shots on the MegaRay pulsed electron accelerator at LLNL. The impulse response may alternatively be obtained using single particle interactions with the detector, at substantially less cost and higher frequency compared to NIF timing shots, which typically occur months apart. Here, the response of a PTOF detector setup is characterized by statistically averaging a large number of single particle waveforms. A high fidelity instrument response function can be constructed in this way. This is confirmed by comparison of the single particle counting-constructed response to the impulse response function measured for the same detector at LLNL's MegaRay facility.

Vertical concentrations gradients and transport of airborne microplastics in wind tunnel experiments

Abstract. Microplastics are an ubiquitous anthropogenic material in the environment, including the atmosphere. Little work has focused on the atmospheric transport mechanisms of microplastic nor its dispersion, despite it being a potential pollutant. We study the vertical transport of airborne microplastics in a wind tunnel, a controllable environment with neutral stability, to identify the necessary conditions for the long-range atmospheric transport of microplastics. An ultrasonic disperser generated airborne water droplets from a suspension of polystyrene microsphere microplastics (MPs) with a diameter of 0.51 µm. The water droplets were injected into the airflow, evaporating and releasing single airborne MPs. The disperser allowed for time-invariant and user-controlled concentrations of MPs in the wind tunnel. MPs were injected at 27, 57, and 255 mm above the ground. A single GRIMM R11 optical particle counter (OPC) and three Alphasense OPCs measured time-averaged MP concentration profiles (27, 57, and 157 mm above the ground). These were combined with turbulent airflow characteristics measured by a hotwire probe to estimate vertical particle fluxes using the flux-gradient similarity theory. The GRIMM R11 OPC measured vertical concentration profiles by moving its sampling tube vertically. The three Alphasense OPCs measured particle concentrations simultaneously at three distinct heights. Results show that maximum concentrations are not measured at the injection height but are rather shifted to the surface by gravitational settling. The MPs experience higher gravitational settling while they are part of the larger water droplets. For the lowest injection at 27 mm, the settling leads to smaller MP concentrations in the wind tunnel, as MPs are lost to deposition. Increasing the wind speed decreases the loss of MPs by settling, but settling is present until our maximum friction velocity of 0.14 m s−1. For the highest injection at 255 mm and laminar flow, the settling resulted in a net MP emission, challenging the expectation of a net MP deposition for high injection. Turbulent flows reverse the MP concentration profile giving a net MP deposition with deposition velocities of 3.7 ± 1.9 cm s−1. Recognizing that microplastics share deposition velocities with mineral particles bridges the gap in understanding their environmental behavior. The result supports the use of existing models to evaluate the transport of microplastics in the accumulation mode. The similar deposition velocities suggest that microplastics transported in the atmosphere can be found in the same places as mineral particles.

A portable deep-learning-assisted digital single-particle counting biosensing platform for amplification-free nucleic acid detection using a lens-free holography microscope

The digital single-particle assay has emerged as a highly promising technology in various detection applications, such as food safety inspection, environmental monitoring, and in vitro diagnosis. Conventional digital assays rely on pre-amplification and expensive equipment, which limits their practical applications to point-of-care testing. Herein, we report a deep-learning-assisted digital single-particle counting biosensing platform for nucleic acid detection without pre-amplification using a portable and low-cost lens-free holography microscope. This device can perceive the number change of signal probes and capture microsphere probe holograms, which is ultra-lightweight (∼ 318 g), and has a low cost (∼ $70) and an ultrawide field of view of 24.396 mm2. The improved YOLOv7-based deep learning algorithm is trained to detect small objects (∼ 10 μm) in high-resolution images with high throughput. As a proof of concept, our strategy has successfully distinguished viable and nonviable Salmonella typhimurium quantitatively with high sensitivity (72 CFU/mL) without pre-amplification using phage-mediated DNA extraction and has been verified in various real samples. It has also been successfully applied to detecting non-nucleic acid targets in real samples, including procalcitonin and chloramphenicol. As a versatile and multi-functional platform, this platform exhibits excellent potential for point-of-care multi-type target detection in resource-limited settings.

