Digital Counting Research Articles

Functional analysis of single enzymes combining programmable molecular circuits with droplet-based microfluidics.

The analysis of proteins at the single-molecule level reveals heterogeneous behaviours that are masked in ensemble-averaged techniques. The digital quantification of enzymes traditionally involves the observation and counting of single molecules partitioned into microcompartments via the conversion of a profluorescent substrate. This strategy, based on linear signal amplification, is limited to a few enzymes with sufficiently high turnover rate. Here we show that combining the sensitivity of an exponential molecular amplifier with the modularity of DNA-enzyme circuits and droplet readout makes it possible to specifically detect, at the single-molecule level, virtually any D(R)NA-related enzymatic activity. This strategy, denoted digital PUMA (Programmable Ultrasensitive Molecular Amplifier), is validated for more than a dozen different enzymes, including many with slow catalytic rate, and down to the extreme limit of apparent single turnover for Streptococcus pyogenes Cas9. Digital counting uniquely yields absolute molar quantification and reveals a large fraction of inactive catalysts in all tested commercial preparations. By monitoring the amplification reaction from single enzyme molecules in real time, we also extract the distribution of activity among the catalyst population, revealing alternative inactivation pathways under various stresses. Our approach dramatically expands the number of enzymes that can benefit from quantification and functional analysis at single-molecule resolution. We anticipate digital PUMA will serve as a versatile framework for accurate enzyme quantification in diagnosis or biotechnological applications. These digital assays may also be utilized to study the origin of protein functional heterogeneity.

Digital coincidence counting with 4πβ(PPC)-γ for the standardization of 60Co

A 4πβ(PPC)-γ coincidence system has been made at KRISS based on a digital DAQ. 60Co sources were measured to verify the system. The maximum detection efficiency for beta particles was estimated to be 96.7 %. Massic activities for sample sources had 0.005 % of the sample variability error, which was well within the expanded standard uncertainty of 0.54 % (k = 2).

Exploring diel vertical migration and spatiotemporal variation of zooplankton backscattering strength using an acoustic Doppler current profiler instrument in the Halmahera Sea, Indonesia

Given its critical role in marine ecosystems, this study comprehensively examined zooplankton distribution and behavior in the Halmahera Sea. The temporal and spatial dynamics of zooplankton acoustic backscatter values were analyzed using a 153.6 kHz vessel-mounted acoustic Doppler current profiler (ADCP). Analysis was supplemented by biological sampling with a bongo plankton net. Further evaluation included the analysis of oceanographic and bathymetric data. The acoustic, oceanographic, and biological sampling data were obtained from the Jala Citra I ?Aurora? survey expedition in 2021, while the bathymetry data were obtained from the General Bathymetric Charts of the Ocean (GEBCO). The raw ADCP data, represented as digital counts, were transformed into mean volume backscattering strength (MVBS) expressed in decibels (dB) using sonar equations to yield a measure proportional to zooplankton biomass. Temporal observations revealed a diel vertical migration (DVM) pattern in zooplankton aggregation, characterized by movements responding to the daily solar cycle. Spatial observations indicated a higher zooplankton density in semi-enclosed waters than in open water. The high values of acoustic backscatter are not attributed to a single species of zooplankton. Biological sampling identified that Oncaea spp. and Oithona spp., a species from the Cyclopoida order, exhibit the highest abundance. The study concludes that the ADCP, based on acoustic backscatter measurements and data sampling, is an effective tool for detecting the presence and behavior of zooplankton.

An Improved Focus-Forming Assay for Determination of the Dengue Virus Titer.

