Spectral Overlap Research Articles

Open‐set recognition of compound jamming signal based on multi‐task multi‐label learning

AbstractIn the increasingly intricate electromagnetic environment, the radar receiver may simultaneously encounter multiple intentional or unintentional jamming signals, which results in temporal and spectral overlap of received signals and forms a composite jamming signal. The nature and extent of interference contained in the received signal are often unknown, while they significantly affect the accuracy of radar detection. AnOpen‐Set Compound Jamming Signal Recognition Framework based on Multi‐Task Multi‐Label (MTML‐OCJR) is proposed. Based on the time–frequency characteristic of compound jamming signals, the proposed framework employs multi‐label classification to identify components of compound jamming signals while incorporating an unknown signal detection task into the classification process. Time–frequency image reconstruction combined with extreme value model estimation is used to detect unknown types of jamming signals, enabling simultaneous signal recognition and anomaly detection. The obtained results show that the proposed approach has superior recognition performance for composite jamming signals in closed‐set environments and high anomaly detection ability for unknown signals in open‐set environments. This method has the potential to significantly enhance the effectiveness and reliability of jamming systems in battlefield scenarios.

Bond-length dependence of attosecond ionization delays in O2 arising from electron correlation to a shape resonance.

We experimentally and theoretically demonstrate that electron correlation can cause the bond-length sensitivity of a shape resonance to induce an unexpected vibrational state-dependent ionization delay in a nonresonant channel. This discovery was enabled by a high-resolution attosecond-interferometry experiment based on a 400-nm driving and dressing wavelength. The short-wavelength driver results in a 6.2-electron volt separation between harmonics, markedly reducing the spectral overlap in the measured interferogram. We demonstrate the promise of this method on O2, a system characterized by broad vibrational progressions and a dense photoelectron spectrum. We measure a 40-attosecond variation of the photoionization delays over the X2Πg vibrational progression. Multichannel calculations show that this variation originates from a strong bond-length dependence of the energetic position of a shape resonance in the [Formula: see text] channel, which translates to the observed effects through electron correlation. The unprecedented energy resolution and delay accuracies demonstrate the promise of visible-light-driven molecular attosecond interferometry.

Spectroscopic investigation and molecular docking analysis of an FRET probe: Trypsin from bovine pancreas@Coumarin 440 dye conjugate

Herein, a systematic investigation of energy transfer from bovine pancreas trypsin to coumarin 440 (C440) dye is presented with an aim to design a FRET probe, as trypsin is an important enzyme for absorption and digestion of nutrients in the small intestine. Conformational changes in trypsin upon interaction with C440 dye are studied by employing both spectroscopic and molecular docking techniques. Steady state and time resolved measurements validate the formation of trypsin-C440 complex via dynamic quenching, that confirms the role of C440 as an inhibitor to suppress the activity of trypsin. Spectral overlap integral, Förster's distance, rate and energy transfer efficiency are calculated from steady state measurements. The observed blue shift in fluorescence suggests conformational changes due to hydrophobicity that arises in trypsin structure on binding with C440 possibly due to their interaction through H-bond (SER195 and GLN192), hydrophobic (GLN192 and HIS57) and π-π stacking bonding (HIS57). The stability of trypsin@C440 bio-conjugate at higher temperature was established from temperature dependent PL studies and the thermodynamic parameters determined agree with second law of thermodynamics. The thermodynamic parameters such as change in enthalpy (ΔH=172.2Jmol−1), change in entropy (ΔS=15.3Jmol−1K−1) and the respective approximately same and unity values of Gibb's free energy (ΔG) and Hill coefficient (n), as well as binding affinities determined from temperature dependent fluorescence studies validate the hydrophobic, spontaneous, exothermic process engaged in trypsin@C440 bio-conjugate.

Quantifying Förster Resonance Energy Transfer from Single Perovskite Quantum Dots to Organic Dyes.

Colloidal quantum dots (QDs) are promising regenerable photoredox catalysts offering broadly tunable redox potentials along with high absorption coefficients. QDs have thus far been examined for various organic transformations, water splitting, and CO2 reduction. Vast opportunities emerge from coupling QDs with other homogeneous catalysts, such as transition metal complexes or organic dyes, into hybrid nanoassemblies exploiting energy transfer (ET), leveraging a large absorption cross-section of QDs and long-lived triplet states of cocatalysts. However, a thorough understanding and further engineering of the complex operational mechanisms of hybrid nanoassemblies require simultaneously controlling the surface chemistry of the QDs and probing dynamics at sufficient spatiotemporal resolution. Here, we probe the ET from single lead halide perovskite QDs, capped by alkylphospholipid ligands, to organic dye molecules employing single-particle photoluminescence spectroscopy with single-photon resolution. We identify a Förster-type ET by spatial, temporal, and photon-photon correlations in the QD and dye emission. Discrete quenching steps in the acceptor emission reveal stochastic photobleaching events of individual organic dyes, allowing a precise quantification of the transfer efficiency, which is >70% for QD-dye complexes with strong donor-acceptor spectral overlap. Our work explores the processes occurring at the QD/molecule interface and demonstrates the feasibility of sensitizing organic photocatalysts with QDs.

