Fluorescence Localization Imaging via a Spectral-Splitting Perovskite Single-Pixel Detector

Fluorescence Localization Imaging via a Spectral-Splitting Perovskite Single-Pixel Detector | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Fluorescence Localization Imaging via a Spectral-Splitting Perovskite Single-Pixel Detector Xueli Chen, Yujin Liu, Hongling Wan, Jingyang Xing, Hao Wang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7217975/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Image-guided surgery systems require precise fusion of fluorescence mapping and structural background imaging, yet current dual-camera system methods suffer from field-of-view misalignment and pixel offsets, risking millimeter-scale surgical errors. To address these challenges, we developed an innovative fluorescence/background fusion single-pixel imaging system based on spectral-splitting perovskite photodetectors (PDs). By incorporating gradient-optimized wide-bandgap perovskite filter layers, the perovskite photodetector can achieve spectrally selective detection through efficient separation of 520 nm fluorescence signals ( S 1 ) from 450 nm backscattered light( S 2 ). With a high signal suppression ratio ( S 1 /S 2 ) of 57 times of the optimized PD, the system achieves a low detection limit of 50 nmol/mL for sodium fluorescein aqueous solution. More importantly, the exceptional active single-pixel imaging architecture ensures perfect field-of-view and pixel-level consistency across multiple detectors’ images, eliminating the need for complex registration algorithms or sophisticated dual-optical path designs. Finally, through in murine tumor experiments, we achieved precise fluorescence-labeled tumor localization imaging, demonstrating the reliability of our developed system in fluorescence/background fusion imaging. This provides a novel fluorescence-targeting approach for medical imaging, demonstrating the innovative utility of single-pixel imaging in advanced diagnostics. Physical sciences/Optics and photonics/Applied optics/Optical sensors Physical sciences/Physics/Techniques and instrumentation/Imaging techniques Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Surgical navigation represents a critical technological advancement for ensuring procedural precision and safety. Conventional systems primarily depend on spatial co-registration between preoperative imaging and intraoperative anatomical landmarks (such as MRI/CT) 1,2 . However, intraoperative tissue deformation, instrument obstruction, and physiological dynamics frequently cause spatial discrepancies between preoperative data and real-time field of view (FOV), significantly compromising clinical utility. Fluorescence imaging provides functional molecular visualization through targeted labeling of critical structures (tumor margins 3 – 7 , neural bundles 6 , 8 – 11 , vascular networks 6 , 12 – 15 ), yet suffers from inherent limitations: absence of anatomical context and vulnerability to ambient light interference, tissue autofluorescence, and nonspecific binding. While backscatter imaging offers structural reference, it lacks sensitivity for pathological discrimination. Consequently, developing a registration-free fluorescence/background fusion imaging system capable of simultaneous molecular punctuation of tumor information and anatomical structure information has emerged as a pivotal solution for advancing surgical navigation capabilities. Current multi-modal fluorescence/background fusion imaging approaches predominantly employ three conventional strategies, each presenting significant limitations for surgical applications:1) Dual-camera synchronous acquisition 7 , 16 : While offering high temporal resolution through beam-splitter optical paths, this method suffers from prohibitive hardware complexity, bulky form factors unsuitable for surgical integration, and substantial cost barriers. 2) Time-multiplexed single-camera imaging 17 : Utilizing filter wheels or broadband spectrometry for alternating signal capture inherently compromises temporal resolution and sacrifices dynamic synchronization, critical parameters for real-time surgical navigation. 3) Spectral-separation single-camera imaging 181920 : Although achieving theoretically synchronous imaging through on-chip filter arrays, this approach faces fundamental constraints including lower pixel resolution, complex microfabrication requirements and unacceptable channel crosstalk. These methods have the common challenges of high cost, difficulty in achieving synchronization, spatial registration accuracy and system complexity. Particularly in dual-camera systems, parallax-induced FOV misalignment necessitates complex post-acquisition registration algorithms a process further compromised by intraoperative tissue deformation and instrument movement. While spectral-splitting techniques circumvent geometric registration, their dependence on computationally intensive unmixing algorithms and vulnerability to ambient light render them unsuitable for robust surgical implementation. Fourier single-pixel imaging (FSPI) emerges as a transformative alternative, leveraging its unique optical architecture and computational imaging paradigm to address these limitations. In this context, FSPI technology offers an innovative solution for operative navigation due to its unique optical architecture and computational imaging algorithm. FSPI combines structured light modulation with single-pixel photodetectors (PDs) and reconstructs images based on the spatial frequency features of Fourier-based structured light patterns 21 , 22 . The fundamental advantage lies in its spatial consistency, the imaging geometry (FOV and pixel dimensions) is solely determined by the encoded spatial light patterns while fluorescence and background signals share the same optical path 23 . This intrinsic property ensures perfect spatial registration when acquiring multi-spectral images using PDs with different spectral responses, completely eliminating alignment errors. Furthermore, single-pixel PDs offer compact size, low cost, and strong interference resistance, overcoming the limitations of conventional optical systems (high cost, complex configuration, and bulky size) while guaranteeing multi-channel synchronization. Building upon these considerations, conventional multimodal imaging systems predominantly utilize array-based image sensors such as charge-coupled devices (CCD) and complementary metal-oxide-semiconductor (CMOS) detectors for fluorescence and background signal acquisition. Beyond the aforementioned limitations in spectral separation, these array sensors suffer from fundamental constraints: 1) the small photosensitive area of individual pixels in high-resolution arrays severely limits detection sensitivity, 2) inadequate photon capture capability for weak fluorescence signals, and 3) substantial readout noise that can obscure faint biological signals, collectively rendering them suboptimal for fluorescence detection applications. Last decade, metal halide perovskites have demonstrated transformative potential across optoelectronic applications including photovoltaics, light-emitting diodes (LEDs) 24 , PDs 25 , 26 , and lasers 27 , owing to their exceptional optoelectronic properties (such as high light absorption coefficient, high mobility, low binding energy, adjustable band gap) coupled with cost-effective solution processability 28 , 29 . For photodetection applications, precise composition engineering enables spectral response tuning to match target fluorescence and background bands. Furthermore, perovskite PDs integrated with single-pixel imaging have demonstrated remarkable potential across various imaging applications, including bionic vision systems 30 , wide-field imaging 31 , and visible-near-infrared dual-modal imaging 32 . More importantly, unlike CMOS arrays where 30–50% of the surface area is occupied by circuitry, perovskite thin films achieve near-unity fill factors (> 90%), offering superior performance for weak fluorescence signal detection. Here, we present an intraoperative fluorescence-background fusion imaging system based on metal halide perovskite photodetectors (PVK PDs) and active-mode single-pixel computational imaging, referred to as the perovskite single-pixel fluorescence-background fusion imaging system (PVK-SPI-FB System). The system architecture incorporates perovskite filter layers (PFLs) for spectral splitting of background and fluorescence signals, enabling synchronous dual-modal acquisition through two PDs. Experimental results demonstrate that the inherent characteristics of FSPI ensure perfect spatial congruence between fluorescence and background images obtained from different detectors, with identical FOV and pixel-level registration, eliminating the need for complex image alignment algorithms and significantly improving workflow efficiency. In murine validation confirms the system's clinical potential, establishing a novel paradigm for intraoperative multi-modal imaging and advancing the application of FSPI in surgical and biomedical fields. 