FLuorescent nanO-agents for super-Resolution Imaging and seNsing

Project Information

FLORIN

Grant agreement ID: 101086142

DOI

10.3030/101086142

EC signature date 26 September 2022

Start date 1 November 2022

End date 31 October 2026

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 791 200,00

Coordinated by

ITA-SUOMEN YLIOPISTO
Finland

Periodic Reporting for period 1 - FLORIN (FLuorescent nanO-agents for super-Resolution Imaging and seNsing)

Reporting period: 2022-11-01 to 2024-10-31

Summary of the context and overall objectives of the project

Since Alzheimer disease (AD) affects up to 50% of individuals above 85, we will witness the three-fold increase in the number of patients by 2050 if no efficient therapy will be found.
The FLORIN offers non-invasive real-time monitoring of key mechanisms involved in the AD pathogenesis by avant-garde bioimaging at temporal resolution less than 1 millisecond and spatial resolution 20 - 50 nm combined with the simultaneous all-optical thermal control with 20 mK accuracy. FLORIN relies on three pillars.
(1) super-resolution optical fluctuation image scanning microscopy (SOFISM) and photon antibunching contrast enhanced super-resolved optical imaging (Q-ISM), providing accurate real-time information on fluorescent nanoparticles delivery, their
organelle-specific targeting on the molecular level, and intracellular distribution with spatial resolution below 20-50 nm;
(2) frequency upconverting quantum emitters that enable convert excitation in the tissue transparency window (from 650 to 1350 nm) to fluorescence in visual spectral range;
(3) biosensing techniques capable to detect tiny changes of temperature in the living tissue with 20 mK accuracy.
With its vision for the project and beyond, FLORIN will facilitate the further development of the devices for the clinical diagnosis and treatment of AD, being fully in line with the EU Joint Programme – Neurodegenerative Disease Research and contributing to UN Sustainable Development Goal “To Ensure healthy lives and promote well-being for all at all ages”.
Uniting 6 well-recognized academic partners from EU and Canada, and 3 hi-tech SMEs, ATOMICUS (Germany), Adamas (USA) and Platformina (Lithuania), FLORIN action as a part of the ‘biophotonics and quantum sensing’ flow will contribute to the rise of the potential of individuals and improve their career perspectives in research and innovations within this strongly networked European and global Photonics, Material science, Quantum technologies and Neuroscience communities.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Three types of upconversion nanoparticles (UCNPs) – NaGdF4, LiYF4, and LiYbF4 – are synthesized with rare earth ions like Yb³⁺, Er³⁺, and Tm³⁺, optimized for size, morphology, and efficiency. Core/shell structures are created to enhance luminescence, improving stability and water dispersibility for biological applications. High-resolution characterization confirms the production of highly crystalline UCNPs with tunable emission properties.

Fluorescent nanodiamonds (FNDs) are produced using plasma-enhanced CVD, with sizes ranging from 50 to 700 nm. FNDs with SiV and NV color centers are created through substrate etching and nitrogen incorporation. A procedure to analyze relaxation times and radical formation in living cells has been developed, enabling spin sensing, high-sensitivity nanothermometry, and miRNA detection. Thermometry and bioimaging are demonstrated with SiV and NV centers in single-crystal diamond needles.

Lithium-based UCNPs generate intense UV emissions, beneficial for light-triggered applications such as drug release and photodynamic therapy. Imaging and sensing were optimized using Fisher information analysis, and the analysis of frame correlations improved SOFI-type super-resolution imaging.

Neuron cell modeling was used to simulate the electrophysiological behavior of neuron cells in 2D and 3D. The model is now capable of analyzing the effects of nanosized light sources, such as UCNPs, on neuron cells through electromagnetic and thermal forces.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Diamonds are synthesized using advanced plasma-enhanced CVD technology, creating color centers like NV, SiV, GeV, Ni, and Cr by optimizing parameters such as gas mixture, substrate material, and solid additives. This process allows precise control over diamond characteristics, including size (0.1 to 300 µm), apex thickness (a few nanometers), and apex angle (~5°), with eight types of color centers for versatile applications.

FLORIN advances nanodiamond surface modification with non-thermal plasma, a method beyond the current state of the art. By introducing partially ionized gas into aqueous colloidal solutions, we optimized surface termination for quantum sensors. The plasma, produced from molecular hydrogen, led to increased O–H and C–H bonds, with significant changes in FTIR spectra. Zeta potential decreased notably, especially for oxidized NDs, and electron spin relaxation times for NV centers improved, with T1 times increasing by 17%–29% and T2 times prolonged by 40%.

We achieved coherent control of NV center-doped nanodiamonds in living cells, enabling intracellular thermodynamic accuracy within 100 mK. Theoretical research on blinking's impact on Fisher information found that the information per detected photon is greater with blinking emitters, and this information increases as the on-time ratio decreases, as seen in techniques like STORM and PALM. A parallelizable software tool for modeling emitter blinking was developed, and simulations revealed that a distribution with heavy tails reduces SOFI image informativeness.

Global market forecasts predict the micro- and nano-diamond market will exceed $370 billion by 2030, with a compound annual growth rate of 19%. However, the biomedical market, including bioimaging and drug delivery, currently accounts for less than 1.8% of the total market due to a lack of suitable diamonds for functionalization. This presents a significant opportunity to commercialize the scalable production of CVD diamond micro- and nano-crystals and their biofunctionalization, enabling new biomedical applications.

3D imaging of a rat neural cell culture labeled with a fluorescent dye – axial resolution improvemen

One of Z axis slice of human brain section (7 um) with labeled Synapsin1 (red) and PSD95 (green).

Characteristic SEM images of synthesized FND with size of 600-700 nm (a, b), 350-450 nm (c,d) and 50

Typical TEM images of NaGdF4:Yb3+, Er3+ core UCNPs (left), NaGdF4:Yb3+, Er3+ core/NaGdF4 shell UCNPs

Scheme of applying an artificial neural network for selection of the optimal SOFI cumulant or combin