Lanthanide-doped upconverting nanoparticles

Abstract

Disclosed herein are lanthanide-doped upconverting nanoparticles and methods of making the same. Also disclosed are methods of imaging target proteins and methods of performing single particle tracking comprising the upconverting nanoparticles of this disclosure.

Description

[0001]Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 LANTHANIDE-DOPED UPCONVERTING NANOPARTICLES RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 553,882, filed February 15, 2024, the entire contents of which are incorporated herein by reference. STATEMENT OF GOVERNMENT SUPPORT This invention was made with government support under Grant No. AG065516 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND Numerous strategies have been developed to reduce fluorophore photobleaching and blinking, such as employing enzymatic oxygen-scavenging systems or addition of triplet-state quenchers either in solution or directly conjugated to the fluorophores. Recently, blinking of quantum dots has been greatly suppressed. However, toxicity remains a concern for using these probes in live cells. Longer single-particle tracking (SPT) was obtained by using biomolecular scaffolds that assemble multiple fluorescent proteins or dyes, but these methods are still limited to the timescale of few minutes. Current multiplex methods require excitation sources of different wavelengths or sequential imaging. Moreover, the broad and featureless emission spectra of reported SPT probes limit the number of colors that can be concurrently imaged. Expanding the array of simultaneous colors would enable the study of interactions and colocalizations on a single-molecule level. This could significantly increase the number of targets in highly multiplexed imaging techniques such as MERFISH, seqFISH, and STARmap. In view of these limitations, there is an unmet need to develop improved, photostable and tunable probes with distinct emission spectra for multiplex SPT. SUMMARY OF THE INVENTION In one aspect, the present disclosure provides an upconverting nanoparticle, comprising: FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 a) a core comprising one or more activator ions and one or more emitter ions; wherein, upon excitation with radiation, the one or more activator ions emit radiation of a wavelength capable of exciting the one or more emitter ions; and b) an optically inert shell enveloping the core. In another aspect, the present disclosure provides methods of performing single-particle tracking, wherein the particle to be tracked is conjugated to an upconverting nanoparticle disclosed herein. In yet another aspect, the present disclosure provides methods of making an upconverting nanoparticle, comprising: a) contacting a salt of one or more activator ions, a salt of one or more emitter ions, and a solvent, thereby forming a reaction mixture; b) contacting the reaction mixture with a halide salt to form a core; and c) contacting the core with a solution comprising a halide source to form a shell around the core. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A shows a 3D schematic rendering of a core-shell UCNP. Green: activator ion (Yb3+); Pink: emitter ion (Er3+or Tm3+); Gray: Inert shell (Y3+and Gd3+). FIG. 1B shows normalized size histograms of shell thickness series showing the meanand standard deviation. FIG.1C shows a representative TEM image of 8% Er UCNP cores. Scale bar: 40 nm. FIG.1D – FIG.1J show representative TEM images of cores in c with increasing shell thickness. The frame color in c-j correspond to the peaks in b. Scale bar measures 40 nm. FIG.2A shows single-particle optical images of 8% Er3+-doped CS UCNPs with 8 nm cores and increasing inactive shell thicknesses (a: Core, b: 0.45 nm, c: 1.00 nm, d: 2.90 nm, e: 4.20 nm). Scale bars: 2 m. FIG.2B shows single-particle optical images of 8% Er3+-doped CS UCNPs with 8 nm cores and 0.45 nm shell thickness. Scale bars: 2 m. FIG.2C shows single-particle optical images of 8% Er3+-doped CS UCNPs with 8 nm cores and 1.00 nm inactive shell thickness. Scale bars: 2 m. FIG.2D shows single-particle optical images of 8% Er3+-doped CS UCNPs with 8 nm cores and 2.90 nm inactive shell thickness. Scale bars: 2 m. FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 FIG.2E shows single-particle optical images of 8% Er3+-doped CS UCNPs with 8 nm cores and 4.20 nm inactive shell thickness. Scale bars: 2 m. FIG.2F shows a zoom-in view highlighted by the yellow box in e. Scale bar: 400 m. FIG.2G shows a SEM image of the same field-of-view in f, Scale bar: 400 nm. FIG.2H shows single-particle emission of 8% Er3+-doped CS UCNPs with increasing inactive shell thicknesses as a function of incident power density. FIG. 2I shows emission of 8% Er3+-doped CS UCNPs as a function of inactive shell thickness at a power density of 30 kW / cm2. Error bars indicate the standard deviation. FIG. 3A shows a single-particle emission of CS UCNPs, NaYb1-xErxF4@ -NaY0.8Gd0.2F4, doped with different Er3+percentages as a function of incident power density. These UCNPs have 8 nm core diameter and 2 nm inactive shell thickness. FIG. 3B shows emission of Er3+-doped CS UCNPs as a function of Er3+doping percentage at a power density of 30 kW / cm2. A doping of 10% Er3+yields the brightest particle. FIG. 4A shows optical images from the Blue and NIR channels of CS UCNPs dopedwith different Tm3+ percentages, (a: NaYb1-xTmxF4@ -NaY0.8Gd0.2F4 (0.04 < x < 0.50); TheseUCNPs have 8 nm core diameter and 2 nm inactive shell thickness. Scale bar measures 1 μm. FIG. 4B shows optical images from the Blue and NIR channels of CS UCNPs dopedwith different Tm3+ percentages, (b: NaY0.7-xYb0.3TmxF4@ -NaY0.8Gd0.2F4 (0.005 < x < 0.02)).These UCNPs have 8 nm core diameter and 2 nm inactive shell thickness. Scale bar measures 1 μm. FIG.4C shows single-particle emission of different doping percentages of Tm3+-doped CS as a function of incident power density (c, f: Blue channel). FIG.4D shows single-particle emission of different doping percentages of Tm3+-doped CS as a function of incident power density (d, g: NIR channel). FIG.4E shows emission of Tm3+-doped CS UCNPs as a function of doping percentageat a power density of for two channels and overall emission. A doping of 2% Tm3+, 30% Yb3+yields the best probe for the blue channel. FIG.4F shows single-particle emission of different doping percentages of Tm3+-doped CS as a function of incident power density (c, f: Blue channel). FIG.4G shows single-particle emission of different doping percentages of Tm3+-doped CS as a function of incident power density (d, g: NIR channel). FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 FIG.4H shows emission of Tm3+-doped CS UCNPs as a function of doping percentage at a power density of for two channels and overall emission. A doping of 15% Tm3+yields the best probe for the NIR channel. FIG.5A shows emission of selected probes with 8 nm core diameter and 1 nm inactive shell thickness as a function of incident power density for channels with non-negligible emission, (a: Blue probe) with insets of the TEM (scalebar: 20 nm). FIG.5B shows emission of selected probes with 8 nm core diameter and 1 nm inactive shell thickness as a function of incident power density for channels with non-negligible emission, (b: Green probe) with insets of the TEM (scalebar: 20 nm). FIG.5C shows emission of selected probes with 8 nm core diameter and 1 nm inactive shell thickness as a function of incident power density for channels with non-negligible emission, (c: NIR probe) with insets of the TEM (scalebar: 20 nm). FIG. 5D shows the same optical FOV in different channels for a sample with the threeprobes at similar concentrations (d: Blue channel). FIG. 5E shows the same optical FOV in different channels for a sample with the threeprobes at similar concentrations (e: Green channel). FIG. 5F shows the same optical FOV in different channels for a sample with the threeprobes at similar concentrations (f: NIR channel). FIG.5G shows a blended optical image with semi-transparent colormaps. FIG. 5H shows a 3D rendering of intensity for the same field-of-view of d-g. Scale bars measure 1 