Dual and multimodal imaging probes are powerful tools for improving detection sensitivity and accuracy in disease diagnosis. In this work, we introduce a novel bimodal dendrimer-based imaging probe that combines magnetic and fluorescent properties for potential use in magnetic resonance imaging (MRI) and fluorescence imaging (FI).

Remarkably, this system is among the few examples where organic radicals, rather than metals, serve as the magnetic source. The use of organic radicals in place of conventional metal-based contrast agents, such as gadolinium (Gd³⁺)-chelates, offers the significant advantage of reducing toxicity risks. Our approach involved an amino-terminated polyamide dendrimer incorporating a 1,8-naphthalimide (Naft) fluorescent core, amino acid-based linkers for enhanced water solubility, and TEMPO organic radicals as terminal groups (G2Naft). A structurally similar dendrimer, without the fluorophore, was also synthesized and functionalized to serve as a reference.

Partial fluorescence quenching caused by the organic radicals was observed, likely due to photoinduced electron transfer (PET), as supported by DFT calculations. Despite this quenching, the probe retained sufficient fluorescence properties, as demonstrated by successful fluorescence imaging of live mesenchymal stem cells (MSCs) using a laser raster-scanning nonlinear optical (NLO) microscope. Progressive cellular internalization of G2Naft was observed over 24 hours, and z-stack analysis confirmed the presence of G2Naft within the cytoplasm.

NLO images of MSCs incubated with G2Naft for 24 h: CCD image, G2Naft fluorescence, Hoechst-stained nuclei, and merged visualization.

Co-staining with Hoechst 33342 further validated the localization: green fluorescence originated from G2Naft in the cytoplasm, while blue emission from Hoechst marked the nuclei, clearly distinguishing cytoplasmic and nuclear regions within the same cells. Simultaneously, the G2Naft dendrimer exhibited an effective r1 relaxivity of 1.3 mM⁻¹s⁻¹, demonstrating its ability to shorten the relaxation time of water protons, a key characteristic of MRI contrast agents. Consequently, G2Naft effectively showcased both magnetic and fluorescent properties in vitro.

"Our experiments demonstrated that the imaging probe effectively emits fluorescence in a biological environment", says dr. Vega Lloveras. "The bimodal magnetic-fluorescent imaging probe holds great potential for future comprehensive diagnostic applications."

These results underscore its potential as a promising bimodal imaging probe for combined FI and MRI in biomedical diagnostics. In addition, molecular dynamics (MD) simulations provided deeper insights into the dendrimer’s structure-function relationships, shedding light on its electron paramagnetic resonance (EPR) properties, relaxivity, and fluorescence behavior.

"NFFA-Europe provided us with an excellent opportunity to integrate cutting-edge NLO microscopy techniques with live-cell imaging and treatment”, concludes dr. José Vidal Gancedo.

Yufei Wu, Vega Lloveras,* Anjara Morgado, Ezequiel Perez-Inestrosa, Eleftheria Babaliari, Sotiris Psilodimitrakopoulos, Yolanda Vida, and José Vidal-Gancedo*. ACS Applied Materials & Interfaces 2024 16 (47), 65295-65306. DOI: 10.1021/acsami.4c13578

To obtain the presented results, several facilities and advanced techniques available through NFFA-Europe at FORTH-Hellas were utilized.

Cell Culture Facilities – C57BL/6 mouse bone marrow mesenchymal stem cells (MSCs) were cultured under controlled conditions using Dulbecco's Modified Eagle's Medium – Low Glucose (DMEM-LG) supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin (PS). Cells were incubated at 37 °C in a 5% CO₂ atmosphere to ensure optimal growth. Planar polyethylene terephthalate (PET) coverslips were used as substrates for cell adhesion, pre-sterilized via UV treatment before seeding. Fluorescence Labeling – To assess cellular uptake and fluorescence properties of the probe, MSCs were incubated with our probe (G2Naft) for 3 h and 24 h. Post-incubation, cells were stained with the nuclear dye Hoechst 33342 to visualize cell nuclei, ensuring precise localization of the fluorescent probe.