High-concentration measurements with optical aerosol spectrometers by signal fluctuation analysis

Optical aerosol measurement systems can be classified into single-particle counting techniques and ensemble techniques. In single-particle counting instruments, such as optical aerosol spectrometers, each particle is analyzed individually. This allows for the accurate measurement of the number concentration and the size distribution of the measured aerosol. Current aerosol spectrometers suffer from a limited concentration range due to coincidence errors. In contrast, ensemble techniques, such as photometers, are suitable for high-concentration measurements. One major drawback of such instruments is that they only provide information about one aerosol property, which is typically the mass concentration. A new ensemble technique for the simultaneous measurement of two aerosol properties, namely particle size and concentration, was developed and experimentally verified. The results show that the median particle size (D50) of a monodisperse aerosol can be determined with a deviation of about 10 %, and the concentration with a deviation of about 30 %. With individual instrument calibration, the accuracy can be further improved to approximately 5 % with respect to particle size and 10 % with respect to concentration. The method may be either implemented as a standalone measurement device or, as an add-on for e. g. optical particle spectrometers in the form of a high-concentration mode.

Instrument with an ultra-wide dynamic detection range for the optical counting and sizing of individual particles in suspensions.

The characterization of dispersions, suspensions, and emulsions is important in a wide range of scientific applications and industries. Samples can consist of different materials and a wide range of particle sizes and concentrations. A single particle sizing and counting instrument with a dynamic detection range of ≥6 decades has been developed to detect single nano- and microparticles in aqueous suspensions based on light scattering measured in two directions. Hydrodynamic focusing is employed for particle separation and to provide stable conditions for light scattering detection. This gives the advantage of size resolution in the nm range, allowing, e.g., number based size distributions, classification of nanomaterials, determination of particle agglomerates, developments for dispersion stability analysis, or cutoff of filter media. In addition, concentration determination is based on sample volume measurement with <20 nl measurement uncertainty. We present results of particle detection in a size range from approximately above 40 nm for gold nanoparticles to 8μm for polystyrene particles using a prototyped instrument of the LUMiSpoc® series produced by LUM GmbH. The data obtained demonstrate the advantages of single-particle detection, particularly for characterizing polydisperse systems, such as precise particle sizing in the nanometer range through light scattering intensity based on Mie scattering theory. In addition, we present particle concentration data based on the integrated measurement of sample volume, which allows particle concentration to be determined with an uncertainty of 2.5% (95% confidence interval). To achieve such small uncertainties, dilution series measurements must be used to correct for coincidence losses and particle adhesion.

Rapid single-particle chemical imaging of nanoplastics by SRS microscopy.

Plastics are now omnipresent in our daily lives. The existence of microplastics (1 µm to 5 mm in length) and possibly even nanoplastics (<1 μm) has recently raised health concerns. In particular, nanoplastics are believed to be more toxic since their smaller size renders them much more amenable, compared to microplastics, to enter the human body. However, detecting nanoplastics imposes tremendous analytical challenges on both the nano-level sensitivity and the plastic-identifying specificity, leading to a knowledge gap in this mysterious nanoworld surrounding us. To address these challenges, we developed a hyperspectral stimulated Raman scattering (SRS) imaging platform with an automated plastic identification algorithm that allows micro-nano plastic analysis at the single-particle level with high chemical specificity and throughput. We first validated the sensitivity enhancement of the narrow band of SRS to enable high-speed single nanoplastic detection below 100 nm. We then devised a data-driven spectral matching algorithm to address spectral identification challenges imposed by sensitive narrow-band hyperspectral imaging and achieve robust determination of common plastic polymers. With the established technique, we studied the micro-nano plastics from bottled water as a model system. We successfully detected and identified nanoplastics from major plastic types. Micro-nano plastics concentrations were estimated to be about 2.4 ± 1.3 × 105 particles per liter of bottled water, about 90% of which are nanoplastics. This is orders of magnitude more than the microplastic abundance reported previously in bottled water. High-throughput single-particle counting revealed extraordinary particle heterogeneity and nonorthogonality between plastic composition and morphologies; the resulting multidimensional profiling sheds light on the science of nanoplastics.