Dengue virus (DENV), a common and prevalent mosquito-borne endemic disease, is caused by four serotypes (DENV-1-4) and has spread rapidly on a global scale over the past decade. A crucial step in the development of antiviral therapeutics requires the utilization of in vitro cell-based techniques, such as plaque assays and focus-forming assays (FFA) for virus quantification. Vero cells have been widely used for FFA and plaque assay; however, there are instances when their efficacy and efficiency in the detection of certain clinical DENV isolates are low. Here, we showed that BHK-21 cells are more sensitive than Vero cells in the detection of all DENV-1-4 plaques and foci. In addition, we developed an improved FFA protocol for the quantification of all four DENV serotypes. Using a pan-flavivirus envelope (E) antibody, we reduce the possibility of false positives by defining a focus to consist of a minimum of eight infected cells. We outlined a protocol using the Operetta® high-content imaging system to automate the digital capture of these infected cells. A pipeline was also designed using the CellProfilerTM automated image analysis software to detect these foci. We then compare the results of the improved FFA with plaque assay. Notably, the improved FFA detected clear foci of the DENV-4 strain that does not form distinct plaques. We subsequently demonstrated the potential application of the improved FFA protocol in antiviral testing, utilizing a nucleoside inhibitor of DENV, NITD008 as a control. The protocol is amenable to a diverse array of applications, including high-throughput compound screening (HTS). Key features • An improved focus-forming assay that has the potential to quantify the DENV-4 strain, which was previously hard to plaque. • Improvements have been made to reduce the possibility of false positives. • Improved workflow using automated digital imaging process and counting of foci. • Applicable to antiviral compounds screening and is amenable to high-throughput screening.

Plasmonic nanoparticle-assisted single-molecule dynamic binding for protein kinase activity digital counting.

In this work, we design a new approach for digital counting-based protein kinase activity assay by using plasmonic nanoparticle-assisted single-molecule dynamic binding. This method breaks the concentration-dependent limitation in single-molecule detection and displays good selectivity and sensitivity with a detection limit as low as 0.0001 U mL-1 for the protein kinase A (PKA) assay, which would find broad applications in drug screening and medical investigations.

Digital counting of force accumulation during mechanotransduction

Digital counting of force accumulation during mechanotransduction

A development of a calibration method for field pyranometers using satellite image data

In this work, a calibration method for deriving a sensitivity of field pyranometers using Himawari satellite image data was developed. The purposed method representing a relation between 10–bit digital counts ranging between 0–1023 from satellite data and global solar irradiance of a standard pyranometer measured at Nakhon Pathom (13.82 °N, 100.04 °E) was investigated and it was found to be an exponential relation. Afterward, this model was applied to estimate the sensitivity of field pyranometers installed at three meteorological monitoring stations located in the main regions of Thailand namely, Chaing Mai in the North (18.78 °N, 98.98 °E), Ubon Ratchathani in the Northeast (15.52 °N, 104.87 °E) and Songkhla in the South (7.20 °N, 100.60 °E) for the validation. The results showed that the sensitivity of field pyranometers derived from satellite data and that obtained from the standard outdoor calibration method of ISO9847 was in a very good agreement with a discrepancy between both datasets of about 1%.

TMIC-11. SPATIAL PROFILING OF TUMOR VASCULATURE AT AUTOPSY IN BRAINSTEM GLIOMAS

Abstract BACKGROUND Diffuse midline glioma (DMG) is a lethal pediatric tumor, and new treatment strategies are urgently needed. To design therapies that can cross the blood-tumor barrier, we designed a pilot study to investigate protein expression on DMG tumor vasculature using spatial profiling, with attention to targetable surface proteins. METHODS We identified 5 patients and 4 controls consented to institutional tissue banking protocols with adequate autopsy tissue from multiple anatomical regions within the brainstem. Portions of formalin-fixed, paraffin-embedded tissue were arranged into tissue microarrays profiled using the nanoString GeoMX platform. Briefly, a multiplexed cocktail of 51 primary antibodies tagged with UV-photocleavable oligonucleotide barcodes and fluorescent morphology markers were applied to tissues and 10-14 regions of interest (ROIs) were segmented by vessel area for each patient. ROIs were processed using UV light and barcodes were quantified using ex situ digital counting. RESULTS Five patients were included in the pilot study with average age 9.8 years (range 8-13), and overall survival was 9.5 months (range 3-18 months). Three tumors were classified as DMG, H3K27-mutant, WHO grade 4; one as high grade glioma without identified histone alteration but with recurrent DMG drivers (ACVR1 and PIK3CA mutations) and one as infiltrating atypical glioma with high proliferative rate and no DMG driver mutations. Controls ranged from 4-15 years and did not have pathological changes in the brain at death. Protein abundance was compared between cases and control and within cases where tumor-adjacent normal tissue was identified. Several proteins were significantly more abundant on tumor vasculature compared to non-tumor adjacent or control brainstem vessels. Validation studies in primary tissue are ongoing. CONCLUSIONS Digital spatial profiling is a promising technology for investigating tumor vasculature and is feasible in autopsy specimens. Work is ongoing to validate candidate biomarkers and expand the cohort to develop a vascular atlas for DMG.