A dual‐responsive luminescent sensor for efficient detection of 3‐nitrotyrosine and dipicolinic acid biomarkers based on copper(II) organic framework

The trace detection of biomarkers in real samples has profound a most important significance in the early diagnosis and daily monitoring of diseases. Based on the ligand pre‐design strategy, a novel 3D CuMOF, with the formula of {[Cu4(BTPB)(μ2‐OH)2(H2O)5]·3H2O}n, was fabricated by using the hexacarboxyl ligand of 1,4‐bis(2,4,6‐tricarboxylpyrid‐5‐yl)benzene (H6BTPB) and tetranuclear {Cu4(COO)4(μ2‐OH)2} SBUs. Benefiting from the robust framework and unique luminescence performance, the prepared CuMOF displays great potential as a dual‐responsive efficient luminescent sensor in “turn‐off” detection of 3‐nitrotyrosine (3‐NT) biomarker and “turn‐on” detecting the anthrax biomarker of dipicolinic acid (DPA), with detection limits (LOD) for 3‐NT and DPA being 110.8 ng/ml and 85.2 ng/ml, respectively. Additionally, the practicality and compatibility of such developed sensors were verified by quantifying 3‐NT and DPA biomarkers in diluted serum and urine samples with satisfactory recoveries. Further, the theoretical calculations of energy levels as well as the spectral overlap between the analytes and CuMOF were conducted to elucidate the possible sensing mechanisms. This work demonstrated that MOFs‐based luminescent sensors are evolving as an efficacious and equable approach for the detection of biomarkers in real samples.

Dual-Encoded Affinity Microbead Signature Combinatorial Profiling for Acute Myocardial Infarction High-Sensitivity Diagnosis.

The early diagnosis of acute myocardial infarction (AMI) is dependent on the combined feedback of multiple cardiac biomarkers. However, it remains challenging to precisely detect multicardiac biomarkers in complex blood early due to the lack of sensitive and specific diagnostic indicators and the low abundance and small size of associated biomarkers with high specificity (such as microRNAs). To make matters worse, spectral overlap significantly limits the multiplex analysis of cardiac biomarkers by fluorescent probes, leading to bias in the diagnosis of myocardial infarction. Herein, we developed a method for simultaneous detection of miRNAs and protein biomarkers using size- and color-coded microbeads that carry signature for target capture. We also constructed a microfluidic chip with different spacer arrays that segregate these microbeads in different chip regions according to their size to produce signature signals, indicating the level of different biomarkers. The signals on the microbeads were hugely amplified by catalytic hairpin assembly and rolling circle amplification. Notably, this strategy enables the simultaneous and in situ sensitive profiling of six kinds of biomarkers via adding two different fluorescent labels, removing the limitations of spectral overlap. We envision that the strategy has great potential for application in clinical diagnosis for AMI.

Advantageous occupation of Cu2+ in the B site of double-perovskite Rb2BB'Br6 (B = K; B′ = Bi) framework for white-light-emission

Research on lead-free double perovskite has demonstrated its potential as a solution to the stability and toxicity issues associated with lead-based inorganic perovskite white light-emitting diodes (WLED). This current investigation was designed to fabricate, examine, and compare the photoluminescence (PL) characteristics of Cu2+-activated Rb2BB'Br6 (B = K and B′ = Bi) double-perovskite (DP) [A2BB'Br6] luminous materials. Bi3+ ions are substituted with Cu2+ ions at their B′ sites. These ions are distributed to the equilibrium valence state and demonstrate minimal variation in ion radius. The microstructure and phase purity of Rb2BB'Br6:Cu2+ DP were assessed by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The Rietveld refinement was also used to identify the crystal structures, and the resulting samples all exhibit cubic crystal structures. The Fm3m space group in Rb2BB'Br6 DP frameworks is significantly impacted by the B sites. The PL spectra were used to thoroughly examine the associated mechanism of the Cu2+ → Bi3+ energy-transfer process. Cu2+ ions can be successfully doped in the Rb2KBiBr6 host with an optical band gap (Eg) of 2.74 eV (RKBBr: 0.025Cu2+). The Rb2KBiBr6:Cu2+ phosphor exhibits distinct emission bands from 450 to 650 nm upon UV excitation at 366 nm. The Cu2+→ Bi3+ energy transfer design allows for systematic luminescence tuning from blue to white due to spectral overlap between the Cu2+ emission and Bi3+ excitation spectra. It has been established that Cu2+ → Bi3+ energy transfer significantly increases the white emission intensity of Bi3+, with an associated colour temperature of 7892 K and an outstanding colour rendering index of 88.8. As a down-conversion luminous material in solid-state illumination for the upcoming generation of display devices, the discovered Rb2KBiBr6: 0.025Cu2+ phosphor exhibits remarkable promise.