Nanoparticle Analyzing Technique Review and Sub-10 nm Nanoparticle Sizing Methods Comparison

This paper provides a comprehensive review of current nanoparticle analysis methods specifically designed for sub-20 nm particles. The techniques are categorized into three main groups: ensemble (I), single particle counting (II), and separating &amp; counting (III) methods. The study further presents a comparative analysis of test results obtained using these three categories of methods on polydisperse polymer nanoparticles. For this investigation, four different tools capable of sub-10 nm particle analysis were utilized. The techniques employed in this study include dynamic light scattering (I), atomic force microscope (II), scanning mobility liquid particle sizer (III), and cryo-electron microscopy (I). The findings indicate that, for particles larger than 8 nm, the three tools—excluding dynamic light diffraction—yield similar results. Conversely, in the small size range (&lt;7 nm), cryo-electron microscopy consistently demonstrates reliable outcomes.

Nile Red staining for nanoplastic quantification: Overcoming the challenge of false positive counts due to fluorescent aggregates

The true extent of plastic particle pollution is largely unknown, particularly for particles <1 µm (nanoplastics), as they are difficult to detect. Here, we expand the Nile Red (NR) staining approach often used to visualize microplastics to the quantification of nanoplastics. Using NR staining for nanoplastic quantification is largely unexplored due to the formation of fluorescent NR aggregates which cause false positive counts in single particle counting. Here, we study the number and size of the NR aggregates formed as a function of NR concentration and show that with decreasing NR concentration, the number and size of the NR aggregates drops. At nanomolar concentrations, the number of NR aggregates is low, while staining of nanoplastics at these concentrations still results in signals that are sufficiently bright for single particle detection. To challenge and verify our approach, we spiked a drinking water sample with known amounts of nanoplastics. After quantification of the total amount of fluorescent nanoparticles and considering NR aggregates and the added amounts of nanoplastics, we find a stable number of NR positive nanoparticles for the drinking water sample, thus verifying our approach. To demonstrate the direct applicability of our method, without any pre-analytical treatment, we determined the number of NR positive nanoparticles in different drinking water samples. In water obtained from plastic bottles and plastic-lined cartons, we detected approximately 250 nanoparticles/nL, which is well above the level of detection of our method. In tap water, we found approximately 10 times fewer NR positive nanoparticles. Our study demonstrates that staining at nM NR concentrations combined with a careful characterization of the number of NR aggregates in solution allows for using single-particle counting for nanoplastic quantification in water samples. We foresee that this approach can contribute to filling the gap in knowledge on the abundance of nanoplastics.

A reconfigurable Micro-channel plate “RSP” detector for wide-range application in charged particle detection

We have developed an MCP detector with reconfigurable read-out which is capable of recording the position and time information for single particle counting and alternatively of operating at high particle flux with optical read-out. This is achieved by a Resistive Screen with embedded Phosphor (RSP) anode. On demand, signal pickup electrodes such as a delay-line array can be attached or removed without interfering with vacuum, allowing easy switching between the different operation modes. We show tests of a prototype.

Development of a neutrino detector capable of operating in space

The $\nu$SOL experiment to operate a neutrino detector close to the Sun is building a small test detector to orbit the Earth to test the concept in space. This detector concept is to provide a new way to detect neutrinos unshielded in space. A double delayed coincidence on Gallium nuclei that have a large cross section for solar neutrino interactions emitting a conversion electron and converting the nuclei into an excited state of Germanium, which decays with a well-known energy and half-life. This unique signature permits operation of the detector volume mostly unshielded in space with a high single particle counting rate from gamma and cosmic ray events. The test detector concept which has been studied in the lab and is planned for a year of operations orbiting Earth which is scheduled for launch in late 2024. It will be surrounded by an active veto and shielding will be operated in a polar orbit around the Earth to validate the detector concept and study detailed background spectrums that can fake the double timing and energy signature from random galactic cosmic or gamma rays. The success of this new technology development will permit the design of a larger spacecraft with a mission to fly close to the Sun and is of importance to the primary science mission of the Heliophysics division of NASA Space Science Mission Directorate, which is to better understand the Sun by measuring details of our Sun's fusion core.