2. Results and Discussion 2.1 PVK-SPI-FB System This study takes sodium fluorescein as an example to study the fusion of fluorescence/background. Figure 1 a illustrates the fundamental principle of fluorescence emission: under excitation at specific wavelengths, electrons in the fluorophore absorb photons and transition to higher energy states, followed by rapid return to the ground state with release of excess energy as photons. During this process, non-radiative relaxation (e.g., internal conversion) dissipates partial energy as heat, resulting in emitted photons with lower energy (longer wavelength) than the excitation photons. This energy difference, known as the Stokes shift, enables spectral separation between excitation and emission wavelengths, allowing distinct acquisition of fluorescence and background signals through spectrally optimized PDs. As shown in Fig. 1 b, conventional approaches typically employ two CCD cameras to simultaneously capture fluorescence and background images. Here, the fluorescence channel camera requires a high-performance short-wave cutoff filter (taking sodium fluorescein’s absorption/emission wavelengths as an example, market research indicates typical unit prices ranging from ¥3,000 to ¥5,000 RMB) to completely block background interference from the excitation wavelength band. Moreover, due to the inherent challenge of achieving consistent viewing angles between cameras, fluorescence and background images exhibit spatial mismatches that preclude precise alignment, fundamentally limiting fluorescence localization imaging accuracy. In addition, due to differing sensitivity requirements between the fluorescence and background cameras, the two systems often employ distinct camera models, resulting in a severe pixel count mismatch between fluorescence and background images. Conventional multi-camera fluorescence-background fusion imaging systems face three inherent challenges: signal crosstalk, perspective misalignment, and pixel resolution disparity. These issues collectively lead to significant image registration errors and degraded fusion quality, as illustrated in Fig. 1 c. To address the inherent image fusion challenges of conventional dual-camera systems, we developed a novel PVK PD-based single-pixel imaging system for fluorescence-background fusion imaging (PVK-SPI-FB System) (Fig. 1 d), which integrates a light source module for simultaneous fluorescence excitation and backscattered signal generation, a digital micromirror device (DMD) for generating pre-encoded Fourier-based patterns carrying spatial information, a perovskite filtered detector (PVKF-PD) specifically for fluorescence signal detection, and a perovskite broadband detector (PVK-PD) for backscattered signal acquisition. The system features p-i-n structured photovoltaic perovskite detectors (PVK PDs) (Fig. 1 e) that offer exceptional response speed and sensitivity for weak fluorescence detection while their self-powered operation significantly simplifies the imaging architecture. The fabrication process of PVK PDs is shown in Fig. S1 . Figure S2 shows the survey XRD spectra of the perovskite PD layer (Cs 0.05 FA 0.79 MA 0.16 PbI 2.5 Br 0.5 ). Building on our previous findings demonstrating the inherent FOV invariance and perfect pixel registration advantages of active-mode single-pixel imaging systems 30 , this integrated solution effectively overcomes the critical fusion limitations between fluorescence and background images. Specifically, active-mode single-pixel imaging is that the image FOV and pixel dimensions are determined solely by the spatial illumination patterns rather than location of PDs. This fundamental characteristic enables the two PVK PDs to simultaneously acquire perfectly registered fluorescence and background images with identical spatial coordinates, eliminating the need for post-acquisition registration algorithms. Fluorescence imaging is typically actively excited as well, making the integration of active-mode single-pixel imaging systems particularly promising for perfectly resolving the fusion and registration challenges between fluorescence and background images, as illustrated in Fig. 1 f. 2.2 Spectral-Splitting mechanism of perovskite single-pixel PDs In addition to adopting single-pixel imaging methodology to resolve image registration challenges, we have developed spectral-splitting PVK PDs that fundamentally addresses the critical issue of fluorescence-background signal separation. Unlike conventional approaches relying on costly optical filters, our innovative solution employs an intrinsic wavelength self-filtering strategy enabled by the unique bandgap tunability of perovskite materials. Through controlled ion-exchange processes, we engineered two functionally distinct perovskite films: The small-bandgap perovskite films can be fabricated into broadband PDs (PVK-PD) specifically optimized for detecting the dominant backscattered signals. By strategically combining the small-bandgap PVK-PD with large-bandgap perovskite films exhibiting short-wavelength cutoff characteristics, we developed a novel narrowband PVK PD (PVKf-PD). This innovative design effectively filters out the shorter-wavelength excitation backscattered light while selectively transmitting the desired fluorescence signals, enabling pure fluorescence detection as demonstrated in Fig. 2 d. However, the intensity of backscattered signals significantly exceeds that of fluorescence signals (approximately 17-fold greater, Fig. 2 i), necessitating the use of deep-cutoff short pass filters to effectively suppress the backscattered components, a key factor contributing to the high cost of conventional solutions. Perovskite materials offer unique advantages in this regard, as their bandgap tunability enables precise optimization of short-wavelength cutoff characteristics through simple precursor concentration modulation. To systematically investigate this property, we developed five distinct large-bandgap PFLs with varying precursor concentrations (1: 0.75 M, 2: 1 M, 3: 1.25 M, 4: 1.5 M, 5: 1.75 M) and characterized their transmission spectra to evaluate the short-wavelength blocking and long-wavelength transmission capabilities. As shown in Fig. 2 e, increasing the precursor concentration leads to enhanced film thickness and improved short-wavelength cutoff performance, albeit at the cost of reduced long-wavelength transmittance. This trade-off prompted a comprehensive optimization study across nine concentration gradients of PFLs, with detailed analysis of their spectral filtering characteristics (Fig. S3). The results reveal a non-monotonic trend in the transmission ratio between 640 nm and 450 nm as the concentration increases, exhibiting an initial rise followed by a decline (Fig. S3(b)). Through comprehensive evaluation of both long-wavelength fluorescence transmission and short-wavelength excitation suppression, PFL5 demonstrates optimal spectral-splitting capability in this study. Figure 2 e presents the transmission curves of five representative concentration-gradient PFLs, along with the spectrum curves of the narrowband PVK PDs, where the intersection region between transmission and absorption curves defines the spectral response window for fluorescence detection. Through systematic optimization of PFLs, we developed six distinct PVK PDs, including broadband perovskite detectors (PVK-PD) and narrowband fluorescence detectors incorporating PFLs (PVKf-PDs). As illustrated in Fig. 2 f, the spectral response curves of these six PDs demonstrate that the incorporation of PFLs significantly suppresses the device's detection capability for short-wavelength backscattered signals. The detailed variations in detector response parameters and corresponding signal suppression ratios are summarized in Table S1 , with PVKf-PD5 exhibiting optimal signal separation performance among all devices. Furthermore, we characterized the transient photoresponse properties of PVK-PD, as shown in Fig. 2 g, which exhibits an exceptionally fast rise time of 44 µs. The linear dynamic range (LDR) of PVK-PD was measured to reach 178.2 dB (Fig. 2 h), enabling stable operation across an irradiance range from 5.3×10 − 6 mW cm − 2 to 4.36×10 3 mW cm − 2 while maintaining excellent detection performance. Additional performance characterizations of the PVK-PD are presented in Figure S4,5. These results collectively demonstrate the outstanding photovoltaic performance of PVK-PD. To evaluate the practical performance of PVK-PDs in separating backscattered and fluorescence signals, we conducted signal measurements under real-world fluorescence imaging conditions. Figure 2 i presents the fluorescence and backscattered signal intensities obtained from sodium fluorescein aqueous solution with varying concentrations (100, 50, 10, and 1 µmol/mL) under 450 nm excitation light at 11.09 mW cm − 2 intensity, with detailed values provided in Table S2. The results clearly demonstrate that the fluorescence signal intensity is significantly weaker than the backscattered signal, highlighting the critical requirement for PVKf-PDs to possess exceptional signal discrimination capability. Figure 2j1 demonstrates the fluorescence and background signal intensities detected by the broadband-responsive PVK-PD, revealing that the PD primarily captures backscattered signals with negligible fluorescence intensity, indicating its suitability for single-pixel background imaging. Figure 2j2-6 illustrate the detection capabilities of PVKf-PDs for both fluorescence and background signals, where progressive optimization of the PFL thickness leads to a complete inversion of signal intensity ratios in PVKf-PD5. This