Abstract 2068: Multi-pass flow cytometry for high-marker panels with minimal spillover spread

Abstract Multiparametric flow cytometry on immune cells is essential in cancer research, disease diagnosis, and treatment monitoring. Collecting data on >10 markers at a time is ideal due to deeper cell characterization and assay sensitivity, however, high-marker panels are subject to significant spillover spread from spectrally overlapping fluorophores that compromise data quality, standardization, and interpretation. Clinical flow cytometry still relies on the use of multi-tube low-marker (<10) panels that minimize spillover spread and ease analysis and interpretation of results. To improve and expand the clinical utility of flow cytometry, there is a need for higher marker measurements without use of highly overlapping fluorophores, which would also serve to streamline the use of samples and reagents. Here, we present the development of a novel barcoding method using micron-sized laser particles (LPs) that confer an optical signature to individual cells at high-throughput, enabling tracking of cells through multiple passes in a flow cytometer. This process is possible using a custom cytometer equipped to detect LPs and capture samples. Sequential staining of samples with releasing or photobleaching antibodies/fluors between cycles enables merging of multiple smaller panels that each have minimal spillover, resulting in a high-marker dataset with drastic reduction in spillover compared to conventional single-acquisition flow cytometry. We demonstrate the utility of this technology through three different assays. Human PBMCs were barcoded with LPs and stained with 1) a panel of releasable antibodies (Miltenyi Biotec, 10 markers, 2 passes), 2) a laboratory-designed panel that utilized photobleaching (32 markers, 3 passes), and 3) a panel of dried and validated DuraClone antibodies that utilized photobleaching (Beckman Coulter, 18 markers, 2 passes). For each assay, cells were stained, acquired, photobleached/released, captured, re-stained, re-acquired and steps were repeated either once (for 2 passes) or twice (for 3 passes). In all cases, we found drastically reduced spillover, better data quality, and identified the same cell subtypes detected by conventional flow cytometry. Reproducibility was high across multiple (3+) replicates with CVs <30% among all populations with >1% frequency of total. New cell populations were identified that would not be possible with separate acquisitions of each pass. Recent advances in flow cytometry now allow more markers to be analyzed together, but at the cost of increasing spectral overlap and spillover spread. Multi-pass flow cytometry confines spillover to individual panels, reducing overall interference and simplifying analysis. This enhances data quality and usability while streamlining resources, facilitating the clinical uptake of complex flow cytometry in cancer research, diagnosis, and treatment. Citation Format: Trevor Brown, Sarah Forward, Emane Rose Assita, Geoffrey Abbott, Marissa Fahlberg, Sheldon J. Kwok. Multi-pass flow cytometry for high-marker panels with minimal spillover spread [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2068.

Abstract 1450: Novel whole slide imaging modes for imaging mass cytometry reveal cellular and structural composition of mouse glioblastoma

