Utilizing lanthanide upconversion nanoparticles to convert near-infrared to visible light presents a potential way for fabricating the next generation of low-cost near-infrared imaging sensors. Integrating microlens arrays with upconversion nanoparticles has been shown to be a promising approach for improving the efficiency of upconversion nanoparticles. However, approaches suitable for prototyping and producing microlens arrays to explore optimal device designs are lacking. In this work, we report an approach to fabricating flexible near-infrared-to-visible upconversion devices incorporating upconversion nanoparticles and microlens arrays, which enables easy adjustment of device structures and lens geometry. This is achieved by fabricating flexible films containing upconversion nanoparticles using molding in combination with femtosecond laser 3D printing of lenses, facilitating rapid prototyping for different application scenarios. By adding the microlens array, the intensities of the green (525 and 540 nm) and red (654 nm) upconversion emission bands were enhanced by a factor of 3 and 10, respectively, potentially leading to much reduced detectable near-infrared light intensity.

To overcome challenges in fluorescence labelling of RNA inside living cells we have recently introduced a direct approach using the fluorescent nucleobase analogue 2CNqA. Here we demonstrate its potential for use in fluorescence lifetime imaging (FLIM) to investigate nucleoside metabolism and for metabolic RNA labelling.

Fluorescence-based single-molecule and fluctuation spectroscopy in the near-IR can open avenues for biomolecular dynamic studies in biological media with suppressed autofluorescence and scattering background. However, further implementation is limited by the lower brightness of NIR fluorophores and available single-photon detector technologies that are still to be explored and adapted. Superconducting nanowire single-photon detectors (snSPDs) have found increasing use in quantum optics and optical communication applications thanks to high sensitivity in the near-infraed (NIR), low dark-counts, no after-pulsing, and high time resolution. Here, we present characterization of fluorescence intensity fluctuations from single vesicles and NIR fluorophores based on fluorescence correlation spectroscopy (FCS), specifically taking advantage of these snSPD properties. We present a concept allowing multiplexed readouts based on only one snSPD, in which the emitted photons are separated by their emission wavelength into different optical paths, thereby translating the emission wavelengths into different arrival times onto the snSPD. This concept allows one-laser-one-detector, dual-color fluorescence cross-correlation spectroscopy (FCCS) measurements, with fluorescence intensity fluctuations of two fluorophore species separately analyzed and cross-correlated. It is shown how two fluorophore species in a sample can be distinguished by their different blinking kinetics, fluorescence lifetimes, and/or diffusion properties. Apart from differences in emission spectra, the presented concept for multiplexing using a single detector can also be applied to distinguish emitters by properties such as polarization, coherence lengths, and fluorescence bunching and antibunching signatures. It can also be generalized to other modalities than FCS, including single-molecule detection, confocal microscopy, and imaging.

This study introduces the concept of photophysical structured illumination velocimetry (PP-SIV), verified through comprehensive numerical simulations. PP-SIV can capture two-dimensional (2D) flow velocity fields from a single snapshot image of the emission pattern from luminescent probes, leveraging the suitable photodynamics of the probes and using the applied excitation field pattern as reference. By eliminating the need for any beam or sample scan, PP-SIV has the potential to significantly accelerate the data acquisition process required for velocity field imaging. Furthermore, with excitation patterns applied at different depths, three-dimensional (3D) flow imaging can be potentially achieved. We propose lanthanide-based upconversion nanoparticles (UCNPs), particularly those capable of both absorbing and emitting within the highly biocompatible and transparent NIR-II window (1000-1700 nm), as promising probe candidates for implementing PP-SIV. This concept holds significant potential to pave the way for rapid, three-dimensional (3D) blood flow imaging at sufficient speeds for real-time monitoring of hemodynamic events in the brain.

Exploring lanthanide light upconversion (UC) has emerged as a promising strategy to enhance the near-infrared (NIR) responsive region of silicon solar cells (SSCs). However, its practical application under normal sunlight conditions has been hindered by the narrow NIR excitation bandwidth and the low UC efficiency of conventional materials. Here, we report the design of an efficient multiband UC system based on Ln3+/Yb3+-doped core-shell upconversion nanoparticles (Ln/Yb-UCNPs, Ln3+= Ho3+, Er3+, Tm3+). In our design, Ln3+ ions are incorporated into distinct layers of Ln/Yb-UCNPs to function as near-infrared (NIR) absorbers across different spectral ranges. This design achieves broad multiband absorption withtin the 1100 to 2200 nm range, with an aggregated bandwidth of ~500 nm. We have identified a synthetic electron pumping (SEP) effect involving Yb3+ ions, facilitated by the synergistic interplay of energy transfer and cross-relaxation between Yb3+ and other ions Ln3+ (Ho3+, Er3+, Tm3+). This SEP effect enhances the UC efficiency of the nanomaterials by effectively transferring electrons from the low-excited states of Ln3+ to the excited state of Yb3+, resulting in intense Yb3+ luminescence at ~980 nm within the optimal response region for SSCs, thus markedly improving their overall performance. The SSCs integrated with Ln/Yb-UCNPs with multiband excitation demonstrate the largest reported NIR response range up to 2200 nm, while enabling the highest improvement in absolute photovoltaic efficiency reported, with an increase of 0.87% (resulting in a total efficiency of 19.37%) under standard AM 1.5 G irradiation. Our work tackles the bottlenecks in UCNP-coupled SSCs and introduces a viable approach to extend the NIR response of SSCs.

