Imaging Applications Research Articles

Red-Emitting Coumarin-Derived fluorescent probe for thiol detection and indirect recognition of β-Lactamase

Biothiols, comprising cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), play a pivotal role in maintaining the delicate redox balance essential for life processes, intricately intertwined with the well-being of living organisms. Deviations in their concentrations can precipitate a range of diseases. Herein, we presented a novel fluorescent probe, DIcou-DNBS, derived from coumarin, which possessed red emission and a substantial Stokes shift (122 nm), tailored for biothiol detection. DIcou-DNBS demonstrated rapid-responsiveness (6 min) towards Cys, Hcy and GSH with detection limits of 0.015 µM, 0.032 µM and 0.059 µM, respectively. Furthermore, its excellent biocompatibility enabled successful applications in biothiol fluorescence imaging of living cells and zebrafish. Beyond biothiol sensing, DIcou-DNBS leveraged its unique property to indirectly detect β-lactamases, making it a valuable tool in inhibitor screening and susceptibility testing of both sensitive (Staphylococcus aureus ATCC 29213) and resistant (Enterobacter cloacae ATCC 13047) bacterial strains. This assay system anticipates to furnish a potent instrument for visualization studies across biological and medical domains.

Optimizing the Therapeutic Index of sdAb-Based Radiopharmaceuticals Using Pretargeting.

Single-domain antibodies (sdAbs) demonstrate favorable pharmacokinetic profiles for molecular imaging applications. However, their renal excretion and retention are obstacles for applications in targeted radionuclide therapy (TRT). Methods: Using a click-chemistry-based pretargeting approach, we aimed to reduce kidney retention of a fibroblast activation protein α (FAP)-targeted sdAb, 4AH29, for 177Lu-TRT. Key pretargeting parameters (sdAb-injected mass and lag time) were optimized in healthy mice and U87MG (FAP+) xenografts. A TRT study in a pancreatic ductal adenocarcinoma (PDAC) patient-derived xenograft (PDX) model was performed as a pilot study for sdAb-based pretargeting applications. Results: Modification of 4AH29 with trans-cyclooctene (TCO) moieties did not modify the sdAb pharmacokinetic profile. A 200-µg injected mass of 4AH29-TCO and an 8-h lag time for the injection of [177Lu]Lu-DOTA-PEG7-tetrazine resulted in the highest kidney therapeutic index (2.0 ± 0.4), which was 5-fold higher than that of [177Lu]Lu-DOTA-4AH29 (0.4 ± 0.1). FAP expression in the tumor microenvironment was validated in a PDAC PDX model with both immunohistochemistry and PET/CT imaging. Mice treated with the pretargeting high-activity approach (4AH29-TCO + [177Lu]Lu-DOTA-PEG7-tetrazine; 3 × 88 MBq, 1 injection per week for 3 wk) demonstrated prolonged survival compared with the vehicle control and conventionally treated ([177Lu]Lu-DOTA-4AH29; 3 × 37 MBq, 1 injection per week for 3 wk) mice. Mesangial expansion was reported in 7 of 10 mice in the conventional cohort, suggesting treatment-related kidney morphologic changes, but was not observed in the pretargeting cohort. Conclusion: This study validates pretargeting to mitigate sdAbs' kidney retention with no observation of morphologic changes on therapy regimen at early time points. Clinical translation of click-chemistry-based pre-TRT is warranted on the basis of its ability to alleviate toxicities related to biovectors' intrinsic pharmacokinetic profiles. The absence of representative animal models with extensive stroma and high FAP expression on cancer-associated fibroblasts led to a low mean tumor-absorbed dose even with high injected activity and consequently to modest survival benefit in this PDAC PDX.

Ultrafast Response and Broad Detection Range of a Ternary Cation Perovskite Single-Crystal Thin Film Photodetector for Imaging.