Abstract Mouse tumors have become widely used models in translational research of brain malignancies, helping to address the difficulties related to studying the human brain. Mouse brain can be regarded as a miniaturized model of the human brain, permitting visualization of the whole tissue to provide spatial cellular context. Imaging Mass Cytometry™ (IMC™) is a highly relevant tool capable of quantitative evaluation of the multiparametric protein composition in brain tumor microenvironment (TME) without the complications of autofluorescence, tissue degradation, and spectral overlap. The Hyperion XTi™ Imaging System (Standard BioTools™) utilizes IMC technology to simultaneously assess more than 40 individual structural and functional markers in tissue, providing insight into the organization and function of the TME. We demonstrate the whole slide imaging (WSI) application using a 40-marker panel composed of the Maxpar OnDemand™ Mouse Immuno-Oncology IMC Panel Kit and the Maxpar® Neuro Phenotyping IMC Panel Kit on mouse normal and glioblastoma (GBM) brain tissue. The panel was composed of mouse-specific antibodies, which highlight tumor and immune components of the mouse TME. We performed imaging using two new features of Hyperion™ XTi. Ultrafast preview mode (PM) was applied to rapidly screen entire brain sections for marker expression signatures associated with various immuno-oncology processes. This enabled biomarker-guided selection of areas in tumor tissue that were imaged using region of interest-based IMC and analyzed using single-cell analysis (SCA). In parallel, a high-throughput tissue mode (TM) was applied to perform a detailed whole-slide scan of normal and GBM mouse brain tissues that were quantified using pixel-based analysis (PBA) to unravel the composition of the TME. Using TM, we were able to successfully visualize the entire coronal and sagittal sections of normal mouse brain as well as mouse GBM tissue. PBA on TM data provided quantitative spatial expression patterns of structural and immune markers across the whole tissue. In normal tissue, we visualized the highly organized structure of the normal brain and detected neurons, oligodendrocytes, vascular-adjacent astrocytes, and axonal tracks. In GBM tissue, necrotic cores, areas with high immune infiltration, extracellular matrix deposits, and activated tumor cells were detected. SCA of GBM tissue demonstrated expansive vascularization, replicating Olig2+ cells, activation of Ras signaling, and high abundance of infiltrating immune cells. Overall, we demonstrate the successful application of two novel WSI modes and highlight the power of IMC technology to simultaneously explore dozens of relevant biological outputs to better understand the TME of GBM and other tumors. For research use only. Not for use in diagnostic procedures. Citation Format: Qanber Raza, Nick Zabinyakov, Thomas D. Pfister, Nikesh Parsotam, David Howell, Liang Lim, Christina Loh. Novel whole slide imaging modes for imaging mass cytometry reveal cellular and structural composition of mouse glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1450.

Abstract 5501: Novel whole slide imaging modes for imaging mass cytometry unveil extensive cellular heterogeneity in human glioma

Abstract Gliomas present a complex form of cancer that is challenging to diagnose and treat, with a median survival of just over one year after diagnosis for primary glioblastoma (GBM). GBM can occur as multi-lesion, remote, or diffuse tumors, and often contains a tumor microenvironment (TME) that is devoid of peripheral immune cells. Additional hallmark features of GBM include necrosis, hemorrhage, and pseudo palisades, making it a highly heterogeneous disease requiring further investigation. Identification of the cellular and spatial level composition of the TME is vital for interpretation of GBM disease origin, progression, prediction, and treatment. A 40-plus-marker neuro-oncology Imaging Mass Cytometry™ (IMC™) antibody panel was used to determine the cellular and structural landscape of the brain TME. We applied the panel on a tissue microarray (TMA) containing dozens of human glioma cores and uncovered the spatial distribution of over 40 distinct molecular markers. We performed imaging using two new features of the Hyperion XTi™ Imaging System that provide whole slide scanning capabilities. Ultrafast preview mode was applied to rapidly screen tumor cores for expression signatures associated with tumor immuno-oncology processes. This enabled biomarker-guided selection of areas in tumor tissue that were imaged at higher resolution and analyzed using single-cell analysis. In parallel, a high-throughput tissue mode was applied to perform a detailed scan of the brain tumor TMA followed by pixel-based analysis to unravel the spatial composition of the TME. Using tissue mode imaging, we successfully mapped the spatial location of cell populations making up human gliomas, such as neurons, astrocytes, microglia, and oligodendrocytes, across the entire TMA. Various tumor cell phenotypes, resident and infiltrating cells, and resting and activated microglia were detected across all TMA cores. Subsequent single-cell analysis of select regions of interest provided a quantitative assessment of the cellular composition of the brain TME. We classified the distinct states of neurons and quantified myeloid and lymphoid immune cell infiltration across normal, astrocytoma, and GBM tissues. Striking cellular and protein heterogeneity was observed between acquired cores, indicating the complexity of each case that is impossible to capture with low-plex visualization techniques. Therefore, IMC is a highly relevant tool capable of quantitative spatial evaluation of the high-plex protein composition in the brain TME without the complications of autofluorescence, tissue degradation, and spectral overlap. Empowered by the neuro-oncology panel and new whole slide imaging modes, IMC accelerates neurological research and provides insights into the spatial complexity of gliomas and other tumors. For research purpose only. Not for use in diagnostic procedures. Citation Format: Nick Zabinyakov, Qanber Raza, Thomas D. Pfister, Nikesh Parsotam, David Howell, Liang Lim, Christina Loh. Novel whole slide imaging modes for imaging mass cytometry unveil extensive cellular heterogeneity in human glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5501.

