Nanothermometry methods with intracellular sensitivities have the potential to make important contributions to fundamental cell biology and medical fields, as temperature is a relevant physical parameter for molecular reactions to occur inside the cells and changes of local temperature are well identified therapeutic strategies. Here we show how the GFP can be used to assess temperature-based on a novel fluorescence peak fraction method. Further, we use standard GFP transfection reagents to assess temperature intracellularly in HeLa cells expressing GFP in the mitochondria. High thermal resolution and sensitivity of around 0.26% °C⁻¹and 2.5% °C⁻¹, were achieved for wt-GFP in solution and emGFP-Mito within the cell, respectively. We demonstrate that the GFP-based nanothermometer is suited to directly follow the temperature changes induced by a chemical uncoupler reagent that acts on the mitochondria. The spatial resolution allows distinguishing local heating variations within the different cellular compartments. Our discovery may lead to establishing intracellular nanothermometry as a standard method applicable to the wide range of live cells able to express GFP.

Elesclomol (ELC) is an anticancer drug inducing mitochondria cytotoxicity through reactive oxygen species. Here, for the first time, we encapsulate the poorly water soluble ELC in monoolein-based cubosomes stabilized with Pluronic F127. Cellular uptake and nanocarrier accumulation close to the mitochondria with sub-micrometer distance is identified via three-dimensional (3D) confocal microscopy and edge-to-edge compartment analysis. To monitor the therapeutic effect of the ELC nanocarrier, we apply for the first time, label-free time-lapse multi-photon fluorescence lifetime imaging microscopy (MP-FLIM) to track NAD(P)H cofactors with sub-cellular resolution on live cells exposed to an anticancer nanocarrier. Improved in vitro cytotoxicity is verified when loading the pre-complexed ELC with copper (ELC-Cu). Importantly, for equivalent copper concentration, cubosomes loaded with ELC-Cu show higher cytotoxicity compared to the free drug. The novel nanocarrier shows promising features for systemic ELC-Cu administration, and furthermore we establish the MP-FLIM technique for the assessment of anticancer drug delivery systems. Open image in new window

Biomimicking biological niches of healthy tissues or tumors can be achieved by means of artificial microenvironments, where structural and mechanical properties are crucial parameters to promote tissue formation and recreate natural conditions. In this work, three-dimensional (3D) scaffolds based on woodpile structures were fabricated by two-photon polymerization (2PP) of different photosensitive polymers (IP-S and SZ2080) and hydrogels (PEGDA 700) using two different 2PP setups, a commercial one and a customized one. The structures’ properties were tuned to study the effect of scaffold dimensions (gap size) and their mechanical properties on the adhesion and proliferation of bone marrow mesenchymal stem cells (BM-MSCs), which can serve as a model for leukemic diseases, among other hematological applications. The woodpile structures feature gap sizes of 25, 50, and 100 μm and a fixed beam diameter of 25 μm, to systematically study the optimal cell colonization that promotes healthy cell growth and potential tissue formation. The characterization of the scaffolds involved scanning electron microscopy and mechanical nanoindenting, while their suitability for supporting cell growth was evaluated with live/dead cell assays and multistaining 3D confocal imaging. In the mechanical assays of the hydrogel material, we observed two different stiffness ranges depending on the indentation depth. Larger gap woodpile structures coated with fibronectin were identified as the most promising scaffolds for 3D BM-MSC cellular models, showing higher proliferation rates. The results indicate that both the design and the employed materials are suitable for further assays, where retaining the BM-MSC stemness and original features is crucial, including studies focused on BM disorders such as leukemia and others. Moreover, the combination of 3D scaffold geometry and materials holds great potential for the investigation of cellular behaviors in a co-culture setting, for example, mesenchymal and hematopoietic stem cells, to be further applied in medical research and pharmacological studies.

Quantum and neuromorphic computational platforms in integrated photonic circuits require next-generation optical functionalities. Often, increasingly complex on-chip light-routing that allow superpositions not attainable by planar technologies are paramount e.g. for artificial neural networks. Versatile 3D waveguides are achievable via two-photon polymerization (TPP)-based microprinting. Here, a 3D morphology prediction tool which considers experimental TPP parameters, is presented, enabling on-chip 3D waveguide performance simulations. The simulations allow reducing the cost-intensive systematic experimental optimization process. Fabricated 3D waveguides show optical transmission properties in agreement with simulations, demonstrating that the developed morphology prediction methodology is beneficial for the development of versatile on-chip and potentially inter-chip photonic interconnect technology.

Nanophotonic spiking neural networks (SNNs) based on neuron-like excitable subwavelength (submicrometre) devices are of key importance for realizing brain-inspired, power-efficient artificial intelligence (AI) systems with high degree of parallelism and energy efficiency. Despite significant advances in neuromorphic photonics, compact and efficient nanophotonic elements for spiking signal emission and detection, as required for spike-based computation, remain largely unexplored. In this invited perspective, we outline the main challenges, early achievements, and opportunities toward a key-enabling photonic neuro-architecture using III–V/Si integrated spiking nodes based on nanoscale resonant tunnelling diodes (nanoRTDs) with folded negative differential resistance. We utilize nanoRTDs as nonlinear artificial neurons capable of spiking at high-speeds. We discuss the prospects for monolithic integration of nanoRTDs with nanoscale light-emitting diodes and nanolaser diodes, and nanophotodetectors to realize neuron emitter and receiver spiking nodes, respectively. Such layout would have a small footprint, fast operation, and low power consumption, all key requirements for efficient nano-optoelectronic spiking operation. We discuss how silicon photonics interconnects, integrated photorefractive interconnects, and 3D waveguide polymeric interconnections can be used for interconnecting the emitter-receiver spiking photonic neural nodes. Finally, using numerical simulations of artificial neuron models, we present spike-based spatio-temporal learning methods for applications in relevant AI-based functional tasks, such as image pattern recognition, edge detection, and SNNs for inference and learning. Future developments in neuromorphic spiking photonic nanocircuits, as outlined here, will significantly boost the processing and transmission capabilities of next-generation nanophotonic spike-based neuromorphic architectures for energy-efficient AI applications. This perspective paper is a result of the European Union funded research project ChipAI in the frame of the Horizon 2020 Future and Emerging Technologies Open programme.