FA-MA-Cs ternary cation perovskite exhibits excellent optoelectronic properties and high stabilities against humidity and light soaking and thus has aroused extensive attention in polycrystalline thin film solar cells. Compared with polycrystalline counterparts, FA-MA-Cs ternary cation perovskite single-crystal thin films (SCTFs) have lower defects, better carrier transport capacity, and stability because of lacking grain boundary defects. However, the immature growth technology of SCTFs restricts digging out its optoelectronic potential. Here, we proposed an improved space-confined method to grow large area FA0.9 MA0.05Cs0.05PbI2.7Br0.3 SCTFs using a tunable heating area to control the nucleation and growth process. Its area reaches 64 mm2 with a thickness of 26 μm. The SCTF exhibits high crystallinity, low defect density, long carrier lifetime, and high moisture resistance stability. Besides, a photosensitive chip based on a planar metal-semiconductor-metal photodetector demonstrates linear response to the three primary colors, with a photosensitive range that is 1.5 times that of protocol 3 wide color gamut. Under high-frequency light sources, the on/off ratio reaches 3.9 × 103, and the response time can be as low as 400 ns. Such ultrafast response speed and broad photosensitive range are successfully achieved for imaging applications.

A dual-band polarization independent dielectric metasurface-based absorber for sensing application in the visible light regime

We present a dual-band, polarization-independent dielectric terahertz absorber exhibiting near-perfect absorption and minimal reflection at frequencies of f 1 = 419 THz and f 2 = 528 THz. Using electromagnetic theory, we modeled the structure to derive the surface electric admittance and magnetic impedance of the metasurface, elucidating the conditions required for perfect absorption in terms of inverse electric and magnetic polarizabilities. The absorber features a tunable symmetrical design, facilitating precise frequency adjustment by modifying structural parameters and ensuring polarization independence for perpendicularly incident electromagnetic waves. This scalable and versatile absorber, constructed from readily available materials, is optimally suited for applications in resource detection, imaging, sensing, and medical diagnostics, attributed to its simplicity, cost-effectiveness, and high performance.

Quantitative metrics commonly derived from diffusion tractography covary with streamline length: a characterization and method of adjustment.

Tractography algorithms are used extensively to delineate white matter structures, by operating on the voxel-wise information generated through the application of diffusion tensor imaging (DTI) or other models to diffusion weighted (DW) magnetic resonance imaging (MRI) data. Through statistical modelling, we demonstrate that these methods commonly yield substantial and systematic associations between streamline length and several tractography derived quantitative metrics, such as fractional anisotropy (FA). These associations may be described as piecewise linear. For streamlines shorter than an inflection point (determined for a group of tracts delineated for each individual brain), estimates of FA exhibit a positive linear relation with streamline length. For streamlines longer than the point of inflection, the association is weaker, with the slope of the relationship between streamline length and FA differing only marginally from zero. As the association is most pronounced for a range of streamline lengths encountered typically in DW imaging of the human brain (less than ~ 100mm), our results suggest that some quantitative metrics derived from diffusion tractography have the potential to mislead, if variations in streamline length are not considered. A method is described, whereby an Akaike information weighted average of linear, Blackman and piecewise linear model predictions, may be used to compensate effectively for the association of FA (and other quantitative metrics) with streamline length, across the entire range of streamline lengths present in each specimen.

Continuously tunable ultrasonic focusing by Moiré metalenses

Tunable ultrasonic focusing holds great significance in both medicine and engineering. Recent advancements in metalenses have introduced approaches for tunable acoustic focusing, but their complex configurations and limited tuning range remain challenges. Here, acoustic Moiré metalenses (AMMs) are proposed to achieve continuously tunable ultrasonic focusing in water. Two cascading metasurfaces that can function as Moiré diffractive elements make up the AMM. By mutually rotating the metasurface, the focal point of the AMM can be continuously tuned in a large range. The focal length can be adjusted continuously from ∼14.3λ0to ∼50λ0for the axial focusing. We further show that the well-designed AMM can achieve the continuously tunable lateral focusing, with the deflection angle of the focal point being tunable between approximately -40°,40°. Both simulation and experimental results confirm the excellent tunable focusing performances of the AMMs. The proposed AMMs with continuously tunable focusing capability may have potential applications in ultrasonic imaging and ultrasound treatment.

Chemoexcited Formation and Radiationless Decay Dynamics of Firefly Chromophore.

