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.

Photoisomerization kinetics of the near-infrared (NIR) fluorophore Sulfo-Cyanine7 (SCy7) was studied by a combination of fluorescence correlation spectroscopy (FCS) and transient state (TRAST) excitation modulation spectroscopy. A photoisomerized state with redshifted emission was identified, with kinetics consistent with a three-state photoisomerization model. Combining TRAST excitation modulation with spectrofluorimetry (spectral-TRAST) further confirmed an excitation-induced redshift in the emission spectrum of SCy7. We show how this red-emissive photoisomerized state contributes to the blinking kinetics in different emission bands of NIR cyanine dyes, and how it can influence single-molecule, super-resolution, as well as Förster resonance energy transfer (FRET) and multicolor readouts. Since this state can also be populated at moderate excitation intensities, it can also more broadly influence fluorescence readouts, also readouts not relying on high excitation conditions. However, this additional red-emissive state and its photodynamics, as identified and characterized in this work, can also be used as a strategy to push the emission of NIR cyanine dyes further into the NIR and to enhance photosensitization of nanoparticles with absorption spectra further into the NIR. Finally, we show that the photoisomerization kinetics of SCy7 and the formation of its redshifted photoisomer depend strongly on local environmental conditions, such as viscosity, polarity, and steric constraints, which suggests the use of SCy7 and other NIR cyanine dyes as environmental sensors. Such environmental information can be monitored by TRAST, in the NIR, with low autofluorescence and scattering conditions and on a broad range of samples and experimental conditions.

Optimization of lanthanide-doped luminescent nanoparticles for use in nanomedicine has encountered some difficulties due to the specific properties of water as a solvent. In this review, the current challenges for the adaptation of lanthanide-doped luminescent nanoparticles to aqueous environments, and promising strategies to optimize their colloidal dispersibility and stability in water and physiological buffers, are summarized. Moreover, the possible luminescence de-excitation paths caused by water molecule vibrations and how they can be prevented under different measurement conditions are discussed. This review also deals with the latest developments in lanthanide-doped luminescent nanoparticle design for nanomedicine, to increase the depth at which they can be monitored, which is mainly limited by the absorption bands of water. Furthermore, the anomalous temperature dependence of water and the different effects it has on lanthanide-doped luminescent nanoparticles in the physiological temperature range are commented on. Finally, a critical opinion on the possible next steps in this field is provided. 

The authors introduce stimulated-emission induced excitation depletion (STExD) nanoscopy using a single pair of low-power, near-infrared, continue-wave lasers. Emission of multichromatic probes is inhibited by cascade amplified depletion in lanthanide upconversion systems induced by manipulating their common sensitizer. Stimulated emission depletion (STED) microscopy is a powerful diffraction-unlimited technique for fluorescence imaging. Despite its rapid evolution, STED fundamentally suffers from high-intensity light illumination, sophisticated probe-defined laser schemes, and limited photon budget of the probes. Here, we demonstrate a versatile strategy, stimulated-emission induced excitation depletion (STExD), to deplete the emission of multi-chromatic probes using a single pair of low-power, near-infrared (NIR), continuous-wave (CW) lasers with fixed wavelengths. With the effect of cascade amplified depletion in lanthanide upconversion systems, we achieve emission inhibition for a wide range of emitters (e.g., Nd3+, Yb3+, Er3+, Ho3+, Pr3+, Eu3+, Tm3+, Gd3+, and Tb3+) by manipulating their common sensitizer, i.e., Nd3+ ions, using a 1064-nm laser. With NaYF4:Nd nanoparticles, we demonstrate an ultrahigh depletion efficiency of 99.3 +/- 0.3% for the 450 nm emission with a low saturation intensity of 23.8 +/- 0.4 kW cm(-2). We further demonstrate nanoscopic imaging with a series of multi-chromatic nanoprobes with a lateral resolution down to 34 nm, two-color STExD imaging, and subcellular imaging of the immunolabelled actin filaments. The strategy expounded here promotes single wavelength-pair nanoscopy for multi-chromatic probes and for multi-color imaging under low-intensity-level NIR-II CW laser depletion.

Lanthanide-doped upconversion nanoparticles (UCNPs) have rich photophysics exhibiting complex luminescence kinetics. In this work, we thoroughly investigated the luminescence response of UCNPs to excitation pulse durations. Analyzing this response opens new opportunities in optical encoding/decoding and the assignment of transitions to emission peaks and provides advantages in applications of UCNPs, e.g., for better optical sectioning and improved luminescence nanothermometry. Our work shows that monitoring the UCNP luminescence response to excitation pulse durations (while keeping the duty cycle constant) by recording the average luminescence intensity using a low-time resolution detector such as a spectrometer offers a powerful approach for significantly extending the utility of UCNPs.