m. While the Blue probe displays signal in the NIR channel, this is overshadowed by the large NIR emission of the NIR probe. The converse is also true for the NIR probe and its blue emission. The Green probe shows negligible emission in the Blue and NIR channels. FIG.6A shows the last frame of the 10-minute three-color merged video of the top of a HeLa cell membrane. Membrane proteins were labeled with the three colors of 10-nm probes and imaged at 21 kW / cm2with 300 ms time resolution. Scale bar measures 5 μM. FIG. 6B shows 10-minute single-particle tracks colored by channel (Blue channel: Blue, Green channel: Green, NIR channel: Magenta). Scale bar measures 5 μM. FIG.6C shows four panels at increasing times of the region highlighted in b. A green- labeled membrane protein and a NIR-labeled membrane protein diffuse close by to each other for the duration of tracking. At t = 2 minutes, a fast-moving, blue-labeled membrane protein approaches and leaves. At t = 5 minutes, both red and green-labeled proteins co-localize, FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 approaching each other to minimum distances of tens of nanometers. Scale bar measures 500 nm. FIG.6D shows an inset from figure 6b, corresponding to the tracks of the NIR, green and blue-labeled proteins described in c. The blue-labeled membrane protein diffuses in and out of focus, making the tracking algorithm fail at some time steps and leaving gaps. Scale bar measures 5 μM. FIG. 6E shows a time-colored track of the green-labeled protein of c and d. It moves upwards of 2.5 um over the span of 10 minutes, and colocalizes with the red-labeled protein around t = 5min. Signal from the particle label does not decrease over the movie length. FIG.7 shows transmission electron micrographs and size distributions for Er3+-doped core and core-shell UCNPs with dopant percentages ranging between 4% and 50%. Scale bar: 20 nm. FIG.8 shows transmission electron micrographs and size distributions for Tm3+-doped core and core-shell UCNPs with dopant percentages ranging between 4% and 50%. Scale bar: 20 nm. FIG.9 shows transmission electron micrographs and size distributions for Tm3+- doped core and core-shell UCNPs with dopant percentages ranging between 0.5% and 2%. Scale bar: 20 nm. FIG.10A – FIG.10D shows X-ray diffraction data for all samples. Background arises from sample holder. Reference data included for hexagonal NaYF (ICDD 04-017-6069). a: Erbium-doped core series; a was acquired using a Smartlab Multipurpose Diffractometer (Rigaku). FIG. 10B shows erbium-doped core-shell series; Taken on the X’Pert X-ray diffractometer (PANalytical B.V.). FIG. 10C shows thulium-doped core series; Taken on the X’Pert X-ray diffractometer (PANalytical B.V.). FIG. 10D shows thulium-doped core-shell series; Taken on the X’Pert X-ray diffractometer (PANalytical B.V.). FIG.10E shows 10 nm probes. Taken on the X’Pert X-ray diffractometer (PANalytical B.V.). FIG. 11A – FIG. 11E show Maxima-normalized spectra of UCNPs under 976 nm excitation at a power density of 3.7 kW / cm2. FIG. 11A shows widefield microscope spectral binning into channels overlaid on emission of an 1% Tm / 30% Yb particle. Emission is split at 510 and 750 nm, with a cutoff at 850 nm. FIG. 11B shows spectra for three UCNPs from the shell thickness series shown in Figure 2. Curves for the core, intermediate thickness and final thickness are shown. The core, even at high concentrations, showed very little signal and was FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 normalized to the noise. FIG.11C shows spectra for increasing concentrations of Er3+doping in 8 nm core, 2 nm shell UCNPs. FIG. 11D shows spectra for increasing concentrations of Tm3+doping in 8 nm core, 2 nm shell UCNPs. FIG. 11E shows spectra for increasing concentrations of Tm3+doping in 8 nm core, 2 nm shell UCNPs with 30% Yb. FIG.11F shows spectra for the three 10 nm probes. Given the lower power density of the spectrometer, the blue emission of the blue probe has not overtaken the NIR emission as shown in single-particle measurements. FIG.12A – FIG.12B show energy level diagrams of Yb3+, Er3+and Tm3+and spectra with features assigned to energy transitions for UCNPs doped with these ions. FIG.12A shows energy level diagrams for the Ln3+ions utilized in the UCNPs discussed in this paper, with highlighted emission transitions. FIG.12B shows individual spectra of the three 10 nm probes with features assigned to highlighted transitions in a. We limited the assignment of transitions to the slope measured in the single-particle measurements of emission versus power density, i.e.3-photon processes for Er-doped UCNPs and 4-photon processes for Tm-doped UCNPs. FIG. 13A – FIG. 13F show correlative optical-SEM images. FIG. 13A shows SEM image of field of view of FIG. 2E. FIG. 13B shows reproduction of FIG. 2B, with the field- of-view of panel (a) overlaid in purple. FIG. 13C shows zoom-ins of single particles in the field of view of c, with borders corresponding to the same particles in (a). FIG. 13D shows zoom-ins of single particles in the field of view of c, with borders corresponding to the same particles in (a). FIG. 13E shows zoom-ins of single particles in the field of view of c, with borders corresponding to the same particles in (a). FIG. 13F shows zoom-ins of a doublet of particles in (a), which creates the doubly intense PSF in the optical image (b). FIG.14A – FIG.14H show histograms of single-particle brightness for the Er-doped UCNP shell thickness. FIG.14A shows brightness histograms for 0.0 nm shell thickness. FIG. 14B shows brightness histograms for 0.7 nm shell thickness. FIG. 14C shows brightness histograms for 1.3 nm shell thickness. FIG.14D shows brightness histograms for 1.8 nm shell thickness. FIG.14E shows brightness histograms for 2.2 nm shell thickness. FIG.14F shows brightness histograms for 2.5 nm shell thickness. FIG. 14G shows brightness histograms for 3.2 nm shell thickness. FIG.14H shows brightness histograms for 3.8 nm shell thickness, with the means and standard deviations denoted. The sigma-over-mu for these histograms decreases from about 20% to 10% as shell thickness increases, roughly double the values of the corresponding particle size histograms from TEM images. This indicates a separate variability in the particles that does not originate from diameter effects. FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 FIG. 15 shows mean-normalized emission trace of the three 10 nm probes over 10 hours at constant exposure to the excitation source at 14 kW / cm2, with histograms of the traces adjacent. All probes were present in the field-of-view as in Figure 5g. Traces are averages of 3 or more particles in the same field of view. For absolute brightness, refer to Figures 5a-c of themain text. The / for each trace are: Blue: 0.07; NIR: 0.07; Green: 0.03. Focus was maintainedover imaging period. FIG. 16A – FIG. 16D show volume-normalized emission of UCNPs ( ) at apower density of 30 kW / cm2. FIG. 16A shows Er-doped UCNPs with increasing doping percentage. FIG.16B shows high Tm-doped UCNPs with increasing doping percentage. FIG. 16C shows low Tm-doped UCNPs with increasing doping percentage containing only 30% Yb, with the remainder comprising Y. FIG.16D shows Green, Blue and NIR 10 nm probes. FIG.17A – FIG.17E show illustration of image registration between channels for the 12 nm overall diameter 6% Tm-doped UCNP. FIG.17A shows the raw image from the camera, with the 3-channel split. The green channel is dimmer than the other two for this particle. FIG. 17B shows the three channels overlaid with colors assigned but misaligned. FIG.17C shows the final aligned result. FIG. 17D shows a 3D rendering of (b) with normalized intensity between channels. FIG.17E shows similar 3D rendering of (c). FIG. 18 shows transmission electron micrographs of silica-coated 10 nm CS nanoparticles of three colors. Silica coated UCNP TEM images: left column, PEGylated UCNP images: right column. Scale bars measure 20 nm. FIG.19A shows Mean-Square-Displacement of NIR-labeled membrane proteins from FIG. 6. The 2D diffusion coefficient (D) is found by linear fit for hand-picked regions