Multistate nonadiabatic dynamics combined with Mixed-Reference Spin-Flip Time-Dependent Density Functional Theory (MRSF-TDDFT) were performed to investigate the chemoexcitation dynamics of firefly dioxetanone (FDO- in S0) to oxyluciferin (OxyLH- in S1) and its subsequent decay dynamics. The formation of oxyluciferin occurs within approximately 100 fs and is primarily controlled by oscillatory CO2 decarboxylation. Unexpected radiationless decay from oxyluciferin was also observed, facilitated by intramolecular rotation. Simulations under three thermal conditions reveal that higher initial thermal energy not only enhances the formation of oxyluciferin but also increases radiationless decay by surpassing barriers to the ground state. Conversely, lower thermal energy conditions reduce oxyluciferin formation but suppress radiationless decay. These findings suggest that optimal conditions for higher chemiluminescence quantum yield involve initial high thermal energy to accelerate CO2 decarboxylation and gradual thermal dissipation to prevent intramolecular rotation of oxyluciferin. This approach could enhance the chemiluminescence quantum yield beyond the current limit of 40%, offering significant potential for applications in biological imaging and analytical chemistry.

Highly specific GSH-triggered bifunctional molecules to enable precise imaging and targeted therapy of cancer

The utilization of diagnostic-integrated molecules can enable targeted delivery and controlled release to significantly enhance therapeutic effectiveness and minimize toxic effects. Herein, we developed a novel class of glutathione (GSH)-activated bifunctional molecules that respond to elevated levels of GSH in tumor microenvironment. These bifunctional molecules retained the pharmacodynamic effects of parent molecules and mitigated cytotoxicity. Meanwhile, controlled release was monitored using fluorescent signals, enabling detection of drug distribution and accumulation in situ and in real time. Moreover, the correlation between GSH levels and fluorescence intensity offers the possibility of monitoring the effectiveness of responsive drugs. In conclusion, bifunctional molecules, as novel diagnostic-integrated molecules with both fluorescence imaging and therapeutic effects, exhibited potential applications in cancer therapy and imaging.

Characterization of a new low-dose and low-energy Gafchromic film LD-V1.

To characterize the dose-response, energy dependence, postexposure changes, orientation dependence, and spatial capabilities of LD-V1, a new low-dose Gafchromic film for low-energy x-ray dosimetry. A single sheet of LD-V1 Gafchromic film was cut into 15 × 20mm2 rectangles with a notch to track orientation. Eight different doses between 5 and 320mGy were delivered by an MXR-160/22 x-ray tube using x-ray beams of 90, 100, and 120kVp filtered with 3mm of Al and 2mm of Ti. The 120kVp films were scanned at 1, 1.5, 2, 3, 12, 24, 48, 72, and 168h postexposure in portrait orientation and additionally scanned in landscape orientation at 24h. The 90 and 100kVp films were scanned at 24h postexposure in portrait orientation. Lastly, a 20 × 200mm2 strip of film was irradiated using a thin-slit imaging collimator and scanned 24h postexposure to test the film performance in an x-ray imaging application. Of the three color channels, the red channel was found to produce a dose-response curve with a large range of net optical density (netOD) values across the considered dose range. A prominent energy dependence was discovered, resulting in dose discrepancies on the scale of 17mGy between 90 and 120kVp for a dose of 80mGy. The measured postexposure changes suggest that the calibration irradiation-to-scan time should be longer than 12h with a±4h scanning time window for dose errors of<0.5%. An average dose difference of 3.4% was found between the two scanning orientations. Lastly, noise of 4% was measured in the thin slit collimator film for a dose of 30mGy. We have characterized the LD-V1 film for low-energy, low-dose x-ray dosimetry. Energy, scan-time, and orientation dependencies should be considered when using this film.

Fluorescence and Circular Dichroism Dual-Mode Probe for Chiral Recognition of Tyrosine and Its Applications in Bioimaging.

Chiral amino acids (AAs) are essential in metabolism and understanding physiological processes, and they could be used as biomarkers for the diagnosis of different diseases. In this study, chiral Cdots@Van were prepared by postmodifying an achiral Cdots core with vancomycin for recognizing and determining the enantiomeric excess (ee) of tyrosine (Tyr) enantiomers. The fluorescence response of Cdots@Van is based on an "on-off" strategy, with different quenching percentages for d- and l-tyrosine. Interestingly, the circular dichroism (CD) spectrum of Cdots@Van responded to only one form of Tyr enantiomer, specifically d-Tyr, and remained nearly unchanged upon the addition of l-Tyr. Quantum mechanical (QM) calculations were in excellent agreement with the experimental results, confirming the stronger binding affinity of Cdots@Van for d-Tyr compared to l-Tyr. We further investigated the chiral recognition ability of the interconnected vancomycin particles, which was synthesized using the EDC/NHS coupling reaction between vancomycin molecules without a Cdots core. Surprisingly, unlike free vancomycin molecules, interconnected vancomycin displayed an enantiomeric recognition ability by CD spectroscopy, similar to what was observed for Cdots@Van. Crucially, this chiral probe has been successfully utilized for cell imaging applications.