INFLUENCE OF THE EXPERIENCE OF DIGITAL NOMAD TOURISTS ON DESTINATION LOYALTY IN CANGGU, BADUNG DISTRICT, BALI

Nomadic tourism fills the gap between a strategy to increase number of tourist and the development of information and communication technology which makes everyone be able to live and working any where as long as they’re connected to the internet. People who are processing their professional activities while having a migrating lifestyle with high mobility are defined as Digital Nomad. Which in Nomadic Tourism terminology, Digital Nomad counts as one of the type of Nomadic tourist as well. Hence, the appearance of Digital Nomad not only relevant in the growing of living and working environment but also relevant in tourism trend. The presence of digital nomad needs to be highlighted since Indonesia, precisely in Canggu, Bali chosen as number one destination for digital nomad. Even so, destination loyalty from digital nomad’s perspective in Canggu is indicated has a low level, therefore it is needed to see if tourist experience is taking part in this since tourist experience is one of the crucial factors that can affect destination loyalty, besides digital nomad has a characteristic to prioritize their experience. The purpose of this study is to display digital nomad’s tourist experience, destination loyalty, and how their experience affects their destination loyalty in Canggu. The method that is used on this study is descriptive quantitative. The result from this study is digital nomad’s tourist experience and destination loyalty are in a good category, and there’s a significant influence from digital nomad’s tourist experience to their destination loyalty in Canggu. A step that can be done by the destination is to explore and increase the distinction value in Canggu

Prognostic importance of mitosis quantification and PHH3 expression in oral epithelial dysplasia.

Oral epithelial dysplasia (OED) is diagnosed and graded using a range of histological features, making grading subjective and challenging. Mitotic counting and phosphohistone-H3 (PHH3) staining have been used for the prognostication of various malignancies; however, their importance in OED remains unexplored. This study conducts a quantitative analysis of mitotic activity in OED using both haematoxylin and eosin (H&E)-stained slides and immunohistochemical (IHC) staining for PHH3. Specifically, the diagnostic and prognostic importance of mitotic number, mitotic type and intra-epithelial location is evaluated. Whole slide images (WSI) of OED (n = 60) and non-dysplastic tissue (n = 8) were prepared for analysis. Five-year follow-up data was collected. The total number of mitosis (TNOM), mitosis type and intra-epithelial location was manually evaluated on H&E images and a digital mitotic count performed on PHH3-stained WSI. Statistical associations between these features and OED grade, malignant transformation and OED recurrence were determined. Mitosis count increased with grade severity (H&E: p < 0.005; IHC: p < 0.05), and grade-based differences were seen for mitosis type and location (p < 0.05). The ratio of normal-to-abnormal mitoses was higher in OED (1.61) than control (1.25) and reduced with grade severity. TNOM, type and location were better predictors when combined with histological grading, with the most prognostic models demonstrating an AUROC of 0.81 for transformation and 0.78 for recurrence, exceeding conventional grading. Mitosis quantification and PHH3 staining can be an adjunct to conventional H&E assessment and grading for the prediction of OED prognosis. Validation on larger multicentre cohorts is needed to establish these findings.

Biphasic decay of intact SHIV genomes following initiation of antiretroviral therapy complicates analysis of interventions targeting the reservoir