remarkable transition demonstrates the detector's full capability for single-pixel fluorescence detection, with the optimized PVKf-PD5 exhibiting superior performance in selectively capturing fluorescence signals while effectively suppressing background interference. The systematic improvement across the device series highlights the critical role of PFL engineering in achieving high-purity fluorescence detection. The quantitative relationship governing this transition is delineated in Fig. 2 k. 2.3 Imaging performance of 2.1 PVK-SPI-FB System Based on the aforementioned research findings, the PVK-PD and PVKf-PD5 are selected as the background signal detector and fluorescence detector, respectively, and subsequently integrated into our perovskite-based single-pixel imaging system for systematic evaluation of its imaging performance. Figure 3 ​presents the performance evaluation of our fluorescence/background dual-modal imaging system based on PVK single-pixel PDs. The two-dimensional images were reconstructed using a four-step phase-shifting Fourier algorithm. Specifically, by measuring the light intensity values D θ (θ = 0, π/2, π, 3π/2) for each structured illumination pattern, we computed the Fourier coefficients α=[D 0 -D π ] + j·[D π/2 - D 3π/2 ] , and subsequently reconstructed the spatial-domain images via inverse Fourier transform (IFT), as illustrated in Fig. 3 a. Figure S6 presents the schematic diagram of the imaging algorithm. In Fourier spectral analysis, the low-frequency components govern the overall image profile, whereas the high-frequency components encode fine structural details. Consequently, the number of projected structured illumination patterns (M), which directly correlates with the sampling rate, determines the resolution of the reconstructed images. Figure 3 b, c demonstrates the system’s imaging performance for sodium fluorescein aqueous solutions (100, 50, 10, and 1 µmol/mL) loaded in Eppendorf (EP) tubes under visible-light illumination. Under visible-light illumination, the fluorescence signals were overwhelmed by backscattered light, resulting in reconstructed images that only retained the outline of the EP tubes. Notably, as M increased, the image resolution improved significantly, with progressively sharper contours - a finding that validates the sampling theorem of FSPI. Figure 3 d schematically illustrates the dual-modal single-pixel fluorescence/background imaging system, which employs a 450 nm excitation light source. The imaging target, identical to that shown in Fig. 3 c, consisted of four EP tubes containing sodium fluorescein aqueous solutions with sequentially decreasing concentrations from left to right. As demonstrated in Fig. 3 e, the PVK-PD and PVKf-PD single-pixel detectors were employed for backscattered signal and fluorescence detection, respectively. The PVK-PD, serving as the background signal PD, failed to reconstruct complete EP tube contours due to severe localized glare signals caused by the smooth tube surfaces, resulting in substantially reduced image contrast. Notably, since the glare signals fundamentally originate from backscattered light, the progressively enhanced filtering capability of PVKf-PDs successfully suppressed these interference artifacts, thereby enabling clear fluorescence imaging. Furthermore, we performed image fusion between the white-light background image obtained in Fig. 3 c and the fluorescence image captured by PVKf-PD5, as demonstrated in Fig. 3 e-7. These results preliminarily illustrate the unique fusion advantages of our developed PVK-SPI-FB System. Notably, in practical biological fluorescence imaging scenarios, glare artifacts are typically absent, enabling fluorescence/background localization imaging under single-excitation illumination conditions - a capability that will be comprehensively demonstrated in Fig. 4 . Figure 3 f displays 3D grayscale intensity mappings of the selected regions (boxed areas in Fig. 3 e), providing quantitative evaluation of PVKf-PDs' glare suppression capability. Figure 3 g​ establishes a quantitative correlation between sodium fluorescein aqueous solution concentration and image grayscale values to evaluate PVK-SPI-FB System sensitivity. Based on the detection limit of our PVKf-PD5-based PVK-SPI-FB system and fitted curve analysis, the system demonstrates a remarkable detection threshold of 0.05 µmol/mL, highlighting its exceptional low-light detection capability. 2.4 Biomedical imaging results of PVK-SPI-FB System ​​ Conventional multi-camera imaging systems​are inherently plagued by spatial registration challenges. As illustrated in Fig. 4 a, b, the inability to maintain strict co-axial alignment among different cameras introduces variations in the optical paths of reflected light received by each device. This inevitably leads to FOV misalignment and spatial information discrepancies in the acquired images, posing significant obstacles for multi-modal image fusion. In stark contrast, our developed PVK-SPI-FB System ensures complete spatial registration and pixel-level correspondence across images acquired by PDs at different positions. Specifically, their spatial information is entirely determined by the pre-defined structured light patterns, rendering them independent of PD positioning. This unique characteristic enables multiple PDs positioned at distinct spatial locations to acquire image data with perfectly matched FOV and pixels alignment (Fig. 4 c, d, Fig. S7). The experiments in Fig. S8 further corroborate the aforementioned conclusions. Experimental results demonstrate that these images can be seamlessly fused through direct superposition, unequivocally validating the distinctive advantages of single-pixel imaging for multi-modal image integration. Building upon these foundations, we designed an in murine model to validate the practical utility of this imaging methodology, The experimental setup is illustrated in Fig. 1 d and Fig. S9. Three tumor-simulating models were established through injections in the left hindlimb, right hindlimb, and dorsal region of the mice, with sodium fluorescein serving as the tumor-marker via local injection. Consequently, the luminescent areas in fluorescence images exclusively represented the tumor-simulating regions. Prior to formal experiments, this study conducted preliminary validation tests using chicken wings, with the results presented in Fig. S10. Our system successfully acquired both diffuse reflectance background images and specific fluorescence images simultaneously (Fig. 4 e-m). As demonstrated in Fig. 4 n-p, the fused images enabled clear discrimination between marked regions and normal tissues. Quantitative analysis of fluorescence signal intensity distributions permitted precise determination of both geometric dimensions and spatial coordinates of the tumor-simulating models, thereby providing reliable data support for surgical navigation applications. 3. Conclusion ​​This study developed a fluorescence-background dual-modal imaging system based on perovskite-based single-pixel PDs, successfully addressing the critical challenge of multi-modal image fusion in conventional surgical navigation. Our single-pixel imaging system inherently overcomes the pixel registration and spatial alignment challenges inherent in conventional dual-camera fluorescence-background fusion imaging, enabling precise tumor fluorescence localization. To address the spectral overlap between fluorescence and background signals, we developed self-filtering PVK-PDs that achieved efficient spectral splitting between fluorescence emission (520 nm) and backscattered excitation light (450 nm). The optimized PVKf-PD demonstrates exceptional performance, with a backscattered light suppression ratio of 57 times (responsivity ratio at 640/450 nm) and a fluorescence sensitivity of 107 mA W − 1 at 520 nm (PVKf-PD5). Ultimately, In murine experiments successfully overcame the detection challenges posed by tissue-induced fluorescence attenuation, enabling precise localization imaging of tumor-simulating models.​This work presents a novel imaging solution for intraoperative real-time navigation, characterized by hardware simplicity, cost-effectiveness, and superior performance, demonstrating significant potential for applications in surgical oncology and precision medicine. 4. Experiment 4.1 PVK PDs preparation: The fabrication process of the Cs 0.05 FA 0.79 MA 0.16 PbI 2.5 Br 0.5 PVK PD was carried out as follows: First, a homogeneous precursor solution was prepared by dissolving 516 mg PbI₂, 171 mg FAI, 23.2 mg MABr, and 73.4 mg PbBr₂ in 1 mL of a DMF/DMSO mixed solvent (3:1 v/v) with continuous stirring at 60°C for 5 h, followed by the addition of 39 μL CsI (390 mg/mL in DMSO) and subsequent overnight stirring at 60°C. The device was fabricated through sequential spin-coating of functional layers: A PTAA hole transport layer was first deposited at 4000 rpm for 30 s, followed by thermal annealing at 100°C for 10 min. The perovskite active layer was then formed via a two-step spin-coating process - initial coating at 500 rpm for 6 s followed by 5000 rpm for 45 s, with 200 μL chlorobenzene anti-solvent dripping during the last 10 s, and subsequent annealing at 110°C for 60 min. Subsequently, a PCBM electron transport layer was spin-coated at 2000 rpm for 30 s and annealed at 65°C for 3 min, followed by deposition of a BCP modification layer at 3000 rpm for 30 s. Finally, a 100 nm-thick Ag electrode was thermally evaporated to complete the device fabrication. By spin-coating MAPbBr 2.4 Cl 0.6 precursor solutions with concentrations of 0.75, 1, 1.26, 1.5, and 1.75 mol/L, PVK filter layers with different optical filtering effects were obtained. 