One-step detection of T4 polynucleotide kinase activity based on single particle-confined enzyme reaction and digital particle counting

T4 polynucleotide kinase (T4 PNK) is a pivotal enzyme for DNA replication, recombination, and DNA damage repair. Herein, a robust single particle counting-based assay has been developed for the high-sensitive determination of T4 PNK activity through only a simple one-step reaction. Taking benefit of the exceptional space-confined enzymatic property of T4 PNK towards DNA substrates on a single nanoparticle, the T4 PNK activity can be precisely determined by counting the fluorescence-positive nanoparticles in a digital manner with a total internal reflection fluorescent microscope (TIRFM). Due to the featured spatial-confined enzymatic property of T4 PNK and the single particle counting-based signal readout, T4 PNK can be effectively differentiated from other interfering enzymes. This facile strategy has been also successfully applied to screen T4 PNK inhibitor and accurately determine T4 PNK activity in complex biological samples, paving a potential avenue for the digital analysis of biomarkers.

Quantification of the Retention and Disassembly of Virus Particles by a PEI-Functionalized Microfiltration Membrane.

Monitoring the performance of polymer-functionalized surfaces that aim at removing and inactivating viruses is typically labor-intensive and time-consuming. This hampers the development and optimization of such surfaces. Here we present experiments of low complexity that can be used to characterize and quantify the antiviral properties of polymer-functionalized surfaces. We showcase our approach on polyethylenimine (PEI)-coated poly(ether sulfone) (PES) microfiltration membranes. We use a fluorescently labeled model virus to quantify both virus removal and inactivation. We directly quantify the log removal of intact viruses by this membrane using single particle counting. Additionally, we exploit the change in photophysical properties upon disassembly of the virus to show that viruses are inactivated by the PEI coating. Although only a small fraction of intact viruses can pass the membrane, a considerable fraction of inactivated, disassembled viruses are found in the filtrate. Fluorescence microscopy experiments show that most of the viruses left behind on the microfiltration membrane are in the inactivated, disassembled state. Combined, our fluorescence microscopy and spectroscopy experiments show that not only does the model virus adsorb to the PEI coating on the membrane but also the interaction with PEI results in the disassembly of the virus capsid.

Quantitative detection of α-Synuclein and Tau oligomers and other aggregates by digital single particle counting

The pathological hallmark of neurodegenerative diseases is the formation of toxic oligomers by proteins such as alpha-synuclein (aSyn) or microtubule-associated protein tau (Tau). Consequently, such oligomers are promising biomarker candidates for diagnostics as well as drug development. However, measuring oligomers and other aggregates in human biofluids is still challenging as extreme sensitivity and specificity are required. We previously developed surface-based fluorescence intensity distribution analysis (sFIDA) featuring single-particle sensitivity and absolute specificity for aggregates. In this work, we measured aSyn and Tau aggregate concentrations of 237 cerebrospinal fluid (CSF) samples from five cohorts: Parkinson’s disease (PD), dementia with Lewy bodies (DLB), Alzheimer’s disease (AD), progressive supranuclear palsy (PSP), and a neurologically-normal control group. aSyn aggregate concentration discriminates PD and DLB patients from normal controls (sensitivity 73%, specificity 65%, area under the receiver operating curve (AUC) 0.68). Tau aggregates were significantly elevated in PSP patients compared to all other groups (sensitivity 87%, specificity 70%, AUC 0.76). Further, we found a tight correlation between aSyn and Tau aggregate titers among all patient cohorts (Pearson coefficient of correlation r = 0.81). Our results demonstrate that aSyn and Tau aggregate concentrations measured by sFIDA differentiate neurodegenerative disease diagnostic groups. Moreover, sFIDA-based Tau aggregate measurements might be particularly useful in distinguishing PSP from other parkinsonisms. Finally, our findings suggest that sFIDA can improve pre-clinical and clinical studies by identifying those individuals that will most likely respond to compounds designed to eliminate specific oligomers or to prevent their formation.