of the MSD curve for both particles. FIG.19B shows Mean-Square-Displacement of green-labeled membrane proteins from FIG. 6. The 2D diffusion coefficient (D) is found by linear fit for hand-picked regions of the MSD curve for both particles. The NIR-labeled membrane protein diffuses much slower than the green-labeled membrane protein by over an order of magnitude. The Green-labeled membrane protein intercalates between a slow-diffusion mode and a fast diffusion mode. Such diffusion curves would have been hard to separate without multi-color probes given the proximity between the labeled membrane proteins at some time-steps. Furthermore, the stability of the probes yields very confident MSD values for time steps in the order of minutes (n ~ 1800 for T = 1 minute). FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 FIG.20A – FIG.20B show tracking of the interaction between Green and NIR labeled membrane proteins from FIG. 6C. FIG. 20A shows trajectories for both particles for frames where both are localized. (inset) Points for 80 seconds centered around the 5-minute mark are highlighted as stars for the NIR probe and circles for the Green probe. The points of the two particles of the same frame are connected by black lines, and their intensity is proportional to the inverse of the particles’ separation. Scale bar: 100 nm. FIG. 20B shows time trace of the separation between particles. The inset corresponds to the highlighted period where the particles localize near to each other. The dotted horizontal line corresponds to the uncertainty from the gaussian fit (10 nm) and the dashed line corresponds to the maximal estimated error of the channel registration (20 nm). FIG.21A – FIG.21D depict affinity-labelled UCNPs. FIG.21A shows schematics of functionalization of UCNPs to enable specific labeling. The core-shell UCNPs are passivated by poly(maleic anhydride-alt-1-octadecene) (PMAO) followed by 4-(Dimethylamino)pyridine (DMAP) to introduce carboxyl groups on the surface of UCNPs. Subsequently, methyl-PEG- Amine and X-PEG-Amine where X represents Halotag ligand (HTL), SNAP tag ligand (STL) or CLIP tag ligand (CTL) are conjugated to the surface of UCNPs using the EDC chemistry. FIG.21B shows TEM image of PMAO-coated UCNPs. FIG.21C shows fluorescence images of U2OS cells expressing Halotag fused epidermal growth factor receptor (EGFR) (top row), SNAP tag fused HER2 (middle row), and CLIP tag fused HER3 (bottom row). EGFR, HER2, and HER3 are labeled by HTL-TMR, BG-SiR, and BC-TMR dyes, respectively (left column). Cell nuclei are labeled by Hochest (middle column). The blended images of labeled receptors and cell nuclei are shown in the right column. FIG.21D shows Single UCNP images of U2OS cells expressing Halotag fused epidermal growth factor receptor (EGFR) (top row), SNAP tag fused HER2 (middle row), and CLIP tag fused HER3 (bottom row). The left two columns are for the positive experiment where cells express fusion tag receptors, and the right two columns are for the control experiments where cell do not express fusion tag receptors. DETAILED DESCRIPTION OF THE INVENTION In one aspect, the present disclosure provides an upconverting nanoparticle, comprising: a) a core comprising one or more activator ions and one or more emitter ions; wherein, upon excitation with radiation, the one or more activator ions emit radiation of a wavelength capable of exciting the one or more emitter ions; and FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 b) an optically inert shell enveloping the core. In certain embodiments, the upconverting nanoparticle has a diameter of about 5-20 nm. In further preferred embodiments, the upconverting nanoparticle has a diameter of about 10 nm. In certain embodiments, the one or more activator ions are lanthanide ions. In further embodiments, the one or more activator ions are selected from Lu3+, Yb3+, Ho3+, Pr3+, Nd3+, Eu3+, Tb3+, Dy3+, Ce3+, Sm3+, and La3+, or a combination thereof. In yet further embodiments, the one or more activator ions are Yb3+. In certain embodiments, the one or more emitter ions are lanthanide ions. In further embodiments, the one or more emitter ions are selected from Lu3+, Er3+, Tm3+, Ho3+, Pr3+, Nd3+, Eu3+, Tb3+, Dy3+, Ce3+, Sm3+, and La3+, or a combination thereof. In yet further embodiments, the one or more emitter ions are Er3+, Tm3+, or a combination thereof. In certain embodiments, the core has an average diameter of about 5-10 nm. In further, preferred embodiments, the core has an average diameter of about 8 nm. In certain embodiments, the optically inert shell has an average thickness of about 0.5-8 nm. In further embodiments, the optically inert shell has an average thickness of about 1 nm. In certain especially preferred embodiments, the optically inert shell comprises -NaY0.8Gd0.2F4. In further preferred embodiments, the core comprises -NaYb1-xLnxF4, wherein Ln is selected from Yb3+, Er3+, and Tm3+, and wherein x is from 0 to 1. In yet further preferred embodiments, the core comprises NaY(1-x-y)Yb(y)Tm(x)F4, and wherein (x+y) = 1. In still further preferred embodiments, the core comprises NaY(1-x-y)Yb(y)Er(x)F4, and wherein (x+y) = 1. In certain embodiments, x is from 0 to 1. In further embodiments, y is from 0 to 1. In yet further embodiments, x is from 0 to 0.5, and y is from 0.5 to 1. In still further embodiments, x is from 0 to 0.25, and y is from 0.75 to 1. In certain preferred embodiments, the core comprises NaYb0.9Er0.1F4. In further preferred embodiments, the core comprises NaYb0.85Tm0.15F4. In yet further preferred embodiments, the core comprises NaY0.69Yb0.30Tm0.01F4. In certain preferred embodiments, the core does not comprise a seed crystal that does not contain activator ions or emitter ions. In certain embodiments, the upconverting nanoparticle emits radiation of a second wavelength when excited with infrared radiation of a first wavelength. In further embodiments, the first wavelength ranges from about 900 nm to about 1400 nm. In yet further especially preferred embodiments, the first wavelength is about 976 nm. In still further embodiments, the second wavelength ranges from about 350 nm to about 770 nm. In certain embodiments, the second wavelength ranges from about 780 nm to about 1500 nm. FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 In certain embodiments, the upconverting nanoparticle continues to emit radiation at a substantially constant intensity after about 15 hours of exposure to radiation at a wavelength of 976 nm and a power density of about 5-30 kW / cm2. In certain embodiments, the upconverting nanoparticle emits substantially continuous radiation during exposure to radiation at a wavelength of 976 nm and a power density of about 5-30 kW / cm2. In certain embodiments, the upconverting nanoparticle further comprises a reactive shell comprising SiO2enveloping the optically inert shell. In further embodiments, the reactive shell has a thickness of about 1-6 nm. In yet further, preferred embodiments, the reactive shell has a thickness of about 2 nm. In certain embodiments, the reactive shell is functionalized with an organic compound (e.g., a fluorophore, a peptide, a nucleic acid, an affinity tag, such as biotin, an enzyme, or a drug). In further embodiments, the organic compound is an affinity tag, such as biotin. In some embodiments, the reactive shell is functionalized with a moiety capable of reacting with a self-labeling protein tag (e.g., a Halotag, a SNAP tag, or a CLIP tag). In certain such embodiments, the moiety capable of reacting with a self-labeling protein tag is wherein represents the point of attachment to the reactive shell. In certain especially preferred embodiments, the present disclosure provides compositions comprising a plurality of upconverting nanoparticles disclosed herein, wherein the plurality of upconverting nanoparticles is substantially uniform in size, composition, shape, and / or molecular weight. In further preferred embodiments, the plurality of upconverting nanoparticles is substantially monodisperse. In yet further embodiments, the polydispersity index of the plurality of upconverting nanoparticles is between about 1% and about 