Abstract 78: Superior resolution of intracellular markers with CyTOF reveals greater functional diversity of human T cells

Abstract Measuring functional signatures of immune cells in a comprehensive manner, spanning the inflammatory (Th1/Th17) and immunosuppressive (Th2/Treg) lineages, would provide key insights into several facets of cancer research and therapy. Determining mechanisms underlying success/failure of checkpoint blockade, defining immunosuppressive activity of the tumor-resident cell subsets, and identifying immunological biomarkers that predict clinical outcomes, could all be achievable if detection of cytokines spanning many lineages among individual cells was in reach. To achieve this goal, powerful cytometric research tools are required. Cell phenotyping panels are expanding and often now include measurement of intracellular markers of functional potential, such as cytokines, phosphorylation events, and transcription factors. Many of these readouts, including cytokines of the Th2/Treg lineages (IL-5, IL-10, and IL-13), have been notoriously difficult to detect in human cells using fluorescence-based cytometry. Large panels with multiplex intracellular markers are subject to challenges with autofluorescence and spillover spread, thus limiting fluorochrome capacity and reducing resolving power for rare subsets even with full spectrum flow cytometers. For such applications, mass cytometry may overcome such limitations and enable better signal resolution. To address this, we evaluated three small (13-plex) panels using a full spectrum flow cytometer and the CyTOF XT™ mass cytometer. Each panel was comprised of surface and intracellular analytes (cytokines, phospho-epitopes, or transcription factors) and designed to minimize potential impact of spectral overlap on the resolution of spectral flow data. PBMCs were stimulated, split, and stained with either fluorochrome- (Cytek® Aurora) or metal-conjugated antibodies (CyTOF XT). Data sets were analyzed by PhenoGraph clustering and visualized with opt-SNE to determine cellular functional diversity. Overall, data collected on the CyTOF XT demonstrated superior resolution for many intracellular readouts, including cytokines IL-5, IL-10, and IL-13. Also, when better signal/noise enabled accurate clustering, a higher number of immune cell clusters possessing distinct functional signatures resulted from mass cytometry datasets as compared with fluorescent counterparts. In summary, the CyTOF XT mass cytometry platform offers superior signal resolution compared to spectral flow for a range of intracellular targets. Use of mass cytometry translates to the identification of more diverse functional subsets and thus a clearer understanding of the immune signatures present in a sample in the context of unbiased analysis. Our findings indicate that the CyTOF XT platform could serve as a catalyst for seminal discoveries in immune profiling to drive therapeutic design and advanced disease monitoring in cancer. Citation Format: Erika L. Smith-Mahoney, Michael J. Cohen, Anna C. Belkina, Thirumahal Selvanantham, David King, Christina Loh, Amedeo J. Cappione, Jennifer E. Snyder-Cappione. Superior resolution of intracellular markers with CyTOF reveals greater functional diversity of human T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 78.

Photoinduced Magnetic Exchange-Jump Promotes Ground State Biradical Electron Spin Polarization.

Photoinduced electron spin polarization (ESP) is reported in the electronic ground states of three Pt(II) complexes comprised of two S = 1/2 nitronyl nitroxide (NN) radicals attached through different length para-phenylethynyl bridges to the 3,6 positions of a catecholate (CAT, donor) and 4,4'-di-tert-butyl-2,2'-bipyridine (bpy, acceptor). Complexes 1-3 have from 17 to 41 bonds separating NN radicals and display cw-EPR spectra consistent with |JNN-NN| ≫ |aN|, |JNN-NN| ≥ |aN|, and |JNN-NN| < |aN|, respectively, where JNN-NN is the magnetic exchange coupling between NN radicals in the electronic ground state, and aN is the isotropic 14N hyperfine coupling constant. Light-induced transient EPR spectra characterized as enhanced ground-state absorption were observed for all three complexes using 532 nm pulsed laser excitation into the ligand-to-ligand charge transfer (LL'CT) band of the (CAT)Pt(bpy) chromophore. The magnitude of the observed ESP increases in the order 1 < 2 < 3 and is inversely correlated with the magnitude of ground-state JNN-NN. In addition to the experimental observation of net absorptive polarization in 1-3, light excitation also produces multiplet polarization in 2. Since the weak dipolar coupling leads to a strong spectral overlap of the absorptive and emissive components, the multiplet polarization is not observed in 1 and 3 and is very weak in 2. The ability to spin-polarize multiple radical spins with a single photon is anticipated to advance new photoinduced multi qubit/qudit ESP protocols for quantum information science applications.