64-channel label-free fluorescence detection and single-particle counting device.

A 64-channel detection system for laser-induced fluorescence (LIF) detection at the cell level is established and applied to single event counting. Generally, fluorescence detection at the cellular level requires a dyeing label to enhance the scattered light intensity for the photodetector. However, the dyeing labels, such as fluorophores, probes, and dyes, complicate sample preparation and increase cytotoxicity in the process. Therefore, label-free detection becomes essential for in vivo research. The presented 64-channel detection system is designed for label-free detection with the ability to record feeble emissions. Two linear photodetector devices are included in the system, extending the wavelength detection range to 366-680 nm with an improved spectral resolution at an average of 4.9 nm. The performance of the system was validated by detecting unlabeled human hepatocytes (L-02) and other cell-level biologic samples. In addition, the 64-channel detection system was also used for particle counting with a quartz microfluidic chip. The counting method is based on fluorescence spectra differs from those of other devices (i.e., flow cytometry and cell-sorting equipment), which use isolated irradiance intensities.

Temporal and spatial variance in pixelated counting detectors with dead time and retrigger capability

In single particle counting detectors, the existence of dead time modifies the statistical distribution of the recorded counts in the time domain. In particular, for increasing incoming event rates it has a regularizing effect as by its nature it prevents events to be recorded within a certain time window after the last one occurred, with non-trivial consequences on the statistical moments of the counting process outcome. Moreover, spatial correlation between neighboring channels – e.g. in pixelated detectors – leads to additional effects on the ensemble properties. In this study we present the derivation of the exact expression of the temporal and spatial mean and variance for multi-channel particle counting systems equipped with retrigger capability, a feature that allows the system to work in non-paralyzable mode, and in presence of spatial cross-correlation among the channels. The analytical derivations are cross-validated with the help of Monte Carlo simulations. The case of non-ideal signals and the possibility to achieve sub-Poisson signal-to-noise ratios for increasing incoming rates are also discussed.

A microreactor sealing method using adhesive tape for digital bioassays.

Digital assays using microreactors fabricated on solid substrates are useful for carrying out sensitive assays of infectious diseases and other biological tests. However, sealing of the microchambers using fluid oil is difficult for non-experts, and thus hinders the widespread use of digital microreactor assays. Here, we propose the physical isolation of tiny reactors with adhesive tape (PITAT) using simple, commercially available pressure-sensitive adhesive (PSA) tape as a separator of the microreactors. We confirmed that PSA tape can effectively seal the microreactors and prevent molecules from diffusing out. By testing several types of adhesive tape, we found that rubber-based adhesives are the most suitable for this purpose. In addition, we demonstrated that single-molecule enzyme assays can be successfully performed inside microreactors sealed with PSA tape. The results obtained using PITAT are quantitatively comparable to conventional oil sealing, although it is quick and cost-effective. Finally, we demonstrated that single-particle virus counting of the influenza virus can be achieved using PITAT. Collectively, our results suggest that PITAT may be suitable for use in the design of sensitive tests for infectious diseases at the point of care, where no sophisticated equipment or machines are available.

Small-Angle Particle Counting Coupled Photometry for Real-Time Detection of Respirable Particle Size Segmentation Mass Concentration.

Respirable particulate matter air pollution is positively associated with SARS-CoV-2 mortality. Real-time and accurate monitoring of particle concentration changes is the first step to prevent and control air pollution from inhalable particles. In this research, a new light scattering instrument has been developed to detect the mass concentration of inhalable particles. This instrument couples the forward small-angle single particle counting method with the lateral group particle photometry method in a single device. The mass concentration of four sizes of inhalable particles in the environment can be detected simultaneously in a large area in real-time without using a particle impactor. Different from the traditional light scattering instrument, this new optical instrument can detect darker particles with strong light absorption, and the measurement results mainly depend on the particle size and ignore the properties of the particles. Comparative experiments have shown that the instrument can detect particles with different properties by simply calibrating the environmental density parameters, and the measurement results have good stability and accuracy.