10%. In still further embodiments, the polydispersity index of the plurality of upconverting nanoparticles is between about 3% and about 7%. In certain preferred embodiments, the polydispersity index of the plurality of upconverting nanoparticles is between 3.8% and FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 6.2%.In another aspect, the present disclosure provides methods of performing single-particle tracking, wherein the particle to be tracked is conjugated to an upconverting nanoparticle disclosed herein. In certain embodiments, the particle to be tracked is a single cell. In further embodiments, the particle to be tracked is a living cell. In yet another aspect, the present disclosure provides methods of imaging a target protein, the methods comprising: a) expressing a fusion of the target protein and a self-labeling protein tag in a cell; b) contacting the target protein with an upconverting nanoparticle of claim 38 or 39; c) exposing the upconverting nanoparticle to radiation at a wavelength of 976 nm and a power density of about 5-30 kW / cm2, thereby producing emitted radiation; and d) detecting the emitted radiation, thereby imaging the target protein. In certain embodiments, the self-labeling protein tag is selected from a Halotag, a SNAP tag, and a CLIP tag. In some embodiments, the cell is a living cell. In still another aspect, the present disclosure provides methods of making an upconverting nanoparticle, comprising: a) contacting a salt of one or more activator ions, a salt of one or more emitter ions, and a solvent, thereby forming a reaction mixture; b) contacting the reaction mixture with a halide salt to form a core; and c) contacting the core with a solution comprising a halide source to form a shell around the core. In certain preferred embodiments, the salt of the one or more activator ions is an acetate salt or a chloride salt. In further preferred embodiments, the salt of the one or more emitter ions is an acetate salt or a chloride salt. In certain embodiments, the reaction mixture further comprises an alkene and an organic acid. In further embodiments, the alkene is a terminal alkene. In yet further preferred embodiments, the terminal alkene is 1-octadecene. In still further embodiments, the organic acid is a carboxylic acid. In certain preferred embodiments, the carboxylic acid is oleic acid. In certain embodiments, the halide salt is a fluoride salt. In further embodiments, the fluoride salt is ammonium fluoride. In certain embodiments, the halide source is a trifluoroacetate salt (e.g., lithium trifluoroacetate, sodium trifluoroacetate, or potassium trifluoroacetate). In further preferred embodiments, the halide source is sodium trifluoroacetate. FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 In certain embodiments, step a) further comprises heating the reaction mixture to a first temperature of about 70-140 °C. In further embodiments, the first temperature is about 110 °C. In certain embodiments, step a) is performed over a first time of about 0.5-2.5 h. In further embodiments, the first time is about 1 h. In certain embodiments, step a) further comprises applying a vacuum to the reaction mixture after the reaction mixture is heated. In certain embodiments, step b) further comprises contacting the reaction mixture with a first base. In further embodiments, the first base is an amine, or a salt thereof. In yet further embodiments, the amine is oleylamine, or a salt thereof. In still further embodiments, step b) further comprises contacting the reaction mixture with a carboxylate salt. In certain embodiments, the carboxylate salt is an oleate salt (e.g., lithium oleate, sodium oleate, or potassium oleate). In further preferred embodiments, the carboxylate salt is sodium oleate. In still further embodiments, step b) further comprises heating the reaction mixture to a second temperature of about 280-340 °C after the vacuum is applied. In certain embodiments, the second temperature is about 310 °C. In still further embodiments, step b) is performed over a second time of about 0.5-2.5 h. In further embodiments, the second time is about 0.8 h. In certain embodiments, step c) is performed at a third temperature of about 50-90 °C. In further embodiments, the third temperature is about 70 °C. In certain embodiments, step c) is performed over a third time of about 0.1-1.5 h. In further embodiments, the third time is about 0.5 h. In certain embodiments, step c) is performed at a fourth temperature of about 250- 340 °C after the third time is elapsed. In further embodiments, the fourth temperature is about 290 °C. In certain embodiments, step c) is performed over a fourth time of about 0.5-3 h. In further embodiments, the fourth time is about 1.5 h. In certain embodiments, the method further comprises epitaxially growing a reactive shell comprising SiO2on the optically inert shell. In some embodiments, method further comprises contacting the reactive shell with an activating agent and a base, thereby forming a functionalized reactive shell. In certain such embodiments, the activating agent is poly(maleic anhydride-alt-1-octadecene) (PMAO) and the base is 4-(dimethylamino)pyridine (DMAP). In certain embodiments, the method further comprises contacting the functionalized reactive shell with a coupling reagent (e.g., an amide coupling reagent) and a reagent comprising a moiety capable of reacting with a self-labeling protein tag. In some such embodiments, the coupling reagent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). In some embodiments, the reagent comprising a moiety capable of reacting with a self-labeling protein tag is selected from FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 methyl-poly(ethylene glycol)n-amine, Halotag ligand-poly(ethylene glycol)n-amine, SNAP tag ligand-poly(ethylene glycol)n-amine, and CLIP tag ligand-poly(ethylene glycol)n-amine; wherein: n is an integer from 1 to 200. In certain embodiments, the reagent comprising a moiety capable of reacting with a self-labeling protein tag is methyl-poly(ethylene glycol)n-amine. In certain embodiments, the reagent comprising a moiety capable of reacting with a self-labeling protein tag is Halotag ligand-poly(ethylene glycol)n-amine. In some embodiments, the reagent comprising a moiety capable of reacting with a self-labeling protein tag is SNAP tag ligand-poly(ethylene glycol)n- amine. In some embodiments, the reagent comprising a moiety capable of reacting with a self- labeling protein tag is CLIP tag ligand-poly(ethylene glycol)n-amine. In another aspect, the present disclosure provides an upconverting nanoparticle produced by the methods disclosed herein. Optical microscopy has been a foundational method to study cellular processes. Notably, single-molecule microscopy has revolutionized the way we investigate biomolecular structure and dynamics by enabling the direct visualization of molecular interactions in real- time and reveals variations in molecular behavior that are hidden in ensemble measurements. Single-particle tracking (SPT) has illuminated a wide range of phenomena on a single molecule basis, including the dynamics of organelle interactions, mitochondrial crista, apoptosis, membrane receptors, motor proteins, and synaptic clefts. This technology allows monitoring of the motions of individual molecules with spatial resolution down to tens of nanometers, depending on the brightness of the probe. Single-particle tracking can be achieved with a wide range of probes, including organic dyes, fluorescent proteins, quantum dots, and metal nanoparticles. However, many probes still face limitations, particularly in photostability, toxicity, and the number of colors one can simultaneously image. Organic dyes and fluorescent proteins photobleach within tens of seconds, while quantum dots suffer from intermittent dark states and can be cytotoxic, depending on their functionalization. Photo-instability of these probes hinders the ability to continuously image the dynamics of single molecules for an extended period, which is essential for uncovering many biological processes. Gold or silver nanoparticles are photostable, but their size is relatively