Abstract 3800: Next generation of spatial biology: High-throughput multiplexed Imaging Mass Cytometry™ with whole slide modes

Abstract Gaining spatial insights into the cellular composition of tumor tissue has tremendous potential to inform clinical and translational researchers about mechanisms behind spatial predictors of immunotherapy success, disease etiology and progression. Imaging Mass Cytometry™ (IMC™) is a high-plex spatial biology imaging technique that enables deep characterization of the diversity and complexity of the tumor microenvironment (TME). IMC supports detailed assessment of cell phenotype and function using 40-plus metal-tagged antibodies simultaneously on a single slide without issues associated with fluorescence-based spectral overlap, tissue autofluorescence or implementation of multiple washing and acquisition cycles. Currently, IMC enables user-defined regions of interest (ROI) in tissues to evaluate cellular and structural composition. To enhance the IMC user experience, we developed two new whole slide imaging (WSI) modes which enable streamlined workflows using ultrafast preview mode (PM) and high-throughput tissue mode (TM). PM samples the entire tissue at predefined spacing to rapidly capture a low-resolution image of all expressed markers in the antibody panel. PM generates an image in minutes to enable informed ROI placements while leaving the stained tissue intact for higher-resolution imaging. PM and TM are designed so acquisitions can easily occur on the same slide without additional processing steps. TM rapidly acquires images of the whole tissue at lower resolution (7 μm pixel size) at a quality that can be used for quantitative analysis of the tissue spatial biology. Specifically designed for high-throughput applications, TM in combination with a newly available 40-slide loader for the Hyperion XTi™ Imaging System permits automated and continuous imaging of more than 40 large tissue samples (400 mm2 per tissue) per week. We showcase the application of WSI modes using the newly developed Maxpar® Human Immuno-Oncology IMC Panel Kit. The 31-marker panel was combined with catalog antibodies to create a 40-plus-marker panel that expands the ability to conduct comprehensive high-plex tumor and immune cell profiling. Tumor tissue microarrays (TMA) and whole tumor tissue sections were stained with the expanded panel. Single-cell analysis of selected ROIs, on whole tumor sections and TMAs, guided by PM data successfully provided quantitative analyses of spatial biology at single-cell resolution. In parallel, TM on whole tumor sections followed by pixel-based analysis provided a spatially resolved quantitative assessment of specific tumor and immune components of the TME. This work demonstrates the expanded capabilities of IMC and establishes it as a reliable high-plex spatial biology imaging platform with high-throughput imaging capabilities ideally suited for translational and clinical applications. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Qanber Raza, Thomas D. Pfister, Jyh Yun Chwee, Liang Lim, David Howell, Nikesh Parsotam, David King, Christina Loh. Next generation of spatial biology: High-throughput multiplexed Imaging Mass Cytometry™ with whole slide modes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3800.

Abstract 91: Comprehensive 27-marker standard panel for immune monitoring of pre-clinical tumor mouse models using spectral analyzer technology

Abstract Within the last decade, major technological advances in flow cytometry and (single cell) RNA-sequencing have deepened our understanding of complex anti-tumoral immune responses. This allows a comprehensive immune monitoring of novel therapies in pre-clinical models. Conventional flow cytometry is reaching its technical limitation. The emitted fluorescence signal of the target population stained with antibodies is evaluated with simple bandpass filters, which leads to spectral overlap and thus the number of parameters that can be analyzed simultaneously is limited. The spectral analyzer technology combines both methods, providing great flexibility in evaluating different immune cell populations. This provides the opportunity to obtain target information by measuring the entire fluorescence spectrum measuring each wavelength individually and thus evaluating the true signal of each fluorochrome unaffected by autofluorescence or spillover. Our standard all-in-one flow cytometry panel uses the full capacity of a conventional BD Fortessa flow cytometer and enables the differentiation of important immune cell populations in tumors, such as T cells (CD4+, CD8+, regulatory T cells), B and NK cells as well as macrophages (M1/M2), MDSCs (granulocytes and monocytes) and dendritic cells. However, it would be of great advantage to gain further insights into activation and effector functions of the immune cells using markers like CD44 &amp; CD62L, CD69, PD-1, Lag-3, Tim-3 and PDL-1, CD80, CD86, CD40 and Ki-67. The Sony ID7000 spectral analyzer enables the simultaneous assessment of 27 markers to evaluate immune phenotypes, with the ability to include up to 4 individual target antibodies. Flow cytometry data from various tumor models are presented, demonstrating the enormous utility and potential of the spectral analyzer, especially with limited and precious tumor sample material, by evaluating different immune cell populations simultaneously without reaching its technical limits. The spectral analyzer technology enables us to mechanistically investigate differences in tumor therapies such as PD-1 treatment or other immune cell modulators not only phenotypically but also with regard to activation and differentiation of immune cell populations. Citation Format: Jonas F. Hummel, Philipp Metzger, Roland Sonntag, Dennis Borrero-Wolff, Cynthia Obodozie, Holger Weber. Comprehensive 27-marker standard panel for immune monitoring of pre-clinical tumor mouse models using spectral analyzer technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 91.