The latent reservoir for HIV-1 in resting CD4+ T cells persists despite antiretroviral therapy (ART) and precludes cure. Reservoir-targeting interventions are evaluated in ART-treated macaques infected with simian immunodeficiency virus (SIV) or simian-human immunodeficiency virus (SHIV). Efficacy is determined by reservoir measurements before and after the intervention. However, most proviruses persisting in the setting of ART are defective. In addition, intact HIV-1 and SIV genomes undergo complex, multiphasic decay observable when new infection events are blocked by ART. Intervention-induced elimination of latently infected cells must be distinguished from natural decay. Here, we address these issues for SHIV. We describe an intact proviral DNA assay that allows digital counting of SHIV genomes lacking common fatal defects. We show that intact SHIV genomes in circulating CD4+ T cells undergo biphasic decay during the first year of ART, with a rapid first phase (t1/2 = 30.1 d) and a slower second phase (t1/2 = 8.1 mo) that is still more rapid that the slow decay observed in people with HIV-1 on long-term ART (t1/2 = 3.7 y). In SHIV models, most interventions are tested during 2nd phase decay. Natural 2nd phase decay must be considered in evaluating interventions as most infected cells present at this time do not become part of the stable reservoir. In addition, for interventions tested during 2nd phase decay, a caveat is that the intervention may not be equally effective in people with HIV on long-term ART whose reservoirs are dominated by latently infected cells with a slower decay rate.

Precise Identification and Profiling of Surface Proteins of Ultra Rare Tumor Specific Extracellular Vesicle with Dynamic Quantitative Plasmonic Imaging.

Specific detection of tumor-derived EVs (tEVs) in plasma is complicated by nontumor EVs and non-EV particles. To accurately identify tEVs and profile their surface protein expression at single tEV resolution directly with clinical plasma is still an unmet need. Here, we present a Dynamic Immunoassay for Single tEV surface protein Profiling (DISEP), a kinetic assay based on surface plasmon resonance microscopy (SPRM) for specific single tEV profiling. DISEP adopts a pair of low-affinity oligonucleotide probes to respectively label EV surface proteins and functionalize an SPRM biosensor interface. tEVs labeled with the oligonucleotide probes possess distinctive binding kinetics from nonspecific particles in plasma, which permits accurate digital plasmonic counting of single EVs. We demonstrate DISEP for recognizing target EVs among 350-fold background plasma particles with high sensitivity (4677 EVs per μL). Clinical plasma samples were analyzed to discriminate between pancreatic cancer patients (n = 40) and healthy donors (n = 45). With a panel of biomarker signatures (EpCAM, HER2, and GPC1), DISEP only requires 10 μL primary sample from each donor to classify tumor patients with an area under the curve of 0.98. DISEP provides a highly specific EV detection and surface protein profiling strategy for early cancer diagnosis.

Short airway telomeres are associated with primary graft dysfunction and chronic lung allograft dysfunction

Primary graft dysfunction (PGD) is a major risk factor for chronic lung allograft dysfunction (CLAD) following lung transplantation, but the mechanisms linking these pathologies are poorly understood. We hypothesized that the replicative stress induced by PGD would lead to erosion of telomeres, and that this telomere dysfunction could potentiate CLAD. In a longitudinal cohort of 72 lung transplant recipients with >6years median follow-up time, we assessed tissue telomere length, PGD grade, and freedom from CLAD. Epithelial telomere length and fibrosis-associated gene expression were assessed on endobronchial biopsies taken at 2 to 4weeks post-transplant by TeloFISH assay and nanoString digital RNA counting. Negative-binomial mixed-effects and Cox-proportional hazards models accounted for TeloFISH staining batch effects and subject characteristics including donor age. Increasing grade of PGD severity was associated with shorter airway epithelial telomere lengths (p=0.01). Transcriptomic analysis of fibrosis-associated genes showed alteration in fibrotic pathways in airway tissue recovering from PGD, while telomere dysfunction was associated with inflammation and impaired remodeling. Shorter tissue telomere length was in turn associated with increased CLAD risk, with a hazard ratio of 1.89 (95% CI 1.16-3.06) per standard deviation decrease in airway telomere length, after adjusting for subject characteristics. PGD may accelerate telomere dysfunction, potentiating immune responses and dysregulated repair. Epithelial cell telomere dysfunction may represent one of several mechanisms linking PGD to CLAD.

Microwell Confined Electro-Coalescence for Rapid Formation of High-Throughput Droplet Array.