large (>30 nm). Numerous strategies have been developed to reduce fluorophore photobleaching and blinking, such as employing enzymatic oxygen-scavenging systems or addition of triplet-state FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 quenchers either in solution or directly conjugated to the fluorophores. Recently, blinking of quantum dots has been greatly suppressed. However, toxicity remains a concern for using these probes in live cells. Longer SPT was obtained by using biomolecular scaffolds that assemble multiple fluorescent proteins or dyes, but these methods are still limited to the timescale of few minutes. Current multiplex methods require excitation sources of different wavelengths or sequential imaging. Moreover, the broad and featureless emission spectra of reported SPT probes limit the number of colors concurrently imaged. Expanding the array of simultaneous colors enables the study of interactions and colocalizations on a single-molecule level. This could significantly increase the number of targets in highly multiplexed imaging techniques such as MERFISH, seqFISH, and STARmap. To overcome these limitations, rare-earth doped Upconverting Nanoparticles (UCNPs) may be utilized, which have been shown to be extremely photostable, bright and non-toxic, which makes them promising bio-imaging probes. UCNPs do not blink or bleach, even after hours of excitation. UCNPs are doped with activator ions such as Yb3+, a lanthanide which absorbs near- infrared excitation at 976 nm. The absorbed energy can be transferred to the co-doped emitter ions such as Er3+and Tm3+via multiphoton (upconversion) processes. Upconversion between near-infrared excitations to visible emission provides nearly background-free imaging by eliminating autofluorescence that is often seen when using visible laser excitations. Furthermore, UCNPs have much sharper emission lines. Spectrally pure UCNPs can be generated by doping UCNPs with different rare-earth ions enabling multiplexed imaging using a single excitation source. Despite UCNPs’ high photostability and anti-stokes shifts, their relatively large size (>20 nm) could perturb the dynamics of the labeled molecules and limit the current applications to studying larger complexes, such as cargo transport. Existing reports of smaller UCNPs show the field has not yet achieved brightness suitable for SPT with high temporal resolution and limited excitation-induced cell damage. Thus, smaller probes may retain effective brightness for SPT at high frame rates and low power densities. Furthermore, these probes are promising tools for illustrative applications such as nucleotide sequencing, or as color sources in display screens (e.g., television screens). More highly multiplexed palettes of UCNPs can be accessed by fine-tuning the emission spectrum of these small UCNPs. These photostable and multiplexed probes open new FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 avenues for numerous and diverse biological applications by enabling imaging of molecular interactions in real time at the single molecule level, and for a prolonged period. Accordingly, the present disclosure describes upconverting nanoparticles with tunable emissive properties, which may be further functionalized and are suitable for a range of applications, including, but not limited to, single-particle tracking, live-cell imaging, nucleotide sequencing, or use in color displays. The present disclosure further describes methods by which to produce said upconverting nanoparticles. The upconverting nanoparticles of this disclosure generally comprise lanthanide ions of the form MLnX4, where Ln is a lanthanide ion, X is a halide ion, and M is an alkali metal ion. In preferred embodiments, Na+or K+ions can be used as the alkali metal ion. The crystal lattice of the UCNPs imparts several important properties upon the UCNPs for facilitating energy transfer. In preferred embodiments, the crystal lattice has a low lattice phonon energy, is highly chemically stable, and has low symmetry. Each of these features raises the efficiency of photon upconversion. In preferred embodiments, the UCNPs of this disclosure comprise ions with similar ionic radii, which improve these properties of the crystal lattice, and in turn improve upconversion. The selection of activator and emitter ions is typically also dependent on the ions’ photophysical properties. Appropriate selections of activator and emitter ion will minimize non-radiative energy loss and maximize photon absorption and energy transfer. The selection of halide is also important; in preferred embodiments, the UCNPs of this disclosure comprise fluoride, as it is chemically stable and has a low phonon energy. In especially preferred embodiments, the UCNPs of this disclosure are core-shell structured, comprising an internal, approximately spherical core, and an external, approximately spherical shell enveloping the core. In preferred embodiments, the cores range in size from about 5 nm in diameter to about 20 nm in diameter, while the external shells may range in thickness from about 0.5 nm to about 8 nm. In certain embodiments, the UCNPs comprise an infrared-inert lanthanide ion such as gadolinium or yttrium, as well as dopant activator and emitter ions as described above. The doping ratio of both activator and emitter ions may vary, such that the preferred empirical formula of a UCNP may range from, for example, NaY(0.20 – 0.80)Yb(0.05 –0.40)Tm(0.01 – 0.15)F4. The optical characteristics of the UCNPs are a function of multiple factors, such as the size of the core, the thickness of the external shell, and of the doping ratios of activator and emitter ions. The particles disclosed herein can be tuned to emit radiation with a wavelength ranging from the visible into the infrared, or from about 400 nm to about 1000 nm when activated with infrared radiation. The photophysical features of the emission spectra are FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 also dependent on the physical characteristics of the UCNPs; for example, a polydisperse sample of particles will have a broader emission spectrum than a monodisperse sample that is otherwise identical in composition, because particles with different physical characteristics will differ in their individual emission characteristics. Similarly, aggregation of the UCNPs can result in broader emission. As a result, the parameters of the synthetic route used to access the UCNPs, which influences the physical characteristics of the particles, also has a strong influence on the optical properties. Accordingly, preferred compositions of UCNPs according to the disclosure will be highly uniform or monodisperse to ensure that the collection of UCNPs, and each UCNP within it, will emit radiation within a narrow band of wavelengths. Methods for achieving substantial monodispersity and low aggregation are disclosed herein. UCNPs can also be coated in a layer of silicon dioxide, which enables the UCNPs to be functionalized with organic compounds. In addition to their photophysical properties, this can impart secondary functionality to the UCNPs, illustrative examples including modifying the solubility of the particles, enabling the particles to target biomolecules, enabling the particles to bind or interact with specific targets, or enabling the particles to label or deliver an organic payload. UCNPs can be characterized and assessed for uniformity by a range of experimental techniques, which may include, but are not limited to, optical microscopy, electron microscopy, zeta potential, dynamic light scattering, dynamic image analysis, static image analysis, laser diffraction, inductively-coupled plasma-mass spectrometry (ICP-MS), or X-ray fluorescence. Definitions Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well known and commonly used in the art. The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g. “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 “Molecular Cell Biology, 4th ed.”, W. H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed.”, W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.”, Sinauer Associates, Inc., Sunderland, MA (2000). Chemistry terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985). All of the above, and any other publications, patents and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control. As used herein, the term “optically inert” refers to the property of lacking substantial interaction with electromagnetic radiation within a desired range of the electromagnetic spectrum. For example, a fluorescent probe with an emission spectrum in the ultraviolet range could be considered optically inert in the infrared range. In certain embodiments, the present disclosure provides materials that are optically inert in the infrared range. As used herein, the terms “upconverting” and “upconversion” refer to a photophysical process of anti-Stokes emission in which at least two lower-energy photons are absorbed by an object, thereby exciting the object, whereupon a single, higher-energy photon is emitted upon relaxation to the ground state. As used herein, the terms “average diameter” and “average thickness” refer to values obtained though transmission electron microscopy by imaging and measuring the area of aminimum of 700 nanoparticles and calculating the diameter (d = 2^(A / )). The mean value(“average diameter”) and standard deviation can then be calculated and used to determine the shell thickness. As used herein, the term “polydispersity index” refers to a quantification of the heterogeneity in average diameter of a plurality of upconverting nanoparticles, as calculated by the standard deviation divided by the average diameter. The standard deviation and average diameter can be obtained through any of the methods described above. As used herein, the term “Halotag” is art-recognized and refers to a self-labeling protein tag derived from Rhodococcus rhodochrous. The Halotag may be used to incorporate a detectable label (e.g., an upconverting nanoparticle, a fluorophore, or a radiolabel) in a biomolecule of interest, such as a protein, that is fused to the Halotag. A fusion protein comprising a protein of interest and a Halotag is capable of reacting with a parent molecule comprising a haloalkyl (e.g., a chloroalkyl) moiety (i.e., a Halotag ligand) to form a covalent FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 bond. One non-limiting example of such a haloalkyl moiety is represents the point of attachment to the parent molecule (e.g., the upconverting nanoparticle). As used herein, the term “SNAP tag” is art-recognized and refers to a self-labeling protein tag derived from human alanine-glyoxylate transaminase (AGT). The SNAP tag may be used to incorporate a detectable label (e.g., an upconverting nanoparticle, a fluorophore, or a radiolabel) in a biomolecule of interest, such as a protein, that is fused to the SNAP tag. A fusion protein comprising a protein of interest and a SNAP tag is capable of reacting with a parent molecule comprising an O6-benzylguanine derivative moiety (i.e., a SNAP tag ligand) to form a covalent bond. One non-limiting example of such an O6-benzylguanine derivativemoiety ; wherein represents the point of attachment to theparent molecule (e.g., the upconverting nanoparticle). As used herein, the term “CLIP tag” is art-recognized and refers to a self-labeling protein tag derived from human alanine-glyoxylate transaminase (AGT). The CLIP tag may be used to incorporate a detectable label (e.g., an upconverting nanoparticle, a fluorophore, or a radiolabel) in a biomolecule of interest, such as a protein, that is fused to the CLIP tag. A fusion protein comprising a protein of interest and a CLIP tag is capable of reacting with a parent molecule comprising an O2-benzylcytosine derivative moiety (i.e., a CLIP tag ligand) to form a covalent bond. One non-limiting example of such an O6-benzylcytosine derivative moiety is represents the point of attachment to the parent molecule (e.g., the upconverting nanoparticle). FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 EXAMPLES The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention. Exemplary Materials Yttrium (III) acetate hydrate (Y(CH3CO2)3·H2O), erbium(III) acetate hydrate (Er(CH3CO2)3·H2O), gadolinium(III) acetate hydrate (Gd(CH3CO2)3·H2O), ytterbium(III) acetate tetrahydrate (Yb(CH3CO2)3·6H2O), ammonium fluoride (NH4F), 90% 1-octadecene (ODE), 90% oleic acid (OA), and 70% oleylamine were purchased from Sigma-Aldrich. Sodium oleate (NaC18H33O2) was purchased from TCI America. All chemicals were used as received. Synthesis of Nanoparticles of the Disclosure The present application discloses UCNPs that are brighter and smaller than previously shown. In these representative embodiments, 8 nm cores are synthesized and the thickness of an inert shell is optimized. For example, by systematically tuning Er3+and Tm3+doping ratios, three bright UCNP probes of 10 nm diameter are achieved that selectively emit in the blue, green, or near-infrared regions for multiplexed imaging while using a single 976 nm laser excitation. Nanoparticle cores were synthesized to be 8 ± 0.5 nm in diameter. Inert shells were introduced via epitaxial growth as previously described. The optimized synthetic method was found to be highly robust and reproducible as shown by the transmission electron microscope (TEM) images (FIG. 1, FIG. 7 – FIG. 9). All UCNPs were confirmed to be in the pure hexagonal -phase via X-ray diffraction (FIG. 10), and their dopant concentrations were measured by inductively coupled plasma optical emission spectroscopy (Table 2, Table 3). To obtain the optimal probes for single-molecule imaging, both the thickness of the shell, as well as the doping percentage of the core of the CS UCNPs, are varied. Various strategies have been developed to increase the brightness of UCNPs such as optimizing the host matrix or the sensitizer and activator ions. Other methods include adding an inactive shell to reduce non-radiative quenching through vibrational modes of the solvent, utilizing organic dye to sensitize the upconversion process, and incorporating plasmonic resonance enhancement. The most effective strategy for brightness enhancement is to dope UCNPs with FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 high Yb3+to significantly increase the absorption cross section of 976 nm excitation, followed by an epitaxial growth of an optically inactive shell which reduces the resonant energy transfer among Yb3+ions to the surface defects and to vibrational modes of the surrounding solvent. Specifically, for Er3+-doped high-Yb3+UCNPs, it was found that ~8% Er3+doping results in the brightest UCNPs (i.e., NaYb0.92Er0.08F4). Therefore, beginning with this doping percentage, the dependance of the single-particle emission on shell thickness was investigated. Using a -NaYb0.92Er0.08F4core with a diameter of 8.0 ± 0.3 nm inert shells were epitaxially grown ranging in thickness from 0.45 to 4.20 nm. A schematic of the Core-Shell architecture is shown in FIG.1A. Representative TEM images of this series are shown in FIG. 1C – FIG.1J. FIG.1B shows the histograms for particle size for UCNPs with increasing shell thickness. For all the samples, the standard deviation over the mean is less than 5%, indicating a highly monodisperse UCNP population. To synthesize the NaLnF4(Ln = Yb3+, Er3+, Tm3+) cores for the CS architecture, 2 mmol of Ln acetate (Ln = Yb3+, Er3+, Tm3+), 18.25 mL of OA, and 20 mL of ODE were combined in a 250 mL three-neck round-bottom flask. The relative percentage of lanthanides were adjusted according to the desired doping percentage of Er3+or Tm3+. The contents were stirred under vacuum and then slowly heated to 110 °C for 1 h. The flask was then cooled and 6.25 mmol sodium oleate, 10 mmol NH4F, 6.25 mL oleylamine, and 8.75 mL ODE were added. Vacuum was pulled again for 1 hour, then the flask was cycled with nitrogen gas three times prior to heating rapidly to 310 °C for 50 min. The flask was then cooled rapidly, and the nanoparticles were washed twice with ethanol and acetone and finally resuspended in 50 mL of hexanes with 0.2% (v / v) oleic acid (100 μL). Shelling precursors were prepared as follows. In a 100 mL three-neck round-bottom flask, 2 mmol of Y acetate, 0.5 mmol Gd acetate, 10 mL of OA, and 