4.2 Imaging system: The schematic diagram of the experimental setup is shown in Fig. 1d, which mainly consists of a spatial light modulator (SLM), two PVK PDs (PVK-PD and PVKf-PD), a data acquisition system, a computer, and a white light source. The excitation blue light (~435–465 nm) was obtained by filtering the white light. A DMD (V-7001 VIS, ViALUX) with a projection rate of up to 22 kHz was used as the SLM. First, a set of four-step phase-shifted sinusoidal patterns was generated by the computer and loaded onto the DMD for structured light modulation. The PVK-PD and PVKf-PD collected and recorded the light intensity values of each structured light pattern through a multi-channel acquisition system. The set of current intensities was referred to as the I-m curve, where m is the pattern number. Finally, the I-m curve was processed using a Fourier transform algorithm to reconstruct the fluorescence and background images. A simple superposition of the two images yielded the fused image. 4.3 Imaging experiments: First, in vitro experiments were conducted using sodium fluorescein aqueous solutions at four concentrations (100, 50, 10, and 1 μmol/mL) as imaging targets. Subsequently, in murine experiments were performed by mixing agar solution sodium fluorescein aqueous solutions at a 1:1 volume ratio. A 150 μL aliquot of the mixture was subcutaneously injected into the right hind limb, left hind limb, and back of mice to establish tumor-mimicking models. Animal experiments were approved by Experimental Animal Ethics Committee of Chengdu Dossy Experimental Animals Company Limited in accordance with the Ethical Guidelines for Animal Care (NO.CDDEACL2024-16). Declarations Supporting Information Supporting Information is available from the Wiley Online Library or from the author. Acknowledgements Authors acknowledge the financial supports from the National Natural Science Foundation of China (62405236, 62275210, 62375210); the National Leading Talent Program, the National Young Talent Program, the Key Research and Development Program of Shaanxi (2024SF2-GJHX-25), the Shaanxi Young Top-notch Talent Program; Xidian University Specially Funded Project for Interdisciplinary Exploration (TZJH2024059, TZJH2024060); Fundamental Research Funds for Central Universities (QTZX24079). Notes The authors declare no competing financial interests. Data availability Data available on request from the authors. References Souzaki R, Ieiri S, Uemura M et al (2013) An augmented reality navigation system for pediatric oncologic surgery based on preoperative CT and MRI images[J]. 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Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYDACdgY2IGkDJBMbQHzGBoJamMFa0kjXchiIExiI02JwmPnZg487zif2sSc3fi5gsJHdcAAogk+LZDObueHMM7eN2XgeNkvPYEgz3nCAzdwAnxZ+Zh42ad6223JsEokN0jwMhxM3HOBhk8CnhQ2k5W/bOaCyxObfPAz/CWsB28LYdgBkSxvQlgOEtQD9YibZ25YM8kubNY9BsvHMw2xmeLUYHG9+JvGzzS5xfnv649s8FXayfSARfFrQTWAAxdQoGAWjYBSMAkoBABu7Pr5WpQnXAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-3898-9892","institution":"Xidian University","correspondingAuthor":true,"prefix":"","firstName":"Xueli","middleName":"","lastName":"Chen","suffix":""},{"id":534102163,"identity":"5d843811-eb7e-4fed-8b8a-bb1374317c8c","order_by":1,"name":"Yujin Liu","email":"","orcid":"","institution":"XIDIAN UNIVERSITY","correspondingAuthor":false,"prefix":"","firstName":"Yujin","middleName":"","lastName":"Liu","suffix":""},{"id":534102164,"identity":"68872da3-4167-47c7-b9c0-4655e653fa01","order_by":2,"name":"Hongling 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12:57:20","extension":"html","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":94757,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7217975/v1/66547558a22062f6ba453383.html"},{"id":94354952,"identity":"2d9b6f6f-b6f4-4f29-b38c-dca1f97d5b13","added_by":"auto","created_at":"2025-10-27 12:56:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":909521,"visible":true,"origin":"","legend":"\u003cp\u003ePerovskite single-pixel fluorescence-background fusion imaging method. \u003cstrong\u003ea.\u003c/strong\u003e The luminescence principle of the fluorescent probe (sodium fluorescein). \u003cstrong\u003eb.\u003c/strong\u003e System architecture of the conventional dual-camera setup for simultaneous fluorescence/background imaging. \u003cstrong\u003ec.\u003c/strong\u003eChallenges in fusing fluorescence and background images acquired by dual-camera systems. \u003cstrong\u003ed.\u003c/strong\u003e Our proposed PVK-SPI-FB System. \u003cstrong\u003ee.\u003c/strong\u003e Schematic diagram of the structure of PVK-PD. \u003cstrong\u003ef.\u003c/strong\u003e PVK-SPI-FB System effectively resolves the fusion challenges between fluorescence and background images.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7217975/v1/aec5979759606b5ad9a4e392.png"},{"id":94354707,"identity":"bf4e3ee4-f599-4bcb-9e73-635109605bca","added_by":"auto","created_at":"2025-10-27 12:56:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":699294,"visible":true,"origin":"","legend":"\u003cp\u003eSpectral-splitting PVK PDs \u003cstrong\u003ea.\u003c/strong\u003e The generation process of fluorescence/backscattered light. \u003cstrong\u003eb.\u003c/strong\u003e The spectral curves of the fluorescence signal and excitation light signal of sodium fluorescein. \u003cstrong\u003ec. \u003c/strong\u003eSchematic illustration of photon absorption and electron-hole separation mechanism in broadband PVK-PD for background signal detection. \u003cstrong\u003ed.\u003c/strong\u003e Working principle of filtered PVKf-PD with additional PKL for background rejection and selective fluorescence detection, showing photon absorption and charge separation processes. \u003cstrong\u003ee.\u003c/strong\u003e The absorption spectrum of PVK film and the transmission spectrum of PVK filter films (Film 1-5 correspond to precursor concentrations ranging from 0.75 to 1.75 M). \u003cstrong\u003ef.\u003c/strong\u003e Experimental wavelength-dependence responsivity curves of PVK-PD and PVKf-PDs.\u003cstrong\u003e g. \u003c/strong\u003eTransient photoresponse curves of PVK-PD. \u003cstrong\u003eh.\u003c/strong\u003e Linear dynamic range (LDR) curve of PVK-PD. \u003cstrong\u003ei. \u003c/strong\u003eMeasured backscattered signal intensity (Signal 1) and fluorescence intensity (Signal 2) from s sodium fluorescein aqueous solution\u003cbr\u003e\ns at varying concentrations under 11.09 mW cm\u003csup\u003e-2\u003c/sup\u003e excitation power, detected by a reference PD at the imaging position.\u0026nbsp; \u003cstrong\u003ej1.\u003c/strong\u003e Fluorescence and background signal intensities detected by broadband PVK-PD across different sodium fluorescein concentrations\u003cbr\u003e\n. \u003cstrong\u003ej2-j6.\u003c/strong\u003e Fluorescence/background signal intensities measured by filtered PVKf-PDs with progressive optimization across different sodium fluorescein concentrations. \u003cstrong\u003ek.\u003c/strong\u003e Complete signal inversion between background and fluorescence intensities in PVKf-PD5 compared to PVK-PD at all tested concentrations, confirming successful development of spectrally-selective PDs.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7217975/v1/8556fbea7de49e1451874546.png"},{"id":94355291,"identity":"8c0b94fc-c07c-44d0-97e2-c46fa69d4488","added_by":"auto","created_at":"2025-10-27 12:57:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1394531,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence and background imaging results based on PVK PDs. \u003cstrong\u003ea.\u003c/strong\u003e FSPI principle. \u003cstrong\u003eb-c. \u003c/strong\u003eInvestigation of the relationship between M and reconstructed image resolution under white excitation light at 477.46 mW cm\u003csup\u003e−2\u003c/sup\u003e irradiance. \u003cstrong\u003ed-e. \u003c/strong\u003eSchematic diagram of imaging device and the imaging results obtained by PDs under 450 nm excitation light at 11.09 mW cm\u003csup\u003e−2\u003c/sup\u003e irradiance. \u003cstrong\u003ef. \u003c/strong\u003e3D intensity mapping of the corresponding boxed area in Fig. e. \u003cstrong\u003eg.\u003c/strong\u003e Functional relationship between the concentration of sodium fluorescein aqueous solutions and the grayscale value of the image.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7217975/v1/d3869a5f76ecdcea77129561.png"},{"id":94354956,"identity":"8bd588a2-6920-4a9c-b8a1-3c545ae27a1f","added_by":"auto","created_at":"2025-10-27 12:56:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2059785,"visible":true,"origin":"","legend":"\u003cp\u003eThe Spatial consistency advantages of the PVK-SPI-FB System and its application in tumor localization imaging via fluorescence-background fusion. \u003cstrong\u003ea-b.\u003c/strong\u003e Schematic diagram of a multi-camera imaging system and its spatial registration results. \u003cstrong\u003ec-d.\u003c/strong\u003e Structural illustration of a multi-detector single-pixel imaging system and corresponding spatial registration outcomes. \u003cstrong\u003ee-g.