Droplet array is widely applied in single cell analysis, drug screening, protein crystallization, etc. This workproposes and validates a method for rapid formation of uniform droplet array based on microwell confined droplets electro-coalescence of screen-printed emulsion droplets, namely electro-coalescence droplet array (ECDA). The electro-coalescence of droplets is according to the polarization induced electrostatic and dielectrophoretic forces, and the dielectrowetting effect. The photolithographically fabricated microwells are highly regular and reproducible, ensuring identical volume and physical confinement to achieve uniform droplet array, and meanwhile the microwell isolation protects the paired water droplets from further fusion and broadens its feasibility to different fluidic systems. Under optimized conditions, a droplet array with an average diameter of 85µm and a throughput of 106 in a 10cm × 10cm chip can be achieved within 5 s at 120 Vpp and 50kHz. This ECDA chip is validated for various microwell geometries and functional materials. The optimized ECDA are successfully applied for digital viable bacteria counting, showing comparable results to the plate culture counting. Such an ECDA chip, as a digitizable and high-throughput platform, presents excellent potential for high-throughput screening, analysis, absolute quantification, etc.

Improving the Accuracy, Robustness, and Dynamic Range of Digital Bead Assays.

We report methods that improve the quantification of digital bead assays (DBA)─such as the digital enzyme-linked immunosorbent assay (ELISA)─that have found widespread use for high sensitivity measurement of proteins in clinical research and diagnostics. In digital ELISA, proteins are captured on beads, labeled with enzymes, individual beads are interrogated for activity from one or more enzymes, and the average number of enzymes per bead (AEB) is determined based on Poisson statistics. The widespread use of digital ELISA has revealed limitations to the original approaches to quantification that can lead to inaccurate AEB. Here, we have addressed the inaccuracy in AEB due to deviations from Poisson distribution in a digital ELISA for Aβ-40 by changing the AEB calculation from a fixed threshold between digital counting and average normalized intensity to a smooth, continuous combination of digital counting and intensity. We addressed issues with determining the average product fluorescence intensity from single enzymes on beads by allowing outlier, high intensity arrays to be removed from average intensities, and by permitting the use of a wider range of arrays. These approaches improved the accuracy of a digital ELISA for tau protein that was affected by aggregated detection antibodies. We increased the dynamic range of a digital ELISA for IL-17A from AEB ∼25 to ∼130 by combining long and short exposure images at the product emission wavelength to create virtual images. The methods reported will significantly improve the accuracy and robustness of DBA based on imaging─such as single molecule arrays (Simoa)─and flow detection.

Digital microfluidics-based digital counting of single-cell copy number variation (dd-scCNV Seq)

Single-cell copy number variations (CNVs), major dynamic changes in humans, result in differential levels of gene expression and account for adaptive traits or underlying disease. Single-cell sequencing is needed to reveal these CNVs but has been hindered by single-cell whole-genome amplification (scWGA) bias, leading to inaccurate gene copy number counting. In addition, most of the current scWGA methods are labor intensive, time-consuming, and expensive with limited wide application. Here, we report a unique single-cell whole-genome library preparation approach based on digital microfluidics for digital counting of single-cell Copy Number Variation (dd-scCNV Seq). dd-scCNV Seq directly fragments the original single-cell DNA and uses these fragments as templates for amplification. These reduplicative fragments can be filtered computationally to generate the original partitioned unique identified fragments, thereby enabling digital counting of copy number variation. dd-scCNV Seq showed an increase in uniformity in the single-molecule data, leading to more accurate CNV patterns compared to other methods with low-depth sequencing. Benefiting from digital microfluidics, dd-scCNV Seq allows automated liquid handling, precise single-cell isolation, and high-efficiency and low-cost genome library preparation. dd-scCNV Seq will accelerate biological discovery by enabling accurate profiling of copy number variations at single-cell resolution.

On-Chip Compressive Sensing with a Single-Photon Avalanche Diode Array.

Single-photon avalanche diodes (SPADs) are novel image sensors that record photons at extremely high sensitivity. To reduce both the required sensor area for readout circuits and the data throughput for SPAD array, in this paper, we propose a snapshot compressive sensing single-photon avalanche diode (CS-SPAD) sensor which can realize on-chip snapshot-type spatial compressive imaging in a compact form. Taking advantage of the digital counting nature of SPAD sensing, we propose to design the circuit connection between the sensing unit and the readout electronics for compressive sensing. To process the compressively sensed data, we propose a convolution neural-network-based algorithm dubbed CSSPAD-Net which could realize both high-fidelity scene reconstruction and classification. To demonstrate our method, we design and fabricate a CS-SPAD sensor chip, build a prototype imaging system, and demonstrate the proposed on-chip snapshot compressive sensing method on the MINIST dataset and real handwritten digital images, with both qualitative and quantitative results.