Recent Advances in Linear Polarization‐Sensitive Photodetectors Based on Perovskites with Different Molecular Dimensions

AbstractThe function of photodetectors is to convert optical signals into current or voltage output. Polarization‐sensitive perovskite photodetectors have attracted significant interest because of their potential applications in imaging and remote sensing technologies. This study provides an overview of the advancements in research on halide perovskite photodetectors, particularly focusing on their capability for linear polarization‐sensitive detection. First, there is a brief description of the background and strengths of linear polarization‐sensitive photodetectors. Then, the core parameters of linear polarization‐sensitive photodetectors are introduced briefly. Third, according to the classification of active layer materials, linear polarization‐sensitive photodetectors are divided into 3D perovskites and low‐dimensional perovskites. The research on linear polarization‐sensitive perovskite detectors are reviewed. Finally, the developments and challenges faced by linear polarization‐sensitive perovskite photodetectors are analyzed.

Generation of circular symmetric Airy vortex beams based on spin-multiplexed metasurface

Given that circular symmetric Airy vortex beam (CSAVB) was found to possess a stable central cavity and an opposing transport process to circular Airy vortex beam, it has been recognized as a potentially powerful tool for particle trapping and optical communication. In order to facilitate broader application scenarios, this paper presents a novel approach to the generation of multiple CASVBs based on a single metasurface. In the proposed method, three independent CSAVBs carrying different new kinds of power-exponent-phase vortices (NPEPVs) can be obtained under two orthogonal circularly polarized or an arbitrary linear polarized incident, respectively. Each of these vortex structures is characterized by a distinct orbital angular momentum, which is jointly determined by two parameters of the NPEPVs. This indicates that the CSAVB and the device have greater freedom than the canonical optical vortex beams and the previous vortex beam generators in controlling the optical vortex. These results have the potential to advance the development of vortex beams and enhance their applications in micromanipulation, optical communication and imaging.

Integrating Digital Imaging and AI in Forensic Odontology: Enhancing Identification Accuracy and Efficiency

Background: The integration of digital imaging and artificial intelligence (AI) in forensic odontology can significantly enhance the accuracy and efficiency of dental identification processes. These technologies provide precise and rapid analysis, which is crucial for forensic investigations. However, implementing such technologies also introduces new ethical challenges that must be addressed. Purpose: To explore the synergistic use of digital imaging and artificial intelligence (AI) in forensic odontology. This section focuses on how these technologies improve identification accuracy and efficiency while discussing the ethical considerations that arise from their implementation. Methodology: This methodology involves a comprehensive review of the existing literature on the applications of digital imaging and AI in forensic odontology. Key studies, clinical trials, and case examples are analyzed to evaluate the effectiveness of these technologies in improving forensic outcomes. The review assesses these technologies’ impact on identification accuracy and efficiency while addressing ethical concerns such as data privacy and AI biases Results: The review highlights that the combination of digital imaging and AI significantly enhances the accuracy and speed of dental identification. Advanced algorithms can process large datasets and identify subtle patterns that are often missed by human examiners, resulting in more reliable forensic outcomes. However, ethical challenges, including data privacy concerns and potential biases in AI algorithms, are critical areas that require careful consideration and regulation. Ongoing research and policy development are essential to address these issues and ensure the ethical application of AI in forensic odontology.