Single-carrier charge collection in thin direct-conversion semiconductor neutron detector: A numerical simulation

Although direct-conversion solid-state neutron detection has been investigated for over five decades, propelling this technology beyond the basic research stage remains an outstanding challenge. This challenge is due to the very small selection of neutron-sensitive isotopes and therefore lack of mature semiconductor materials available for this technology. Given these constraints, there is a reason to investigate materials with less-than-optimal charge transport properties, which could include low charge carrier mobility/lifetime and/or single-carrier transport (i.e., order of magnitude or greater difference between electron and hole mobility). Such materials are potentially best-suited to a thin-film configuration, which provides not only leniency in terms of charge transport requirements, but also processing flexibility and integration advantages. Single-carrier transport in detectors with thicknesses less than or comparable to radiation penetration depth can lead to partial and position-dependent charge collection effects not treated in the general case of direct-conversion neutron detection. Here, we have developed a theory to include the effect of single-carrier charge collection and the possible mismatch between carrier transit time and integration time to study the performance of thin neutron detectors. Taking a boron carbide (B4C) direct-conversion thermal neutron detector as an example, we use custom Monte Carlo simulations to study the effects of a range of mobility, lifetime, thickness, and integration time values on detection efficiency and pulse height spectra. We discuss the interplay between the traditional mobility–lifetime product (μτ) metric and the integration time to carrier transit time ratio (ti/ttr), which takes into account mobility (μ) specifically, and their effect on detection efficiency. We describe the effect of these parameters on pulse height spectra and show how, although single-carrier transport leads to a loss of spectral resolution when signal current is fully integrated, using integration times shorter than carrier transit time allows for recovery of spectral features. We additionally present two methods to extract the mobility–lifetime product of a single-carrier device, with the first being based on the steady-state current as a function of electric field under a steady-state radiation detection mode, and the second being based on the shift of spectral peaks as a function of electric field under a single-particle radiation counting mode, both using modified Hecht equations that do not require either surface or uniform radiation absorption conditions. Finally, we discuss the performance of a hypothetical single-carrier 5 μm thick B4C neutron detector, which can provide a maximum intrinsic neutron detection efficiency of 14% with a set lower level discriminator value of 25% of the total energy deposited.

Optimizing fluorophore density for single virus counting: a photophysical approach

In health and environmental research, it is often necessary to quantify the concentrations of single (bio) nanoparticles present at very low concentrations. Suitable quantification approaches that rely on counting and tracking of single fluorescently labelled (bio) nanoparticles are often challenging since fluorophore self-quenching limits the maximum particle brightness. Here we study how the number of labels per nanoparticle influences the total brightness of fluorescently labelled cowpea chlorotic mottle virus (CCMV). We analyze in detail the photophysical interplay between the fluorophores on the virus particles. We deduce that the formation of dark aggregates and energy transfer towards these aggregates limits the total particle brightness that can be achieved. We show that by carefully selecting the number of fluorescent labels per CCMV, and thus minimizing the negative effects on particle brightness, it is possible to quantify fluorescently labelled CCMV concentrations down to fM concentrations in single particle counting experiments.

Delivery, Beam and Range Monitoring in Particle Therapy in a Highly Innovative Integrated Design

The design of a particle therapy system that integrates an innovative beam delivery concept based on a static toroidal gantry and an imaging configuration suitable for beam and online range monitoring is proposed and discussed. Such approach would provide a compact and cost-effective layout, with a highly flexible and fast beam delivery, single particle counting capability for fast measurement of beam fluence and position and a precise real time verification of the compliance between the treatment delivery and its prescription. The gantry configuration is discussed, presenting an analysis of the residual magnetic field in the bore and of the feasibility of irradiating a realistic target volume. Moreover, the expected performance of the PET-based range monitor is assessed through Monte Carlo simulations, showing a precision in the reconstruction of the activity distribution from a clinical treatment plan better than the state-of-the-art devices. The feasibility of the proposed design is then discussed through an assessment of the technological improvements required to actually start the construction and commissioning of a system prototype.