Microscopic inspection and tracking of single upconversion nanoparticles in living cells

Nanoparticles have become new tools for cell biology imaging 1


S1 and
), 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 microscopy 19 .
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 microscopy 9 or the recently reported MINFLUX method 20 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 (Supplementary Information Section 5). Note that the processed computer data compensate for the non-uniform excitation field and provide an accurate single identification accuracy (100% accuracy, Supplementary Fig. S6). Remarkably, due to the background-free detection with the NIR excitation, non-blinking and non-bleaching features of the UCNPs, the human eye can also identify this threshold and recognize single UCNPs during microscopic inspection (Figure 2a, eye vision).
The importance of the real-time observation of single cellular event comes from the detection of sub-cellular vesicles and protein movements and understanding their interactions in the complex cellular function. There are myriad of models 21 that propose different sub-cellular functions, including cytoskeleton re-arrangement 22 , protein dynamics, organelle movement and cooperation 23,24 , but until now, options for the real-time observation of these models in living cells have been limited. As Figure 2b shows, in the three typical 2D images taken at different heights, the high brightness of the photostable UCNPs is detectable not only in a dark room but also under bright-field illumination, providing the ability to identify the position of a single nanoparticle within a living cell and establishing a powerful tool for examining intercellular re-organization and trafficking.
Herein, by recording the data and offline analysis of nanoparticles inside living cells, the 3D trajectories of seven spots of single particles (# 2, 3, 4 and 6) and clusters (# 1, 5 and 7) within a single cell for an observation period of 21 s (Figure 2c) clearly show the heterogeneous dynamics of each single nanoparticle and cluster, highlighting the ability to precisely distinguish the transition between different dynamic phases of the transport of a single nanoparticle. The cumulative displacements ( Figure 2d) and their corresponding mean-square displacement figures (Figure 2e) show that most of the particles exhibit 'confined' particle diffusion, which is most likely associated with nanoparticle movement inside the sub-cellular vesicle. Interestingly, particle #2 demonstrates a slightly higher motility as well as a higher diffusion coefficient of 0.18 μm 2 s − 1 (Figure 2f). Recently, Liu et al. 10 established that the motility of gold nanoparticles in early . The photon counts used in this paper are the photon counts that reach a human cornea (I eye ). It can be calculated according to Supplementary Equation S2 (I eye = I con /R c2e , Supplementary Information Section 3), where Icon is the photon counts measured by SPAD and R c2e is the converting ratio.
Letter endosomes is slightly higher than that in late endosomes/lysosomes. Our observation corresponds with those results well, as particles #1, 4, 5, 6 and 7 are all located in the perinuclear area that is similar to the location of late endosomes/lysosomes, while particle #2 is closer to the cell membrane (early endosome).
Remarkably, particle #3 moves relatively fast and exhibits two-phase particle movement, reaching a speed of 3 μm s − 1 during phase II with a specific direction. Such a movement is associated with the active migration of molecular motor proteins on microtubules or actin filaments 8,10,25 . Figure 2f further illustrates that during phase II, particle #3 reaches a diffusion coefficient as large as 0.52 ± 0.04 μm 2 s − 1 , suggesting that its movement occurs within a transport channel. The much lower diffusion coefficients of the other particles and clusters are in good agreement with the values reported for the random diffusion of nanoparticles inside cells 7,26 .
Our approach further enables the quantitative study of the localized environment viscosity, the knowledge of which provides powerful insight into protein dynamics, as the local viscosity contributes to the specific functioning of intracellular proteins 21 . Figure 2g shows that particles #4 and #6 reside inside late endosomes/lysosomes and exhibit low motility, which also corresponds to a higher viscosity inside those organelles (166 ± 13 and 184 ± 14 cP, respectively). The viscosity of early endosomes, where particle #2 is presumably located, is lower (41 ± 4 cP) 27 , which is associated with more a dilute environment. In contrast to confined nanoparticles, particle #3 exhibits a high motility that corresponds to a lower viscosity (11.6 ± 0.9 cP) in its vicinity (cytoplasm). The lower viscosity in the cytoplasm promotes higher hydration, which leads to higher protein functionality.
To further demonstrate the power of our approach in resolving single nanoparticles, we focused on tracking a diffraction-limit spot containing multiple UCNPs. Letter two individual single nanoparticles (P1, P2). We further calculated Pearson's correlation coefficient (see Supplementary Information Section 6) and obtained a value of 0.46 (Po0.05), which is considered to be a moderate positive correlation, indicating that the two UCNPs are not aggregated but have a degree of independency. Figure 3f and g show that both the UCNPs are first retained in a confined area, possibly between actin filaments. After separation into singles nanoparticles (phase I), they proceed to move with a speed similar to that before separation. During phase II, both separated nanoparticles begin to move faster.
Interestingly, the diffusion coefficient analysis (Figure 3h) reveals that the diffusion coefficients for P1&P2, P1 in phase I (P1 I ) and P2 in phase I (P2 I ) have similar values, which also suggests that the two separate nanoparticles are confined in the same location; an aggregate with a larger size should have a smaller value. The viscosity calculation ( Figure 3i) shows a very high viscosity value for P1 and P2, which most likely does not reflect the local viscosity but rather reflects the immobilized state of the nanoparticles associated with cell structural components, i.e., trapping between actin filaments.
The UCNPs presented in this work are not only bright but also display excitation-power-dependent properties, and Supplementary Figure S3 shows that high concentrations of Tm 3+ -doped UCNPs only turn on at relatively high excitation power densities 28 . This result offers a new dimension for the simultaneous imaging and tracking of multiple kinds of single nanoparticles. Figure 4 demonstrates the potential of this fifth untapped dimension independent of the conventional colour channels for optical multiplexed tracking of single nanoparticles in a 3D cellular environment, which is useful because it gives the ability of colour-blind observers to use upconversion fluorescence microscopes.  showing the transformation from a diffraction-limit cluster (red) to two independent nanoparticles (blue for particle 1 and light blue for particle 2). (e) 2D pathways of the two tracked single nanoparticles from their confinement to their escape from their local environment over time (colour coded). (f) Cumulative displacement and (g) MSD analysis of the cluster and its separation into two single nanoparticles, each of which later experienced two phases of movement at different times. (h) Calculated diffusion coefficient and (i) resultant local viscosity measurements for the two separated single UCNPs.