Fourier-based beamforming for 3D plane wave imaging and application in vector flow imaging using selective compounding

Objective. Ultrafast ultrasound imaging using planar or diverging waves for transmission is a promising approach for efficient 3D imaging with matrix arrays. This technique has advantages for B-mode imaging and advanced techniques, such as 3D vector flow imaging (VFI). The computation load of the cross-beam technique is associated with the number of transmit anglesmand receive anglesn. The full velocity vector is obtained using the least square fashion. However, the beamforming is repeatedm × ntimes using a conventional time-domain delay-and-sum (DAS) beamformer. In the 3D case, the collection and processing of data from different beams increase the amount of data that must be processed, requiring more storage capacity and processing power. Furthermore, the large computation complexity of DAS is another major concern. These challenges translate into longer computational times, increased complexity in data processing, and difficulty in real-time applications.Approach. In response to this issue, this study proposes a novel Fourier domain beamformer for 3D plane wave imaging, which significantly increases the computational speed. Additionally, a selective compounding strategy is proposed for VFI, which reduces the beamforming process fromm × ntom(wheremandnrepresent the number of transmission and reception, respectively), effectively shortening the processing time. The underlying principle is to decompose the receive wavefront into a series of plane waves with different slant angles. Each slant angle can produce a sub-volume for coherent or selective compounding. This method does not rely on the assumption that the plane wave is perfect and the results show that our proposed beamformer is better than DAS in terms of resolution and image contrast. In the case of velocity estimation, for the Fourier-based method, only Tx angles are assigned in the beamformer and the selective compounding method produces the final image with a specialized Rx angle.Main results. Simulation studies andin vitroexperiments confirm the efficacy of this new method. The proposed beamformer shows improved resolution and contrast performance compared to the DAS beamformer for B-mode imaging, with a suppressed sidelobe level. Furthermore, the proposed technique outperforms the conventional DAS method, as evidenced by lower mean bias and standard deviation in velocity estimation for VFI. Notably, the computation time has been shortened by 40 times, thus promoting the real-time application of this technique. The efficacy of this new method is verified through simulation studies andin vitroexperiments and evaluated by mean bias and standard deviation. Thein vitroresults reveal a better velocity estimation: the mean bias is 2.3%, 3.4%, and 5.0% forvx,vy, andvz, respectively. The mean standard deviation is 1.8%, 1.7%, and 3.4%. With DAS, the evaluated mean bias is 9.8%, 4.6%, and 6.7% and the measured mean standard deviation is 7.5%, 2.5%, and 3.9%.Significance. In this work, we propose a novel Fourier-based method for both B-mode imaging and functional VFI. The new beamformer is shown to produce better image quality and improved velocity estimation. Moreover, the new VFI computation time is reduced by 40 times compared to conventional methods. This new method may pave a new way for real-time 3D VFI applications.

Corrigendum to "In Vivo Stem Cell Imaging Principles and Applications".

Corrigendum to "In Vivo Stem Cell Imaging Principles and Applications".

Retraction Note: Application of optical super-resolution imaging based on GPU parallel computing in AI motion training system

Retraction Note: Application of optical super-resolution imaging based on GPU parallel computing in AI motion training system

Efficient Broadband Near‐Infrared Emitters in Lead‐Free Metal Halides with Record Photoluminescence Quantum Yield Under Blue Light Excitation

AbstractRealizing ultra‐efficient broadband near‐infrared (NIR) luminescence, especially under blue light excitation, remains an enormous challenge in lead‐free metal halides. Herein, the efficient NIR emission under blue light excitation is achieved in Sb3+‐doped 0D (ETPP)2ZnCl4xBr4‐4x (x = 0–1) (ETPP+ = (Ethyl)triphenylphosphonium) through coordination structure modulation and halogen substitution. Compared with the visible light emission of Sb(III)‐based compounds, the emission in Sb3+‐doped (ETPP)2ZnCl4xBr4‐4x shifts to the NIR region due to the large excited state lattice distortion. Parallelly, the excitation bands gradually shift from 376 to 450 nm as Br gradually replaces Cl, and the emission bands can further shift from 702 to 763 nm. Thus, the broadband NIR emission at 763 nm with a record luminous efficiency of 55.4% under 450 nm excitation can be achieved in Sb3+‐doped compounds. Moreover, the large‐scale synthesis technique of Sb3+‐doped (ETPP)2ZnBr4: NIR phosphors at room temperature is further developed and this compound exhibits impressive thermal and chemical‐stabilities. Finally, a high‐performance NIR light‐emitting‐diode is fabricated by combining a commercial blue chip and (ETPP)2ZnBr4:10%Sb3+ phosphors, which shows the most advanced photoelectric efficiency (17.8%) and output power (67.7 mW) in lead‐free metal halides. Thus, the as‐fabricated device is further demonstrated in the applications of night vision and biomedical imaging.