15 mL of ODE were combined to form a Y / Gd solution. The flask was stirred and heated to 110 °C under vacuum, held at 110 °C for 15 min, and then heated to 160 °C under nitrogen gas. After all precursors were dissolved, a brown solution formed. The flask was cooled to 110 °C and vacuum was pulled once more, followed by cooling to room temperature. For the sodium trifluoroacetate solution, 6 mmol sodium trifluoroacetate was added to 15 mL of OA in a three-neck 50 mL round-bottom flask and stirred under vacuum at room temperature until dissolved. The shells of the CS architecture were made as follows. In a 50 mL round-bottom flask, 2.7 mL of stock NaLnF4 (Ln = Yb3+, Er3+, Tm3+) nanoparticles were combined with 4 mL OA and 6 mL ODE. The solution was stirred under vacuum and then heated to 70 °C. After 30 min, the flask was FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 filled with nitrogen gas and heated to 290 °C. Injections of precursors were then cycled, with each cycle beginning with injecting Y / Gd solution, waiting 15 min, and then ending with injecting sodium trifluoroacetate solution. For the CS structure, 4 injection cycles were completed to achieve a 2 nm shell (see Table 1). Volumes of the shelling precursors were adjusted to achieve other shell thicknesses. After the last injection, the flask was held at 290 °C for 30 min and then cooled rapidly to room temperature. The nanoparticles were washed in the same way as the NaLnF4(Ln = Yb3+, Er3+, Tm3+) core nanoparticles and were finally resuspended in 5 mL of hexanes. For the synthesis of the low Tm-doped UCNPs (below 3% Tm) the same procedure was followed with the exception of a limit on the Yb acetate hydrate to make the Yb3+percentage 30%. The remainder was substituted by Yttrium (III) acetate hydrate. To functionalize the CS nanoparticles, the reverse microemulsion method was used to grow a 4-nm shell of silicon dioxide on the 10-nm CS nanoparticles as follows.682.5 g IGEPAL CO-520 were added to a 20 mL scintillation vial along with 10 mL hexane and sonicated for10 minutes. The solution was then stirred for 15 minutes at room temperature. 100 μL of stockCS nanoparticles were added dropwise. After 15 minutes of stirring, 200 μL NH4OH were added dropwise. After an additional 15 minutes, 10 μL TEOS diluted in 200 μL hexane were added dropwise. The solution was stirred for 3 hours. The silica-coated CS nanoparticles were washed with ethanol, purified through centrifugation, and resuspended in 4 mL ethanol. Table 1: Shell Precursor Injections: Selection of Doping Concentrations Using the architecture of an 8 nm core and a 2 nm shell, -NaYb1-xErxF4@ -NaY0.8Gd0.2F4UCNPs with different doping concentrations (0.04 < x < 0.50) were synthesized to investigate their brightness (FIG. 7). FIG. 11C shows the spectra for the various doping concentrations. The green to red ratio increases as Er3+percentage increases. However, ensemble measurements are not reliable for absolute brightness comparison. FIG. 3A displays single- particle emission versus power-density for different doping concentrations ranging from 4% to FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 50% Er. In general, the brighter UCNPs remain brighter across the entire range of power densities used, except for 4% Er3+-doped UCNPs. FIG. 3B compares the single-particle emission of these Er3+-doped UCNPs at . For all measured power densities, 10% Er-doped CS UCNPs were found to be the brightest, with ~10 kpps at The optimal 10% Er3+ doping for the 12 nm NaYb1-xErxF4@ - NaY0.8Gd0.2F4 UCNPs differsfrom the optimal Er3+doping previously found for UCNPs of different sizes and architectures, highlighting the complex energy transfer between the lanthanide ions in UCNPs and necessitating the need to optimize UCNPs for various applications. To enable multicolor tracking, probes were developed in two other color channels, Blue and NIR. Tm3+-doped UCNPs were characterized as a source of two more emission colors. High percentages of Tm-doping (more than 5%) have been associated with emission at NIR wavelengths whereas low percentages of Tm-doping (less than 5%) have been associated with emission at blue wavelengths. In the search for a NIR-emitting probe, -NaYb1-xTmxF4@ -NaY0.8Gd0.2F4 UCNPs withdifferent doping concentrations (0.04 < x < 0.50) were synthesized. (TEM images in FIG.8). FIG.4A shows optical images of different Tm-doping percentages at an incident power density of 30 kW / cm2from the NIR and Blue channels. The NIR channel detects the 803 nm transition from3H4to3H6while the Blue channel detects the 452 nm transition from1D2to3F4and the 477 nm transition from1G4to3H6(Spectra in FIG.12D. As the Tm3+doping increases, while the NIR emission remains strong, the blue emission decreases significantly. FIG. 4C – FIG. 4D show the emission curves for these Tm3+-doped UCNPs. FIG. 4E compares the emission in both channels as well as their sum at . Different Tm3+doping percentages were screened for the UCNPs with the highest ratio of NIR to blue emission (FIG. 4E). 15% Tm3+-doped CS UCNPs were found to fulfill these criteria. Although brighter NIR emissions can be obtained with UCNPs with lower Tm3+- doping such as 6%, their strong emissions in the blue channel could confound multiplexed imaging. The NIR emission from the 15% Tm3+-doped UCNPs (~ 5000 pps) can still provide very high signal-to-noise ratio in single-molecule imaging. The slopes of the emission curves in the blue channel are close to 4, indicating a 4-photon process, while the slopes in the NIR channel are close to 2, showing that the NIR emission is mostly a 2-photon process. The saturation intensity increases as the Tm3+doping percentage increases. FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 To search for a blue-emitting probe, -NaY0.7-xYb0.3TmxF4@ -NaY0.8Gd0.2F4 UCNPs withdifferent doping concentrations (0.005 < x < 0.02) were synthesized. Increasing Yb3+concentration in Yb3+ / Tm3+co-doped UCNPs was found to enhance the NIR emission, therefore, unlike the previous UCNPs with high Yb3+content, the doping of Yb3+was kept low to optimize for the blue-emitting probe. A percentage of 30% Yb3+was selected, as used in the canonical Tm3+-doped UCNPs (FIG.9). FIG. 4B shows optical images of three low Tm3+-doped UCNPs at 30 kW / cm2in the NIR and Blue channels. FIG.4F – FIG.4G display the single-particle emission curves and Figure 4h compares the emission in both channels as well as their sum at 30 kW / cm2. The slope of the blue emission of the 2% Tm3+-doped UCNPs was larger than that of the 0.5% and 1% Tm3+-doped UCNPs (FIG.4F), indicating more concentration quenching at low power densities. Nevertheless, at power densities > , 2% Tm3+-doped UCNPs have stronger blue emission compared to the other two UCNPs. The slopes for the NIR emission for all three UCNPs are below 2, indicating that at the power densities explored, the upconversion has saturated. The data indicate that UCNP doped with 2% Tm3+- and 30% Yb3+is a strong blue-emitting probe. Although its emission is not as pure as that of the optimal NIR-emitting probe (NaYb0.85Tm0.15F4@ NaY0.8Gd0.2F4), at its ~3500 pps blue emission is larger than that of the NIR-probe (~1000 pps) and its ~2000 pps NIR emission is weaker than that of the NIR-probe (~5000 pps). Having established the optimal doping compositions for Blue, Green, and NIR-emitting UCNPs, the corresponding 10 nm UCNPs were synthesized, as their reduced size can further increase the number of biological applications. It was previously demonstrated that the diffusion behavior of sub-15 nm gold nanoparticles is similar to that of organic dyes, especially when cross-linking is minimized. As shown in FIG. 2I, reducing the shell thickness to 1 nm can still result in single-particle luminescence with exceptional signal-to-noise ratio. CS UCNPs were synthesized with 8 nm cores and 1 nm shell to obtain 10 nm overall diameter probes of three colors: NaY0.8Gd0.2F4, and NaYb0.85Tm0.15F4@ NaY0.8Gd0.2F4for Blue, Green and NIR, respectively. Their TEM images are shown in the insets of Figure 5a-c. FIG.5A – FIG.5C show the emission curves for the 10 nm probes of different colors and