Microscopic inspection and tracking of single upconversion nanoparticles in living cells

Nanoparticles have become new tools for cell biology imaging1, sub-cellular sensing2, super-resolution imaging3,4 and drug delivery5. Long-term 3D tracking of nanoparticles and their intracellular motions have advanced the understanding of endocytosis and exocytosis as well as of active transport processes6–8. The sophisticated operation of correlative optical-electron microscopy9,10 and scientific-grade cameras is often used to study intercellular processes. Nonetheless, most of these studies are still limited by the insufficient sensitivity for separating a single nanoparticle from a cluster of nanoparticles or their aggregates8,11,12. Here we report that our eyes can track a single fluorescent nanoparticle that emits over 4000 photons per 100 milliseconds under a simple microscope setup. By tracking a single nanoparticle with high temporal, spectral and spatial resolution, we show the measurement of the local viscosity of the intracellular environment. Moreover, beyond the colour domain and 3D position, we introduce excitation power density as the fifth dimension for our eyes to simultaneously discriminate multiple sets of single nanoparticles. By introducing thousands of photon sensitizers (i.e., Yb3+ ions) and activators ions (i.e., Tm3+ and Er3+ ions) to form an energy transfer network within a single nanoparticle, upconversion nanoparticles (UCNPs) can up-convert low-energy near-infrared (NIR) photons into high-energy visible emissions13. UCNPs emit tunable multicolour emissions under single-wavelength excitation for multiplexed sensing with low cytotoxicity and high chemical/photostabilities for biomedical applications14. Non-bleaching and non-blinking UCNPs are among the best probes for long-term tracking studies7, autofluorescence-free biomolecular sensing15, super-resolution microscopy imaging4, in vivo bio-imaging16 and light-triggered nanomedicine applications17. UCNPs are the most efficient materials for ultra-low power multiphoton microscopy and deep-tissue imaging18. Here we show a series of monodispersed UCNPs with a brightness that already meets the requirement for our eyes to observe single nanoparticles through a microscope. Figure 1a shows an upconversion fluorescence system built for the purpose of observing single nanoparticles. We performed a definitive vision test (Supplementary Table S1 and Figure 1b), where we individually tested 14 volunteers (overall, 28 eyes) to determine the number of emitted photons from single nanoparticle required to be distinguished by a human eye. We identified that at least 4186 photons per 100 ms are required for all tested eyes to see two separate blue nanoparticles (Figure 1b, region R1). In region R2, 17 eyes failed to recognize the blue colour, but the two particles were still distinguishable. In region R3, 21 eyes barely distinguished the spatially separated two nanoparticles, while region R4 has been identified as an insufficient number of photons to differentiate the two nanoparticles. Figure 1c shows a series of different batches of UCNPs purposely synthesized to cover a large range of representative sizes and emission properties (see Supplementary Information Section 2 for the details of characterization). Remarkably, their emission is highly uniform, which provides the foundation for this work in distinguishing single UCNPs from their clusters, either from images recorded by a camera or through real-time observation by the eyes. Notably, single UCNPs can be detected under low excitation power densities. As shown in Figure 1b, there are 78 photons per 100 ms detected from the 4-photon upconversion emission band (455 nm) under an excitation power density as low as 320 W cm− 2, and even the achieved intensity of 4186 photons per 100 ms for naked eye inspection requires a power density of approximately only 12 kW cm− 2, which is almost four orders of magnitude smaller than the excitation power required in two-photon microscopy19. Due to the optical diffraction limit, conventional far-field fluorescence microscopy does not have sufficient resolution to determine the number of nanoparticles when they are too close to each other. Approaches such as correlative electron microscopy9 or the recently reported MINFLUX method20 can be applied to improve the resolution. A high-level of uniformity in the UCNPs (Figure 1c) provides the ability for observers to identify a threshold intensity for single-UCNP detection. The emitters with a brightness higher than this threshold value will be identified as several nanoparticles within the diffraction limit region (e.g., labelled by orange dots in Figure 2a). The threshold value measured by the system (Figure 1a) enables automatic single-nanoparticle detection (Figure 2a, processed data) in real-time by computer processing of a wide-field fluorescence image