Manipulating topological arrangement is a powerful tool for tuning energy migration in natural photosynthetic proteins and artificial polymers. Here, we report an inorganic optical nanosystem composed of NaErF4 and NaYbF4, in which topological arrangement enhanced upconversion luminescence. Three architectures are designed for considerations pertaining to energy migration and energy transfer within nanoparticles: outside-in, inside-out, and local energy transfer. The outside-in architecture produces the maximum upconversion luminescence, around 6-times brighter than that of the inside-out at the single-particle level. Monte Carlo simulation suggests a topology-dependent energy migration favoring the upconversion luminescence of outside-in structure. The optimized outside-in structure shows more than an order of magnitude enhancement of upconversion brightness compared to the conventional core-shell structure at the single-particle level and is used for long-term single-particle tracking in living cells. Our findings enable rational nanoprobe engineering for single-molecule imaging and also reveal counter-intuitive relationships between upconversion nanoparticle structure and optical properties.

Different from ensemble measurements, singleparticle imaging could offer us a clean structural design versus optical property relationship without the interferences arising from heterogeneity of nanoparticle size and formula, particle-particle interaction and discrepancy of nanoparticle concentration. Moreover, a wider-range of excitation irradiance could be applied in single-particle measurement easily, which is helpful for studying the powerdependent performance.Despite the importance of singleparticle imaging, only limited studies have investigated the optical properties of single UCNPs. [25][26][27][28][29][30] One of the major hurdles is their weak luminescence. Efforts have been made to improve the single UCNPs brightness, such as employing high doping level of sensitizer or emitter ions. [25,[31][32][33][34][35] In particular, higher Er 3+ doping is optimal for the higher power densities at single particle level; enriched sensitizer Yb 3+ in the interior could significantly enhance upconversion brightness. [25,31] In these highly doped UCNPs design, inert shell is indispensable considering that surface quenching is the dominant nonradiative pathway, [36] which could stop the energy transfer from active ions to surface quenchers efficiently, leading to a significantly increased quantum yield. [36][37][38] However, inert shell also results in undesired volume increasing for single molecule imaging, given that bigger particles would have a heavier influence on the labeled-target behavior. Besides, it is still less clear about the effect of the inert shell on single particle brightness in a volume-constrained UCNP.To understand the potential sources of energy loss associated with the nanocrystal surface and obtain optimal volumeconstrained single particle brightness, we systemically investigated the shell effect on upconversion luminescence in the highly doped UCNPs. By tuning the doping concentration of sensitizer Yb 3+ in the outmost shell, we demonstrated a power-dependent performance where Yb 3+ doped outmost shell could outperform inert shell under higher power densities at the single particle level. Best performing active-shelled UCNPs at higher powers turned out to be less-luminescent at the excitation powers typically used in ensemble measurement. Inert shells shown to significantly increase UCNP brightness at low powers are likely dispensable at higher powers. These Lanthanide-based upconversion single molecule imaging (u-SMI) is of great interest due to its excellent photostability and background-free detection. However, the dim brightness strongly hampers its application in quantitative studies. An inert shell can effectively enhance the upconversion brightness, especially for highly doped upconversion nanocrystals in which surface quenching is the dominant nonirradiative pathway. Here, it is discovered that inert shell is actually a drawback for u-SMI in a volume-constrained system and sensitizer Yb 3+ doped outmost shell outperforms inert shell in single mole cule imaging condition, which indicate...

Single molecule localization microscopy based on photoactivation is a powerful tool for investigating the ultrastructure of cells. We developed a general strategy for photoactivatable fluorophores, using 2,3dihydro-1,4-oxathiine group (SO) as a tag to attach to various skeletal structures, including coumarin, BODI-PY, rhodamine, and cyanine. The conjugation of SO resulted in a significant loss of fluorescence due to photoinduced electron transfer (PeT). Under the irradiation of excitation light, singlet oxygen generated by the fluorophores converted the SO moiety into its ester derivative, terminated the PeT process, and restored the fluorescence. Single molecule localization imaging was achieved using a dual functional illuminating beam in the visible, acting as both the activating and the exciting source. We successfully applied these photoactivatable probes for time-lapse super-resolution tracking in living cells and super-resolution imaging of microtubule structures in neurons.

Dye‐sensitization can enhance lanthanide‐based upconversion luminescence, but is hindered by interfacial energy transfer from organic dye to lanthanide ion Yb3+. To overcome these limitations, we propose modifying coordination sites on dye conjugated structures and minimizing the distance between fluorescence cores and Yb3+ in upconversion nanoparticles (UCNPs). Our specially designed near‐infrared (NIR) dye, disulfo‐indocyanine green (disulfo‐ICG), acts as the antenna molecule and exhibits a 2413‐fold increase in luminescence under 808 nm excitation compared to UCNPs alone using 980 nm irradiation. The significant improvement is attributed to the high energy transfer efficiency of 72.1% from disulfo‐ICG to Yb3+ in UCNPs, with majority of energy originating from triplet state (T1) of disulfo‐ICG. Shortening the distance between the dye and lanthanide ions increases the probability of energy transfer and strengthens the heavy atom effect, leading to enhanced T1 generation and improved dye‐triplet sensitization upconversion. Importantly, this approach also applies to 730 nm excitation Cy7‐SO3 sensitization system, overcoming the spectral mismatch between Cy7 and Yb3+ and achieving a 52‐fold enhancement in luminescence. Furthermore, we demonstrate the enhancement of upconversion at single particle level through dye‐sensitization. This strategy expands the range of NIR dyes for sensitization and opens new avenues for highly efficient dye‐sensitized upconversion systems.This article is protected by copyright. All rights reserved

Single molecule localization microscopy based on photoactivation is a powerful tool for investigating the ultrastructure of cells. We developed a general strategy for photoactivatable fluorophores, using 2,3dihydro-1,4-oxathiine group (SO) as a tag to attach to various skeletal structures, including coumarin, BODI-PY, rhodamine, and cyanine. The conjugation of SO resulted in a significant loss of fluorescence due to photoinduced electron transfer (PeT). Under the irradiation of excitation light, singlet oxygen generated by the fluorophores converted the SO moiety into its ester derivative, terminated the PeT process, and restored the fluorescence. Single molecule localization imaging was achieved using a dual functional illuminating beam in the visible, acting as both the activating and the exciting source. We successfully applied these photoactivatable probes for time-lapse super-resolution tracking in living cells and super-resolution imaging of microtubule structures in neurons.