Automatically digital extracellular vesicles analyzer for size‐dependent subpopulation analysis in surface plasmon resonance microscopy

AbstractThe heterogeneity in extracellular vesicles (EVs) introduces an extra level of complexity in small EV‐based liquid biopsy for cancer diagnosis. Surface plasmon resonance microscopy (SPRM) offers sensitive and multifunctional analysis for single tiny EVs. However, the EV SPRM image analysis has been extremely laborious and time‐consuming due to the low contrast and bad signal‐to‐noise ratio. Herein, we present a digital EV analyzer (DEA), a software package, for the automatic analysis of EV data in the SPRM platform. This package enabled fully automated single EV identification, digital counting, sizing EV, and quantification of dwell time. Integrated with a classification algorithm and intelligent searching algorithm for subpopulation analysis, DEA was able to accurately group EVs with five different origins. We hope this software tool would largely promote the application of SPRM technology into cancer diagnosis with EVs.

A photonic resonator interferometric scattering microscope for label-free detection of nanometer-scale objects with digital precision in point-of-use environments

Label-free detection and digital counting of nanometer-scaled objects such as nanoparticles, viruses, extracellular vesicles, and protein molecules enable a wide range of applications in cancer diagnostics, pathogen detection, and life science research. Here, we report the design, implementation, and characterization of a compact Photonic Resonator Interferometric Scattering Microscope (PRISM) designed for point-of-use environments and applications. The contrast of interferometric scattering microscopy is amplified through a photonic crystal surface, upon which scattered light from an object combines with illumination from a monochromatic source. The use of a photonic crystal substrate for interferemetric scattering microscopy results in reduced requirements for high-intensity lasers or oil-immersion objectives, thus opening a pathway toward instruments that are more suitable for environments outside the optics laboratory. The instrument incorporates two innovative elements that facilitate operation on a desktop in ordinary laboratory environments by users that do not have optics expertise. First, because scattering microscopes are extremely sensitive to vibration, we incorporated an inexpensive but effective solution of suspending the instrument’s main components from a rigid metal framework using elastic bands, resulting in an average of 28.7 dBV reduction in vibration amplitude compared to an office desk. Second, an automated focusing module based on the principle of total internal reflection maintains the stability of image contrast over time and spatial position. In this work, we characterize the system's performance by measuring the contrast from gold nanoparticles with diameters in the 10–40 nm range and by observing various biological analytes, including HIV virus, SARS-CoV-2 virus, exosome, and ferritin protein.

Turning Noise Into Data: Characterization of the Van Allen Radiation Belt Using SDO Spikes Data

The Solar Dynamics Observatory (SDO) is a solar mission in an inclined geosynchronous orbit. Since commissioning, images acquired by Atmospheric Imaging Assembly (AIA) instrument on-board the SDO have frequently displayed “spikes,” pixel regions yielding extreme number of digital counts. These are theorized to occur from energetic electron collisions with the instrument detector system. These spikes are regularly removed from AIA Level 1.0 images to produce clean and reliable data. A study of historical data has found over 100 trillion spikes in the past decade. This project correlates spike detection frequency with radiation environment parameters in order to generate an augmented data product from SDO. We conduct a correlation study between SDO/AIA data and radiation belt activity within the SDO's orbit. By extracting radiation “spike” data from the SDO/AIA images, we produce a comprehensive data product which is correlated not only with geomagnetic parameters such as Kp, Ap, and Sym-H but also with the electron and proton fluxes measured by the GOES-14 satellite. As a result, we find that AIA spikes are highly correlated with the GOES-14 electrons detected by the magnetospheric electron detector and energetic proton, electron and alpha detectors instruments at the equator (where the two satellites meet) with Spearman's Correlation values of ρ = 0.73 and ρ = 0.53, respectively, while a weaker correlation of ρ = 0.47 is shown with magnetospheric proton detector protons for the 2 year period where both missions returned data uninterruptedly. This correlation proves that the SDO spike data can be proven useful for characterizing the Van Allen radiation belt, especially at areas where other satellites cannot.