\u003c/strong\u003e Commercial camera-captured images of tumor-simulating models injected in the left hindlimb, right hindlimb, and dorsal region of mice.\u003cstrong\u003e h-p.\u003c/strong\u003e Background imaging results(h-j), Fluorescence imaging results(k-m), Dual-modal fused images(n-p) of our designed PVK-SPI-FB Syetem. \u003cstrong\u003eq-s.\u003c/strong\u003e 3D intensity mapping corresponding to the fluorescent regions in images of k-m.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7217975/v1/4cf10aff516f830dbab6cec9.png"},{"id":94440744,"identity":"86a602b0-c252-475e-850e-45e90fda36a4","added_by":"auto","created_at":"2025-10-27 14:24:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6323179,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7217975/v1/a097e4b8-39d4-4567-b00f-31b89055c094.pdf"},{"id":94355284,"identity":"d5366442-5735-4b37-be51-ac72ca9c4ee5","added_by":"auto","created_at":"2025-10-27 12:57:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1614425,"visible":true,"origin":"","legend":"Fluorescence Localization Imaging via a Spectral-Splitting Perovskite Single-Pixel Detector","description":"","filename":"SI.docx","url":"https://assets-eu.researchsquare.com/files/rs-7217975/v1/346a45682664a167276d98fe.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Fluorescence Localization Imaging via a Spectral-Splitting Perovskite Single-Pixel Detector","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSurgical navigation represents a critical technological advancement for ensuring procedural precision and safety. Conventional systems primarily depend on spatial co-registration between preoperative imaging and intraoperative anatomical landmarks (such as MRI/CT)\u003csup\u003e1,2\u003c/sup\u003e. However, intraoperative tissue deformation, instrument obstruction, and physiological dynamics frequently cause spatial discrepancies between preoperative data and real-time field of view (FOV), significantly compromising clinical utility. Fluorescence imaging provides functional molecular visualization through targeted labeling of critical structures (tumor margins\u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, neural bundles\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, vascular networks\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e), yet suffers from inherent limitations: absence of anatomical context and vulnerability to ambient light interference, tissue autofluorescence, and nonspecific binding. While backscatter imaging offers structural reference, it lacks sensitivity for pathological discrimination. Consequently, developing a registration-free fluorescence/background fusion imaging system capable of simultaneous molecular punctuation of tumor information and anatomical structure information has emerged as a pivotal solution for advancing surgical navigation capabilities.\u003c/p\u003e\u003cp\u003eCurrent multi-modal fluorescence/background fusion imaging approaches predominantly employ three conventional strategies, each presenting significant limitations for surgical applications:1) Dual-camera synchronous acquisition\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e: While offering high temporal resolution through beam-splitter optical paths, this method suffers from prohibitive hardware complexity, bulky form factors unsuitable for surgical integration, and substantial cost barriers. 2) Time-multiplexed single-camera imaging\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e: Utilizing filter wheels or broadband spectrometry for alternating signal capture inherently compromises temporal resolution and sacrifices dynamic synchronization, critical parameters for real-time surgical navigation. 3) Spectral-separation single-camera imaging\u003csup\u003e181920\u003c/sup\u003e: Although achieving theoretically synchronous imaging through on-chip filter arrays, this approach faces fundamental constraints including lower pixel resolution, complex microfabrication requirements and unacceptable channel crosstalk. These methods have the common challenges of high cost, difficulty in achieving synchronization, spatial registration accuracy and system complexity. Particularly in dual-camera systems, parallax-induced FOV misalignment necessitates complex post-acquisition registration algorithms a process further compromised by intraoperative tissue deformation and instrument movement. While spectral-splitting techniques circumvent geometric registration, their dependence on computationally intensive unmixing algorithms and vulnerability to ambient light render them unsuitable for robust surgical implementation.\u003c/p\u003e\u003cp\u003eFourier single-pixel imaging (FSPI) emerges as a transformative alternative, leveraging its unique optical architecture and computational imaging paradigm to address these limitations. In this context, FSPI technology offers an innovative solution for operative navigation due to its unique optical architecture and computational imaging algorithm. FSPI combines structured light modulation with single-pixel photodetectors (PDs) and reconstructs images based on the spatial frequency features of Fourier-based structured light patterns\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The fundamental advantage lies in its spatial consistency, the imaging geometry (FOV and pixel dimensions) is solely determined by the encoded spatial light patterns while fluorescence and background signals share the same optical path\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. This intrinsic property ensures perfect spatial registration when acquiring multi-spectral images using PDs with different spectral responses, completely eliminating alignment errors. Furthermore, single-pixel PDs offer compact size, low cost, and strong interference resistance, overcoming the limitations of conventional optical systems (high cost, complex configuration, and bulky size) while guaranteeing multi-channel synchronization.\u003c/p\u003e\u003cp\u003eBuilding upon these considerations, conventional multimodal imaging systems predominantly utilize array-based image sensors such as charge-coupled devices (CCD) and complementary metal-oxide-semiconductor (CMOS) detectors for fluorescence and background signal acquisition. Beyond the aforementioned limitations in spectral separation, these array sensors suffer from fundamental constraints: 1) the small photosensitive area of individual pixels in high-resolution arrays severely limits detection sensitivity, 2) inadequate photon capture capability for weak fluorescence signals, and 3) substantial readout noise that can obscure faint biological signals, collectively rendering them suboptimal for fluorescence detection applications.\u003c/p\u003e\u003cp\u003eLast decade, metal halide perovskites have demonstrated transformative potential across optoelectronic applications including photovoltaics, light-emitting diodes (LEDs)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, PDs\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, and lasers\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, owing to their exceptional optoelectronic properties (such as high light absorption coefficient, high mobility, low binding energy, adjustable band gap) coupled with cost-effective solution processability\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. For photodetection applications, precise composition engineering enables spectral response tuning to match target fluorescence and background bands. Furthermore, perovskite PDs integrated with single-pixel imaging have demonstrated remarkable potential across various imaging applications, including bionic vision systems\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, wide-field imaging\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, and visible-near-infrared dual-modal imaging\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. More importantly, unlike CMOS arrays where 30\u0026ndash;50% of the surface area is occupied by circuitry, perovskite thin films achieve near-unity fill factors (\u0026gt;\u0026thinsp;90%), offering superior performance for weak fluorescence signal detection.\u003c/p\u003e\u003cp\u003eHere, we present an intraoperative fluorescence-background fusion imaging system based on metal halide perovskite photodetectors (PVK PDs) and active-mode single-pixel computational imaging, referred to as the perovskite single-pixel fluorescence-background fusion imaging system (PVK-SPI-FB System). The system architecture incorporates perovskite filter layers (PFLs) for spectral splitting of background and fluorescence signals, enabling synchronous dual-modal acquisition through two PDs. Experimental results demonstrate that the inherent characteristics of FSPI ensure perfect spatial congruence between fluorescence and background images obtained from different detectors, with identical FOV and pixel-level registration, eliminating the need for complex image alignment algorithms and significantly improving workflow efficiency. In murine validation confirms the system's clinical potential, establishing a novel paradigm for intraoperative multi-modal imaging and advancing the application of FSPI in surgical and biomedical fields.\u003c/p\u003e"},{"header":"2. Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 PVK-SPI-FB System\u003c/h2\u003e\u003cp\u003eThis study takes sodium fluorescein as an example to study the fusion of fluorescence/background. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea illustrates the fundamental principle of fluorescence emission: under excitation at specific wavelengths, electrons in the fluorophore absorb photons and transition to higher energy states, followed by rapid return to the ground state with release of excess energy as photons. During this process, non-radiative relaxation (e.g., internal conversion) dissipates partial energy as heat, resulting in emitted photons with lower energy (longer wavelength) than the excitation photons. This energy difference, known as the Stokes shift, enables spectral separation between excitation and emission wavelengths, allowing distinct acquisition of fluorescence and background signals through spectrally optimized PDs. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, conventional approaches typically employ two CCD cameras to simultaneously capture fluorescence and background images. Here, the fluorescence channel camera requires a high-performance short-wave cutoff filter (taking sodium fluorescein\u0026rsquo;s absorption/emission wavelengths as an example, market research indicates typical unit prices ranging from \u0026yen;3,000 to \u0026yen;5,000 RMB) to completely block background interference from the excitation wavelength band. Moreover, due to the inherent challenge of achieving consistent viewing angles between cameras, fluorescence and background images exhibit spatial mismatches that preclude precise alignment, fundamentally limiting fluorescence localization imaging accuracy. In addition, due to differing sensitivity requirements between the fluorescence and background cameras, the two systems often employ distinct camera models, resulting in a severe pixel count mismatch between fluorescence and background images. Conventional multi-camera fluorescence-background fusion imaging systems face three inherent challenges: signal crosstalk, perspective misalignment, and pixel resolution disparity. These issues collectively lead to significant image registration errors and degraded fusion quality, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo address the inherent image fusion challenges of conventional dual-camera systems, we developed a novel PVK PD-based single-pixel imaging system for fluorescence-background fusion imaging (PVK-SPI-FB System) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed), which integrates a light source module for simultaneous fluorescence excitation and backscattered signal generation, a digital micromirror device (DMD) for generating pre-encoded Fourier-based patterns carrying spatial information, a perovskite filtered detector (PVKF-PD) specifically for fluorescence signal detection, and a perovskite broadband detector (PVK-PD) for backscattered signal acquisition. The system features p-i-n structured photovoltaic perovskite detectors (PVK PDs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee) that offer exceptional response speed and sensitivity for weak fluorescence detection while their self-powered operation significantly simplifies the imaging architecture. The fabrication process of PVK PDs is shown in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Figure S2 shows the survey XRD spectra of the perovskite PD layer (Cs\u003csub\u003e0.05\u003c/sub\u003eFA\u003csub\u003e0.79\u003c/sub\u003eMA\u003csub\u003e0.16\u003c/sub\u003ePbI\u003csub\u003e2.5\u003c/sub\u003eBr\u003csub\u003e0.5\u003c/sub\u003e). Building on our previous findings demonstrating the inherent FOV invariance and perfect pixel registration advantages of active-mode single-pixel imaging systems\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, this integrated solution effectively overcomes the critical fusion limitations between fluorescence and background images. Specifically, active-mode single-pixel imaging is that the image FOV and pixel dimensions are determined solely by the spatial illumination patterns rather than location of PDs. This fundamental characteristic enables the two PVK PDs to simultaneously acquire perfectly registered fluorescence and background images with identical spatial coordinates, eliminating the need for post-acquisition registration algorithms. Fluorescence imaging is typically actively excited as well, making the integration of active-mode single-pixel imaging systems particularly promising for perfectly resolving the fusion and registration challenges between fluorescence and background images, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Spectral-Splitting mechanism of perovskite single-pixel PDs\u003c/h2\u003e\u003cp\u003eIn addition to adopting single-pixel imaging methodology to resolve image registration challenges, we have developed spectral-splitting PVK PDs that fundamentally addresses the critical issue of fluorescence-background signal separation. Unlike conventional approaches relying on costly optical filters, our innovative solution employs an intrinsic wavelength self-filtering strategy enabled by the unique bandgap tunability of perovskite materials. Through controlled ion-exchange processes, we engineered two functionally distinct perovskite films: The small-bandgap perovskite films can be fabricated into broadband PDs (PVK-PD) specifically optimized for detecting the dominant backscattered signals. By strategically combining the small-bandgap PVK-PD with large-bandgap perovskite films exhibiting short-wavelength cutoff characteristics, we developed a novel narrowband PVK PD (PVKf-PD). This innovative design effectively filters out the shorter-wavelength excitation backscattered light while selectively transmitting the desired fluorescence signals, enabling pure fluorescence detection as demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed. However, the intensity of backscattered signals significantly exceeds that of fluorescence signals (approximately 17-fold greater, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei), necessitating the use of deep-cutoff short pass filters to effectively suppress the backscattered components, a key factor contributing to the high cost of conventional solutions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePerovskite materials offer unique advantages in this regard, as their bandgap tunability enables precise optimization of short-wavelength cutoff characteristics through simple precursor concentration modulation. To systematically investigate this property, we developed five distinct large-bandgap PFLs with varying precursor concentrations (1: 0.75 M, 2: 1 M, 3: 1.25 M, 4: 1.5 M, 5: 1.75 M) and characterized their transmission spectra to evaluate the short-wavelength blocking and long-wavelength transmission capabilities. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, increasing the precursor concentration leads to enhanced film thickness and improved short-wavelength cutoff performance, albeit at the cost of reduced long-wavelength transmittance. This trade-off prompted a comprehensive optimization study across nine concentration gradients of PFLs, with detailed analysis of their spectral filtering characteristics (Fig. S3). The results reveal a non-monotonic trend in the transmission ratio between 640 nm and 450 nm as the concentration increases, exhibiting an initial rise followed by a decline (Fig. S3(b)). Through comprehensive evaluation of both long-wavelength fluorescence transmission and short-wavelength excitation suppression, PFL5 demonstrates optimal spectral-splitting capability in this study. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee presents the transmission curves of five representative concentration-gradient PFLs, along with the spectrum curves of the narrowband PVK PDs, where the intersection region between transmission and absorption curves defines the spectral response window for fluorescence detection.\u003c/p\u003e\u003cp\u003eThrough systematic optimization of PFLs, we developed six distinct PVK PDs, including broadband perovskite detectors (PVK-PD) and narrowband fluorescence detectors incorporating PFLs (PVKf-PDs). As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, the spectral response curves of these six PDs demonstrate that the incorporation of PFLs significantly suppresses the device's detection capability for short-wavelength backscattered signals. The detailed variations in detector response parameters and corresponding signal suppression ratios are summarized in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, with PVKf-PD5 exhibiting optimal signal separation performance among all devices. Furthermore, we characterized the transient photoresponse properties of PVK-PD, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg, which exhibits an exceptionally fast rise time of 44 \u0026micro;s. The linear dynamic range (LDR) of PVK-PD was measured to reach 178.2 dB (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh), enabling stable operation across an irradiance range from 5.3\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e mW cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e to 4.36\u0026times;10\u003csup\u003e3\u003c/sup\u003e mW cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e while maintaining excellent detection performance. Additional performance characterizations of the PVK-PD are presented in Figure S4,5. These results collectively demonstrate the outstanding photovoltaic performance of PVK-PD.