Ratiometric electrochemiluminescence sensing and intracellular imaging of ClO- via resonance energy transfer.

Electrochemiluminescence resonance energy transfer (ECL-RET) is a versatile signal transduction strategy widely used in the fabrication of chem/biosensors. However, this technique has not yet been applied in visualized imaging analysis of intracellular species due to the insulating nature of thecell membrane. Here, we construct a ratiometric ECL-RET analytical method for hypochlorite ions (ClO-) by ECL luminophore,with a luminol derivative (L-012) as the donor and afluorescence probe (fluorescein hydrazide) as the acceptor. L-012 can emit astrong blue ECL signal and fluorescein hydrazide has negligible absorbance and fluorescence signal in the absence of ClO-. Thus, theECL-RET process is turned off at this time. In the presence of ClO-, however, the closed-loop hydrazide structure in fluorescein hydrazide is opened via specific recognition with ClO-, accompanied with intensified absorbance and fluorescence signal. Thanks to the spectral overlap between theECL spectrum of L-012 and theabsorption spectrum of fluorescein, theECL-RET effect isgradually recovered with the addition of ClO-. Furthermore, the ECL-RET system has been successfully applied to image intracellular ClO-. Although the insulating nature of thecell itself can generate ashadow ECL pattern in the cellular region, extracellular ECL emission penetrates thecell membrane and excites intracellular fluorescein generated by the reactions between fluorescein hydrazide and ClO-. The cell imaging strategy via ECL-RET circumvents the blocking of thecell membrane and enables assays of intracellular species. The importance of theECL-RET platform lies in calibrating the fluctuation from theexternal environment and improving the selectivity by using fluorescent probes. Therefore, this ratiometric ECL sensor has shown broad application prospects in the identification of targets in clinical diagnosis and environmental monitoring.

Highly emissive pyrrolo[1,2-a]quinoxaline based narrow band fluorescent sensors for detection of nitroaromatics and their application in spot paper test

In the modern day, explosive detection is essential to public safety, and fluorescent small molecules have gained popularity for their quick and accurate detection. Herein, we present the design and synthesis of a series of pyrrolo[1,2-a]quinoxaline-based conjugated small organic fluorophores (PQ-Ph-OH, PQ-Ph-NMe, PQ-Ph-OMe, PQ-Ph-Me, and PQ-Ph-CN) as fluorescence-based sensors for explosive molecules. The sensor molecules were synthesized in good to excellent yields using a dehomologative functionalization strategy. They exhibited excellent photophysical characteristics, which was tunable based on the substituent on the phenyl linker at position 3. DFT studies showed that the pyrroloquinoxaline moiety could act as an on-demand electron donor or acceptor, depending on the phenyl substituent. The molecules also exhibited solid state emission due to twisted geometry around the CC single bond between the heterocyclic scaffold and the phenyl derivative. This resulted in low band intense emission for the synthesized molecules. In the presence of electron-deficient nitroaromatic explosives, all the fluorophores showed excellent fluorescence quenching. Detection limits in the range of 10−4 M were obtained for all the fluorophores. Stern-Volmer plots show high values of binding constants, indicating good interaction between the fluorophores and the explosive molecules. The mechanism of sensing was confirmed by UV-PL spectral overlap and fluorescence lifetime measurements, which suggest a static quenching based on the formation of a ground state complex. Owing to solid state emissions in some of the derivatives, the synthesized molecules were applied to paper strips for spot detection of explosive molecules.

A Gaussian-mixture-model based peak splitting method in X-ray fluorescence for high concentration ratio solution

Simultaneous detection of multi-heavy metal ions in zinc sulfate solution facilitates the zinc hydrometallurgy process control. X-ray fluorescence spectrometry is widely used for composition detection due to its high detection speed and no sample pre-treatment. However, the high matrix ion-to-trace impurity ion ratio in zinc sulfate causes severe spectral overlap, hindering pure peak area acquisition—a vital feature in ion concentration detection. To address this, a peak-splitting method based on the Gaussian mixture model was proposed. Initially, two ion mixing spectra were obtained with optimized reagents. Subsequently, Gaussian models were established for the characteristic peak regions with varying distributions. Finally, the proposed model was combined with Chaotic Sparrow Search Algorithm to handle the overlapping peaks. The experimental results demonstrated superior stability and additivity compared to other methods. All coefficients of determination between single peak areas and ion concentrations exceeded 0.99, confirming the method's efficacy in resolving heavily masked overlapping peaks.