Fluorescence Imaging (FI) via Nonlinear Optical (NLO) Microscopy – A custom-built femtosecond (fs) laser raster-scanning nonlinear optical (NLO) microscope was used to measure fluorescence signals from live cells. A custom-built multiphoton microscope was used to simultaneously excite two-photon fluorescence (2p-F) from G2Naft and three-photon fluorescence (3p-F) from Hoechst 33342 in live cells using a single 1030 nm fs laser source. The system includes galvanometric mirrors for beam steering and an inverted microscope for focusing the laser onto the sample. Emitted fluorescence is collected through the same objective, filtered to remove laser light, and detected using photomultiplier tubes (PMTs) with specific band-pass filters for 2p-F (562 ± 20 nm) and 3p-F (458 ± 32 nm). Image acquisition is controlled via custom-built LabVIEW software. This technique enabled high-resolution imaging and detailed assessment of fluorescence emission within the biological environment.

Dr. Vega Lloveras got her Ph.D. in Materials Science from the Autonomous University of Barcelona (UAB) in 2006, conducting her research at the Institute of Materials Science of Barcelona (ICMAB-CSIC). Her work focused on conjugated organic molecules containing organic radicals as molecular wires, and the selective detection of metal ions through molecular recognition. Then, she completed a six-month postdoctoral fellowship at ICMAB, followed by a short postdoctoral stay at the University of York (UK). There, she explored gold nanoparticles (AuNPs) functionalized with organic radicals, contributing to the development of novel hybrid materials with unique magnetic and optical properties. Additionally, she played a key role in pioneering research on surface-confined molecular switches. She has also conducted specialized measurements at leading international research facilities, including the Istituto Officina dei Materiali (Italy) and FORTH-Hellas (Greece). Along her career, she has conducted research on intramolecular electron transfer phenomenon, molecular wires, molecular switches, multifunctional surfaces, spin-labeled AuNPs, and the development of novel radicals and diradicals as polarization agents for dynamic nuclear polarization (DNP). In recent years, her work has focused on biomedical applications, particularly on the development of metal-free contrast agents for magnetic resonance imaging (MRI) to address the known toxicity associated with Gd-based chelates. This research has led to both in vitro and in vivo demonstrations of highly effective and non-toxic water-soluble radical dendrimers, i.e. dendrimers fully functionalized by organic radicals, as MRI contrast agents (Biomacromolecules 2022, Acta Biomaterialia 2025). Recently appointed as a Tenured Scientist at ICMAB, she was previously responsible for the spectroscopy service at ICMAB, particularly overseeing the Electron Paramagnetic Resonance (EPR) Laboratory. In addition, she is also researcher at the Networking Research Center on Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN). Beyond her research, Dr. Lloveras is actively involved in scientific dissemination, peer review, and evaluation committees for major European research infrastructures. She is a member of various scientific societies, is the secretary of the Spanish Electron Paramagnetic Resonance Group (GERPE), and is a permanent member of the European Federation of EPR Groups. Dr. Lloveras has co-authored 63 scientific articles and one book chapter, accumulating around 2,700 citations (h-index: 26). She has supervised 2 Ph.D. students (with 4 ongoing), 3 Master’s theses, 7 final degree projects, and 10 exchange students. Additionally, she holds 9 patents and has contributed to the creation of a spin-off company.

Dr. José Vidal Gancedo got his Ph.D. in Chemistry from the University of Barcelona (UB) in 1993, followed by postdoctoral short stays at renowned European institutions, including the University of Strasbourg, CNRS Grenoble, the University of Innsbruck, and the University of York. He is currently a CSIC Research Scientist at the Institute of Materials Science of Barcelona (ICMAB-CSIC) and the Networking Research Center on Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN). He is also the Head of the Molecular Nanoscience and Organic Materials Department (NANOMOL) at ICMAB and is the President of the Spanish Electron Paramagnetic Resonance Group (GERPE). Dr. Vidal Gancedo's research focuses on molecular functional materials based on organic radicals, with significant contributions to molecular magnetism, charge transfer salts, intramolecular electron transfer phenomenon, molecular wires, molecular switches, multifunctional surfaces, spin-labeled gold nanoparticles (AuNPs), and the development of novel polarization agents for dynamic nuclear polarization (DNP). In recent years, his work has been dedicated to biomedical applications, particularly on the development of metal-free contrast agents for magnetic resonance imaging (MRI) based on organic radicals, as alternative to Gd-based contrast agents. Dr. Vidal Gancedo has co-authored over 150 scientific articles and 5 book chapters, accumulating more than 5,000 citations (h-index: 40). He has supervised 8 Ph.D. students, 6 Postdoctoral researchers (including a Juan de la Cierva fellow), 7 Master's theses, 9 final degree projects, and over 25 short-term researchers, including predoctoral and postdoctoral fellows, interns, and exchange students. He holds 9 patents and has contributed to the creation of a spin-off company.