\u003c/p\u003e\u003cp\u003eTo evaluate the practical performance of PVK-PDs in separating backscattered and fluorescence signals, we conducted signal measurements under real-world fluorescence imaging conditions. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei presents the fluorescence and backscattered signal intensities obtained from sodium fluorescein aqueous solution with varying concentrations (100, 50, 10, and 1 \u0026micro;mol/mL) under 450 nm excitation light at 11.09 mW cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e intensity, with detailed values provided in Table S2. The results clearly demonstrate that the fluorescence signal intensity is significantly weaker than the backscattered signal, highlighting the critical requirement for PVKf-PDs to possess exceptional signal discrimination capability. Figure\u0026nbsp;2j1 demonstrates the fluorescence and background signal intensities detected by the broadband-responsive PVK-PD, revealing that the PD primarily captures backscattered signals with negligible fluorescence intensity, indicating its suitability for single-pixel background imaging. Figure\u0026nbsp;2j2-6 illustrate the detection capabilities of PVKf-PDs for both fluorescence and background signals, where progressive optimization of the PFL thickness leads to a complete inversion of signal intensity ratios in PVKf-PD5. This remarkable transition demonstrates the detector's full capability for single-pixel fluorescence detection, with the optimized PVKf-PD5 exhibiting superior performance in selectively capturing fluorescence signals while effectively suppressing background interference. The systematic improvement across the device series highlights the critical role of PFL engineering in achieving high-purity fluorescence detection. The quantitative relationship governing this transition is delineated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Imaging performance of 2.1 PVK-SPI-FB System\u003c/h2\u003e\u003cp\u003eBased on the aforementioned research findings, the PVK-PD and PVKf-PD5 are selected as the background signal detector and fluorescence detector, respectively, and subsequently integrated into our perovskite-based single-pixel imaging system for systematic evaluation of its imaging performance. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e​presents the performance evaluation of our fluorescence/background dual-modal imaging system based on PVK single-pixel PDs. The two-dimensional images were reconstructed using a four-step phase-shifting Fourier algorithm. Specifically, by measuring the light intensity values \u003cem\u003eD\u003c/em\u003e\u003csub\u003e\u003cem\u003eθ\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e(θ\u0026thinsp;=\u0026thinsp;0, π/2, π, 3π/2)\u003c/em\u003e for each structured illumination pattern, we computed the Fourier coefficients \u003cem\u003eα=[D\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-D\u003c/em\u003e\u003csub\u003e\u003cem\u003eπ\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e]\u0026thinsp;+\u0026thinsp;j\u0026middot;[D\u003c/em\u003e\u003csub\u003e\u003cem\u003eπ/2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e- D\u003c/em\u003e\u003csub\u003e\u003cem\u003e3π/2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e]\u003c/em\u003e, and subsequently reconstructed the spatial-domain images via inverse Fourier transform (IFT), as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea. Figure S6 presents the schematic diagram of the imaging algorithm.\u003c/p\u003e\u003cp\u003eIn Fourier spectral analysis, the low-frequency components govern the overall image profile, whereas the high-frequency components encode fine structural details. Consequently, the number of projected structured illumination patterns (M), which directly correlates with the sampling rate, determines the resolution of the reconstructed images. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c demonstrates the system\u0026rsquo;s imaging performance for sodium fluorescein aqueous solutions (100, 50, 10, and 1 \u0026micro;mol/mL) loaded in Eppendorf (EP) tubes under visible-light illumination. Under visible-light illumination, the fluorescence signals were overwhelmed by backscattered light, resulting in reconstructed images that only retained the outline of the EP tubes. Notably, as M increased, the image resolution improved significantly, with progressively sharper contours - a finding that validates the sampling theorem of FSPI.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed schematically illustrates the dual-modal single-pixel fluorescence/background imaging system, which employs a 450 nm excitation light source. The imaging target, identical to that shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, consisted of four EP tubes containing sodium fluorescein aqueous solutions with sequentially decreasing concentrations from left to right. As demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, the PVK-PD and PVKf-PD single-pixel detectors were employed for backscattered signal and fluorescence detection, respectively. The PVK-PD, serving as the background signal PD, failed to reconstruct complete EP tube contours due to severe localized glare signals caused by the smooth tube surfaces, resulting in substantially reduced image contrast. Notably, since the glare signals fundamentally originate from backscattered light, the progressively enhanced filtering capability of PVKf-PDs successfully suppressed these interference artifacts, thereby enabling clear fluorescence imaging. Furthermore, we performed image fusion between the white-light background image obtained in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and the fluorescence image captured by PVKf-PD5, as demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-7. These results preliminarily illustrate the unique fusion advantages of our developed PVK-SPI-FB System. Notably, in practical biological fluorescence imaging scenarios, glare artifacts are typically absent, enabling fluorescence/background localization imaging under single-excitation illumination conditions - a capability that will be comprehensively demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef displays 3D grayscale intensity mappings of the selected regions (boxed areas in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee), providing quantitative evaluation of PVKf-PDs' glare suppression capability. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg​ establishes a quantitative correlation between sodium fluorescein aqueous solution concentration and image grayscale values to evaluate PVK-SPI-FB System sensitivity. Based on the detection limit of our PVKf-PD5-based PVK-SPI-FB system and fitted curve analysis, the system demonstrates a remarkable detection threshold of 0.05 \u0026micro;mol/mL, highlighting its exceptional low-light detection capability.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Biomedical imaging results of PVK-SPI-FB System\u003c/h2\u003e\u003cp\u003e​​ Conventional multi-camera imaging systems​are inherently plagued by spatial registration challenges. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b, the inability to maintain strict co-axial alignment among different cameras introduces variations in the optical paths of reflected light received by each device. This inevitably leads to FOV misalignment and spatial information discrepancies in the acquired images, posing significant obstacles for multi-modal image fusion. In stark contrast, our developed PVK-SPI-FB System ensures complete spatial registration and pixel-level correspondence across images acquired by PDs at different positions. Specifically, their spatial information is entirely determined by the pre-defined structured light patterns, rendering them independent of PD positioning. This unique characteristic enables multiple PDs positioned at distinct spatial locations to acquire image data with perfectly matched FOV and pixels alignment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d, Fig. S7). The experiments in Fig. S8 further corroborate the aforementioned conclusions. Experimental results demonstrate that these images can be seamlessly fused through direct superposition, unequivocally validating the distinctive advantages of single-pixel imaging for multi-modal image integration.\u003c/p\u003e\u003cp\u003eBuilding upon these foundations, we designed an in murine model to validate the practical utility of this imaging methodology, The experimental setup is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and Fig. S9. Three tumor-simulating models were established through injections in the left hindlimb, right hindlimb, and dorsal region of the mice, with sodium fluorescein serving as the tumor-marker via local injection. Consequently, the luminescent areas in fluorescence images exclusively represented the tumor-simulating regions. Prior to formal experiments, this study conducted preliminary validation tests using chicken wings, with the results presented in Fig. S10. Our system successfully acquired both diffuse reflectance background images and specific fluorescence images simultaneously (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee-m). As demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003en-p, the fused images enabled clear discrimination between marked regions and normal tissues. Quantitative analysis of fluorescence signal intensity distributions permitted precise determination of both geometric dimensions and spatial coordinates of the tumor-simulating models, thereby providing reliable data support for surgical navigation applications.