Excitation of hybrid modes in plasmonic nanoantennas coupled with GeSiSn/Si multiple quantum wells for the photoresponse enhancement in the short-wave infrared range

Hybrid structures represented by a plasmonic nanoantenna array located on the surface of GeSiSn/Si multiple quantum wells (MQWs) grown on a silicon-on-insulator (SOI) substrate by molecular-beam epitaxy were developed for applications in the short-wave infrared range. Using numerical modeling methods, the electromagnetic characteristics of metasurfaces based on aluminum nanoantennas located on the surface of GeSiSn/Si nanoheterostructures were studied. It is shown that the spectral and spatial overlap of localized plasmons in nanoantennas and waveguide modes in the semiconductor film leads to the formation of hybrid modes with a Fano-type resonance profile. The possibility of significant photoluminescence (PL) enhancement of GeSiSn/Si MQWs through the use of hybrid plasmonic structures is demonstrated. The PL enhancement reached to 250 % at the wavelength of 1.9 μm, coinciding with the maximum of the reflectance spectrum for the hybrid structure. Based on the hybrid system, represented by the plasmonic nanoantenna array on the top part of GeSiSn/Si MQWs, p-i-n photodiodes were fabricated. The photocurrent enhancement was approximately 7 times at wavelengths of 1.73 µm and 1.8 µm. The excitation of separately localized plasmons and waveguide modes is demonstrated, and their hybridization is also studied.

Facile Tailoring of Metal-Organic Frameworks for Förster Resonance Energy Transfer-Driven Enhancement in Perovskite Photovoltaics.

Förster resonance energy transfer (FRET) has demonstrated its potential to enhance the light energy utilization ratio of perovskite solar cells by interacting with metal-organic frameworks (MOFs) and perovskite layers. However, comprehensive investigations into how MOF design and synthesis impact FRET in perovskite systems are scarce. In this work, nanoscale HIAM-type Zr-MOF (HIAM-4023, HIAM-4024, and HIAM-4025) is meticulously tailored to evaluate FRET's existence and its influence on the perovskite photoactive layer. Through precise adjustments of amino groups and acceptor units in the organic linker, HIAM-MOFs are synthesized with the same topology, but distinct photoluminescence (PL) emission properties. Significant FRET is observed between HIAM-4023/HIAM-4024 and the perovskite, confirmed by spectral overlap, fluorescence lifetime decay, and calculated distances between HIAM-4023/HIAM-4024 and the perovskite. Conversely, the spectral overlap between the PL emission of HIAM-4025 and the perovskite's absorption spectrum is relatively minimal, impeding the energy transfer from HIAM-4025 to the perovskite. Therefore, the HIAM-4023/HIAM-4024-assisted perovskite devices exhibit enhanced EQE via FRET processes, whereas the HIAM-4025 demonstrates comparable EQE to the pristine. Ultimately, the HIAM-4023-assisted perovskite device achieves an enhanced power conversion efficiency (PCE) of 24.22% compared with pristine devices (PCE of 22.06%) and remarkable long-term stability under ambient conditions and continuous light illumination.

A highly sensitive electrochemical sensing probe incorporating classical Berthelot's reaction and glassy carbon electrode for measuring ultra-trace levels of ammonia/ NH4+ in water

In quantifying NH3 and/or NH4+ ions, the wavelength-based spectrochemical methods involving formation of colored products of the Berthelot’s or Nessler reactions might be challenging due to auto-self absorbance, spectral overlap, and background scattering noise. Thus, the current study reports a renewable glassy carbon electrode (GCE) sensing probe combined with Berthelot’s reaction (indophenol formation) and adsorptive square - wave-anodic stripping voltammetry (Ads SWSV) at pH = 10 for detection of NH3 and/or NH4+ in water. The redox characteristics and the high surface coverage of the oxidation product of indophenol on the sensing platform suggested its use for NH3 and/or NH4+ detection. The electrochemical sensing probe for NH4+ displayed good linear relationship between 5.56 nM and 55.6 μM of NH4+ with limits of detection (LOD) and quantitation (LOQ) of 4.83 × 10-9 and 1.47 × 10-8 M, and sensing probe sensitivity of 1.27μA/μM−1 cm−2,respectively. The probe was applied for measuring NH3/NH4+in fresh and seawater samples, and the results were validated using standard ion chromatography (IC) and micro spectrophotometry assays. The assembled probe was also tolerably selective against interfering of other contaminants in a comparable potential window. Additionally, the probe has exceptional selectivity, long-term stability, and repeatability, and has good capacity to detect NH3 and/or NH4+ ions with high accuracy (recovery range = 97.14 ± 4.12–102.9 ± 4.7) in environmental water samples. The calculated Student texp and Fexp values (n = 5) were less than the tabulated ttab (2.78) and Ftab (6.39) at 95 % probability (P = 0.05, n = 5). The study offer high reliability towards NH3 and/or NH4+ detection in complex environments.