PelviNet: A Collaborative Multi-agent Convolutional Network for Enhanced Pelvic Image Registration.

PelviNet introduces a groundbreaking multi-agent convolutional network architecture tailored for enhancing pelvic image registration. This innovative framework leverages shared convolutional layers, enabling synchronized learning among agents and ensuring an exhaustive analysis of intricate 3D pelvic structures. The architecture combines max pooling, parametric ReLU activations, and agent-specific layers to optimize both individual and collective decision-making processes. A communication mechanism efficiently aggregates outputs from these shared layers, enabling agents to make well-informed decisions by harnessing combined intelligence. PelviNet's evaluation centers on both quantitative accuracy metrics and visual representations to elucidate agents' performance in pinpointing optimal landmarks. Empirical results demonstrate PelviNet's superiority over traditional methods, achieving an average image-wise error of 2.8 mm, a subject-wise error of 3.2 mm, and a mean Euclidean distance error of 3.0 mm. These quantitative results highlight the model's efficiency and precision in landmark identification, crucial for medical contexts such as radiation therapy, where exact landmark identification significantly influences treatment outcomes. By reliably identifying critical structures, PelviNet advances pelvic image analysis and offers potential enhancements for broader medical imaging applications, marking a significant step forward in computational healthcare.

A tunable chiral metasurface useable in terahertz imaging and wavefront shaping

We proposed a tunable chiral metasurface comprising a reflective bottom layer of gold, a dielectric layer of polyimide, and a structural top layer of gold-graphene. Its main properties were studied via numerical simulations conducted using CST Studio Suite. The results indicate that, based on the chiral metasurface, we achieved dual-band circular dichroism of −0.5 and 0.77 at 0.9 THz and 1.06 THz, respectively, and complementary near-field imaging applications were realized by tuning the Fermi level (E f ) of graphene. Subsequently, exploiting the exceptional selective characteristics of circularly polarized waves using a chiral metasurface, eight chiral phase-gradient metasurfaces were constructed by rotating the chiral structure. Moreover, based on the Pancharatnam-Berry phase principle, tunable wavefront shaping applications were further realized, including anomalous reflection, vortex beams, and focusing. In anomalous reflection, the reflection angles for left-circularly polarized (LCP) and right-circularly polarized (RCP) incidences are opposite when adjusting the E f of graphene. For example, when the graphene E f is 0 eV and the LCP wave is incident at 0°, the reflection angle is −18°. Conversely, when the graphene E f is 1 eV and the RCP wave is incident at 0°, the reflection angle is 18°. In the application of vortex beams, by adjusting the E f of graphene, we achieved vortex beams with opposite topological charges under different circularly polarized incidences. In the focusing application, the incident LCP and RCP can achieve focusing and defocusing, respectively. And the graphene E f can dynamically control the focusing efficiency at the incident LCP, increasing it from 13.63% to 44.84%.

Calibration of FRET-based biosensors using multiplexed biosensor barcoding.

Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) is widely used in the design of genetically encoded fluorescent biosensors, which are powerful tools for monitoring the dynamics of biochemical activities in live cells. FRET ratio, defined as the ratio between acceptor and donor signals, is often used as a proxy for the actual FRET efficiency, which must be corrected for signal crosstalk using donor-only and acceptor-only samples. However, the FRET ratio is highly sensitive to imaging conditions, making direct comparisons across different experiments and over time challenging. Inspired by a method for multiplexed biosensor imaging using barcoded cells, we reasoned that calibration standards with fixed FRET efficiency can be introduced into a subset of cells for normalization of biosensor signals. Our theoretical analysis indicated that the FRET ratio of high-FRET species relative to non-FRET species slightly decreases at high excitation intensity, suggesting the need for calibration using both high and low FRET standards. To test these predictions, we created FRET donor-acceptor pairs locked in "FRET-ON" and "FRET-OFF" conformations and introduced them into a subset of barcoded cells. Our results confirmed the theoretical predictions and showed that the calibrated FRET ratio is independent of imaging settings. We also provided a strategy for calculating the FRET efficiency. Together, our study presents a simple strategy for calibrated and highly multiplexed imaging of FRET biosensors, facilitating reliable comparisons across experiments and supporting long-term imaging applications.