Letter
In summary, here, we have realized a library of UCNPs that allows the human eye to distinguish single nanoparticles in living cells through a microscope. This unique capability further enables the accurate measurement of localized intercellular environment viscosities for functional super-resolution imaging. Time-resolved imaging of lifetime-tunable UCNPs 29 will enable multiplexed imaging and super-resolution imaging of sub-cellular structures. With both their excitation and emission bands in the NIR optically transparent biological window, the UCNPs demonstrated in this work make single nanoparticle tracking in deep tissue feasible. and with the same settings of the CCD camera, the 8 mol% Tm 3+ UCNPs start to emit a comparable amount of upconversion luminescence to that of the 1 mol% Tm 3+ UCNPs. (g-i) Under a relatively high excitation intensity, e.g., 0.6851 MW cm − 2 and above, the 8 mol% Tm 3+ UCNPs become so bright that they saturate the camera; thus, a reduced gain with a value of 2 and an exposure time of 100 ms are applied for the image recording. Under this condition, the signals from each single 1 mol% Tm 3+ UCNP become lower than the detection threshold of the camera (orange dotted lines), making it undetectable compared with the brightly emitting 8 mol% UCNPs. (b, e, h) are the background-free upconversion images. (c, f, i) are the upconversion images in a living cell when brightfield illumination is on. To compare the recorded emission intensity of each UCNP, each upconversion image is also presented as a 3D intensity plot below each image. Letter