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Conclusion","content":"\u003cp\u003e​​This study developed a fluorescence-background dual-modal imaging system based on perovskite-based single-pixel PDs, successfully addressing the critical challenge of multi-modal image fusion in conventional surgical navigation. Our single-pixel imaging system inherently overcomes the pixel registration and spatial alignment challenges inherent in conventional dual-camera fluorescence-background fusion imaging, enabling precise tumor fluorescence localization. To address the spectral overlap between fluorescence and background signals, we developed self-filtering PVK-PDs that achieved efficient spectral splitting between fluorescence emission (520 nm) and backscattered excitation light (450 nm). The optimized PVKf-PD demonstrates exceptional performance, with a backscattered light suppression ratio of 57 times (responsivity ratio at 640/450 nm) and a fluorescence sensitivity of 107 mA W\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 520 nm (PVKf-PD5). Ultimately, In murine experiments successfully overcame the detection challenges posed by tissue-induced fluorescence attenuation, enabling precise localization imaging of tumor-simulating models.​This work presents a novel imaging solution for intraoperative real-time navigation, characterized by hardware simplicity, cost-effectiveness, and superior performance, demonstrating significant potential for applications in surgical oncology and precision medicine.\u003c/p\u003e"},{"header":"4. Experiment","content":"\u003cp\u003e\u003cstrong\u003e4.1 PVK PDs preparation:\u0026nbsp;\u003c/strong\u003eThe fabrication process of the Cs\u003csub\u003e0.05\u003c/sub\u003eFA\u003csub\u003e0.79\u003c/sub\u003eMA\u003csub\u003e0.16\u003c/sub\u003ePbI\u003csub\u003e2.5\u003c/sub\u003eBr\u003csub\u003e0.5\u003c/sub\u003e PVK PD was carried out as follows: First, a homogeneous precursor solution was prepared by dissolving 516 mg PbI₂, 171 mg FAI, 23.2 mg MABr, and 73.4 mg PbBr₂ in 1 mL of a DMF/DMSO mixed solvent (3:1 v/v) with continuous stirring at 60°C for 5 h, followed by the addition of 39 μL CsI (390 mg/mL in DMSO) and subsequent overnight stirring at 60°C. The device was fabricated through sequential spin-coating of functional layers: A PTAA hole transport layer was first deposited at 4000 rpm for 30 s, followed by thermal annealing at 100°C for 10 min. The perovskite active layer was then formed via a two-step spin-coating process - initial coating at 500 rpm for 6 s followed by 5000 rpm for 45 s, with 200 μL chlorobenzene anti-solvent dripping during the last 10 s, and subsequent annealing at 110°C for 60 min. Subsequently, a PCBM electron transport layer was spin-coated at 2000 rpm for 30 s and annealed at 65°C for 3 min, followed by deposition of a BCP modification layer at 3000 rpm for 30 s. Finally, a 100 nm-thick Ag electrode was thermally evaporated to complete the device fabrication. By spin-coating MAPbBr\u003csub\u003e2.4\u003c/sub\u003eCl\u003csub\u003e0.6\u003c/sub\u003e precursor solutions with concentrations of 0.75, 1, 1.26, 1.5, and 1.75 mol/L, PVK filter layers with different optical filtering effects were obtained.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2 Imaging system:\u0026nbsp;\u003c/strong\u003eThe schematic diagram of the experimental setup is shown in Fig. 1d, which mainly consists of a spatial light modulator (SLM), two PVK PDs (PVK-PD and PVKf-PD), a data acquisition system, a computer, and a white light source. The excitation blue light (~435–465 nm) was obtained by filtering the white light. A DMD (V-7001 VIS, ViALUX) with a projection rate of up to 22 kHz was used as the SLM. First, a set of four-step phase-shifted sinusoidal patterns was generated by the computer and loaded onto the DMD for structured light modulation. The PVK-PD and PVKf-PD collected and recorded the light intensity values of each structured light pattern through a multi-channel acquisition system. The set of current intensities was referred to as the \u003cem\u003eI-m\u003c/em\u003e curve, where \u003cem\u003em\u003c/em\u003e is the pattern number. Finally, the \u003cem\u003eI-m\u003c/em\u003e curve was processed using a Fourier transform algorithm to reconstruct the fluorescence and background images. A simple superposition of the two images yielded the fused image.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3 Imaging experiments:\u0026nbsp;\u003c/strong\u003eFirst, in vitro experiments were conducted using sodium fluorescein aqueous solutions at four concentrations (100, 50, 10, and 1 μmol/mL) as imaging targets. Subsequently, in murine experiments were performed by mixing agar solution sodium fluorescein aqueous solutions at a 1:1 volume ratio. A 150 μL aliquot of the mixture was subcutaneously injected into the right hind limb, left hind limb, and back of mice to establish tumor-mimicking models. Animal experiments were approved by Experimental Animal Ethics Committee of Chengdu Dossy Experimental Animals Company Limited in accordance with the Ethical Guidelines for Animal Care (NO.CDDEACL2024-16).\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupporting Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupporting Information is available from the Wiley Online Library or from the author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors acknowledge the financial supports from the National Natural Science Foundation of China (62405236, 62275210, 62375210); the National Leading Talent Program, the National Young Talent Program, the Key Research and Development Program of Shaanxi (2024SF2-GJHX-25), the Shaanxi Young Top-notch Talent Program; Xidian University Specially Funded Project for Interdisciplinary Exploration (TZJH2024059, TZJH2024060); Fundamental Research Funds for Central Universities (QTZX24079).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData available on request from the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSouzaki R, Ieiri S, Uemura M et al (2013) An augmented reality navigation system for pediatric oncologic surgery based on preoperative CT and MRI images[J]. 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Photonics Res 12(12):2873\u0026ndash;2880\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7217975/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7217975/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eImage-guided surgery systems require precise fusion of fluorescence mapping and structural background imaging, yet current dual-camera system methods suffer from field-of-view misalignment and pixel offsets, risking millimeter-scale surgical errors. To address these challenges, we developed an innovative fluorescence/background fusion single-pixel imaging system based on spectral-splitting perovskite photodetectors (PDs). By incorporating gradient-optimized wide-bandgap perovskite filter layers, the perovskite photodetector can achieve spectrally selective detection through efficient separation of 520 nm fluorescence signals (\u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e) from 450 nm backscattered light(\u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e). With a high signal suppression ratio (\u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e/S\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e) of 57 times of the optimized PD, the system achieves a low detection limit of 50 nmol/mL for sodium fluorescein aqueous solution. More importantly, the exceptional active single-pixel imaging architecture ensures perfect field-of-view and pixel-level consistency across multiple detectors\u0026rsquo; images, eliminating the need for complex registration algorithms or sophisticated dual-optical path designs. Finally, through in murine tumor experiments, we achieved precise fluorescence-labeled tumor localization imaging, demonstrating the reliability of our developed system in fluorescence/background fusion imaging. This provides a novel fluorescence-targeting approach for medical imaging, demonstrating the innovative utility of single-pixel imaging in advanced diagnostics.\u003c/p\u003e","manuscriptTitle":"Fluorescence Localization Imaging via a Spectral-Splitting Perovskite Single-Pixel Detector","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-24 14:56:00","doi":"10.21203/rs.3.rs-7217975/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2dc4b700-88ab-44cc-8279-f93f4990948f","owner":[],"postedDate":"October 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":56791893,"name":"Physical sciences/Optics and photonics/Applied optics/Optical sensors"},{"id":56791894,"name":"Physical sciences/Physics/Techniques and instrumentation/Imaging techniques"}],"tags":[],"updatedAt":"2026-03-27T05:41:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-24 14:56:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7217975","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7217975","identity":"rs-7217975","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}