Antibodies, disruptive potent therapeutic agents against pharmacological targets, face a barrier in crossing immune systems and cellular membranes. To overcome these, various strategies have been explored including shuttling via liposomes or biocamouflaged nanoparticles. Here, we demonstrate the feasibility of loading antibodies into exosome-mimetic nanovesicles derived from human red-blood-cell membranes, which can act as nanocarriers for intracellular delivery. Goat-antichicken antibodies are loaded into erythrocyte-derived nanovesicles, and their loading yields are characterized and compared with smaller dUTP-cargo molecules. Applying dual-color coincident fluorescence burst analyses, the loading yield of nanocarriers is rigorously profiled at the single-vesicle level, overcoming challenges due to size-heterogeneity and demonstrating a maximum antibody-loading yield of 38-41% at the optimal vesicle radius of 52 nm. The achieved average loading yields, amounting to 14% across the entire nanovesicle population, with more than two antibodies per loaded vesicle, are fully comparable to those obtained for the much smaller dUTP molecules loaded in the nanovesicles after additional exosome-spin-column purification. The results suggest a promising new avenue for therapeutic delivery of antibodies, potentially encompassing also intracellular targets and suitable for large-scale pharmacological applications, which relies on the exosome-mimetic properties, biocompatibility, and low-immunogenicity of bioengineered nanocarriers synthesized from human erythrocyte membranes.

Reversible dark state transitions in fluorophores represent a limiting factor in fluorescence-based ultrasensitive spectroscopy, are a necessary basis for fluorescence-based super-resolution imaging, but may also offer additional, largely orthogonal fluorescence-based readout parameters. In this work, we analyzed the blinking kinetics of Cyanine5 (Cy5) as a bar-coding feature distinguishing Cy5 from rhodamine fluorophores having largely overlapping emission spectra. First, fluorescence correlation spectroscopy (FCS) solution measurements on mixtures of free fluorophores and fluorophore-labeled small unilamellar vesicles (SUVs) showed that Cy5 could be readily distinguished from the rhodamines by its reversible, largely excitation-driven trans-cis isomerization. This was next confirmed by transient state (TRAST) spectroscopy measurements, determining the fluorophore dark state kinetics in a more robust manner, from how the time-averaged fluorescence intensity varies upon modulation of the applied excitation light. TRAST was then combined with wide-field imaging of live cells, whereby Cy5 and rhodamine fluorophores could be distinguished on a whole cell level as well as in spatially resolved, multiplexed images of the cells. Finally, we established a microfluidic TRAST concept and showed how different mixtures of free Cy5 and rhodamine fluorophores and corresponding fluorophore-labeled SUVs could be distinguished on-the-fly when passing through a microfluidic channel. In contrast to FCS, TRAST does not rely on single-molecule detection conditions or a high time resolution and is thus broadly applicable to different biological samples. Therefore, we expect that the bar-coding concept presented in this work can offer an additional useful strategy for fluorescence-based multiplexing that can be implemented on a broad range of both stationary and moving samples.

Lanthanide-doped upconversion nanoparticles (UCNPs) have attractive emission properties but suffer from weak light-absorbing capacities and thereby relatively low brightnesses. This motivates using strongly absorbing dye molecules as antennas and sensitizers. However, despite much effort, understanding of this dye-UCNP interplay is still limited. Major sensitization mechanisms are still under discussion, largely because there is a lack of effective means to observe key factors such as dark state transitions within the dyes. Here, a combined spectroscopic procedure is established to systematically investigate the photophysics behind the dye-UCNP interaction, embracing fluorescence-based transient-state excitation-modulation, lifetime and correlation spectroscopy, and spectrofluorometry/spectrophotometry. With this procedure the heptamethine cyanine dye IR806, a typical UCNP sensitizer is studied, its photophysical model is established, its photophysics in UCL-sensitization-related environments is deciphered, and the energy transfer from the IR806 singlet excited state to Yb³⁺ (UCNP sensitizer ion) can be identified as the dominant sensitization mechanism. These studies suggest that IR806 can form non-emissive H-aggregates at the nanoparticle surfaces, which can be dissociated after certain light excitation duration (typically>100 µs). Moreover, buildup of a non-fluorescent, photo-redox state of IR806 after longer irradiation times (10–100 ms) can deleteriously affect its UCL sensitization effect, inferring an optimal excitation duration for dye-sensitized UCNPs, relevant for, e.g., optical imaging applications.

This work details the synthesis of paramagnetic upconversion nanoparticles doped with Fe3+ in various morphologies via the thermal decomposition method, followed by comprehensive characterization of their structures, optical properties and magnetism using diverse analytical techniques. Our findings demonstrate that by precisely modulating the ratio of oleic acid to octadecene in the solvent, one can successfully obtain hexagonal nanodiscs with a consistent and well-defined morphology. Further adjustments in the oleic acid to octadecene ratio, coupled with fine-tuning of the Na+/F− ratio, led to the production of small-sized nanorods with uniform morphology. Significantly, all Fe3+-doped nanoparticles displayed pronounced paramagnetism, with magnetic susceptibility measurements at 1 T and room temperature of 0.15 emu g−1 and 0.14 emu g−1 for the nanodiscs and nanorods, respectively. To further enhance their magnetic properties, we replaced the Y-matrix with a Gd-matrix, and by fine-tuning the oleic acid/octadecene and Na+/F− ratios, we achieved nanoparticles with uniform morphology. The magnetic susceptibility was 0.82 emu g−1 at 1 T and room temperature. Simultaneously, we could control the nanoparticle size by altering the synthesis temperature. These upconversion nanostructures, characterized by both paramagnetic properties and regular morphology, represent promising dual-mode nanoprobe candidates for optical biological imaging and magnetic resonance imaging.