FIG. 15 shows their single-particle emission stability over 10 hours of continuous excitation. Surface quenching can significantly reduce emission intensity no longer follows FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 ideal volumetric scaling. Remarkably, near ideal volumetric scaling was retained when the brightness of the three-color probes was scaled from ~12 nm to ~10 nm (FIG.16). Compared to the previous sub-20 nm UCNPs, the 10 nm UCNPs doped with 10% Er3+are significantly brighter with ~5000 pps at . Transmission Electron Microscopy Transmission electron microscopy (TEM) images were taken on a FEI Tecnai (G2 Spirit TWIN) Electron Microscope operated at 120 kV. Samples were prepared by dropcasting approximately 4 L of a dilute solution of the sample in hexanes onto an ultrathin carbon type- B, 200 mesh, copper grid from Ted Pella, Inc. TEM was used to image the synthesized nanoparticles. A large number of transmission electron micrographs were taken to use for measuring the size of each sample. The size distribution of each core and core-shell sample was determined by measuring the area of a minimum of 700 nanoparticles and calculating the diameter. The mean value and standard deviation was then calculated and used to determine the shell thicknesses. FIG.7 – FIG.9 show representative TEM images for core and core-shell nanoparticles, as well as their size distributions, for samples with various dopant percentages. FIG.19 shows representative images of silica-coated nanoparticles and their sizes. Single-Particle Measurements The optimization of the UCNP brightness requires a comparison at the single-particle level because a precise determination of the UCNP concentration in solution is nontrivial and could be easily biased by particle aggregation and precipitation. To measure the optical properties of UCNPs at the single-particle level, nM dilutions of UCNP samples were dropcast onto poly-L-lysine-treated glass coverslips and imaged with a home-built widefield microscope. Samples were excited with a 976 nm diode laser, and emission was split into threechannels based on wavelength: Blue ( < 510 nm), Green (510 nm < < 705 nm) and NIR (705nm < < 850 nm). Single-particle images of 8% Er3+-doped UCNPs with increasing shell thickness at power density of are shown in FIG. 2A – FIG. 2E. To ensure the optical measurements are on single particles, the diffraction limited fluorescent spots in optical images are co-localized to the clearly resolved individual nanoparticles in scanning electron microscope (SEM) images (FIG.2F – FIG.2G, FIG.13). FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 Glass coverslips were treated by dropping 100 L of 1% (w / v) poly-L-lysine as a small droplet to the surface. The droplet was washed away with 1 ml of hexane after 1 minute. Samples were dropcast onto the treated coverslips, and single particle optical characterization was performed using a home-built widefield microscope with a Nikon 60X oil objective (NA 1.49) and a Thorlabs 976 nm fiber coupled laser (P.N. BL976-PAG900) able to provide power densities between 1 and 30 kW / cm2. Custom Python-based code was used to identify single point-spread functions for each particle and perform a 2D Gaussian fit to determine the particle emission in units of photons per second (pps). Only point-spread functions located in the central 128 x 128-pixel region of the camera were registered and analyzed for emission to account for the Gaussian fall off of the beam near the edges. For correlative SEM, nanoparticles were prepared in the same way as for optical measurements, with the addition of a V-shaped glass scratch made by a diamond scribe before drop casting the sample onto the treated coverslip. The sample was imaged by the widefield microscope and nanoparticle locations were registered in relation to the tip of the scratch. The sample was then sputtered with 2 nm of Au-Pd to prepare for SEM imaging. Nanoparticles were imaged using a Gemini 450 Zeiss Scanning Electron Microscope and inLens secondary electron detection, and their positions were correlated to optical images using relative positions to the tip of the scratch on the coverslip. Emission Spectra Emission spectra were obtained with a Thorlabs CCD spectrometer (P.N. CCS175) at a power density of 3.7 kW / cm2. Samples were taken from stock, placed in a 300 L cuvette and irradiated along a 1 cm path length by the 976 nm laser redirected from the widefield microscope path. The fiber leading to the CCD spectrometer observed the transverse direction to the excitation beam path and excitation light was blocked by a 950 nm shortpass filter. Cell Culture The Human Hela cells were purchased from ATCC (CCL-2). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies) containing 10% fetal bovine serum (FBS) (Gibco) and 1× penicillin / streptomycin (Life Technologies). Hela cells were plated on 96-well pretreated glass bottom wells and cultured in the humidified incubatorat 37 °C with 5% CO2.FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 Live-cell Multiplexed Single-Particle Tracking In summary, UCNPs with a diameter of 10 nm that show 3 distinct colors have been optimized for ultralong-term single-particle tracking in live cells. The single-particle optical properties of UCNPs doped with different Er3+and Tm3+concentrations were systematically characterized. For the 12 nm CS UCNPs, particles doped with 2%Tm3+ / 30% Yb3+, 10% Er3+ / 90%Yb3+, and 15% Tm3+ / 85% Yb3+represent the optimal probes for Blue, Green, and NIR emission, respectively. Further, all three selected UCNP compositions remain bright and easily distinguishable with thinner shell thicknesses of 1 nm. Multicolor, ultralong-term single-particle imaging of these three 10 nm probes in live cells is demonstrated by tracking single particles bound to membrane proteins diffusing over 10 minutes, excited by a single laser source. Cultured cells were imaged on 18-well slides (Ibidi 81816).10 μL of 1 μM of biotin- WGA (Sigma L5142) was added to each well having 40 μL of cell culture media for 10 minutes and then washed with 100 μL of cell culture media 3 times. Then, 10 μL of 1 μM of streptavidin was added to each well having 40 μL of cell culture media for 5 minutes followed by washing with 100 μL of cell culture media 5 times.1 nM of biotinylated UCNPs in 1x PBS were added and cells were incubated at 37oC with 5% CO2for 5 min to allow for binding to membrane proteins, followed by a wash with 50 μL of cell culture media. Living cells were imaged for 10 minutes at 300 ms time resolution at room temperature (22 °C) and a power density of 21 kW / cm2. The focal plane was set to the top of the cell membrane and kept there for the duration of the experiment. Custom Python and matlab-based code were used for tracking of single particles on the. Tracked particles were registered between different channels and then merged. X-Ray Diffraction Analysis Thin films of each sample were prepared for x-ray diffraction (XRD) by allowing 600uL of sample to dry and redissolving in 50uL of hexanes. This concentrated solution wasdrop cast onto a glass slide and allowed to dry. XRD data were collected using a Cu K 1 x-raysource ( = 1.54056 ˚A) on an X’Pert X-ray diffractometer (PANalytical B.V.) or SmartlabMultipurpose Diffractometer (Rigaku), as noted. Data was collected in Bragg-Brentanogeometry via a 2 - scan. Diffraction peaks were matched to references from the InternationalCentre for Diffraction Data (ICDD) and confirm hexagonal phase NaLnF4for all samples (FIG.10). FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 Chemical Composition The lanthanide content of each sample was measured using inductively-coupled plasma optical emission spectroscopy (ICP-OES). Approximately 5 mg of each sample was prepared assuming 50% of the mass was organics. Each sample was prepared at a concentration of 10 mg / mL by dissolving the sample in 3% aqueous nitric acid (made using ICP-grade nitric acid). ICP standards for each element were purchased from Agilent and used to create standard solutions for calibration. The absolute concentration of each lanthanide was measured (in ppm) and converted to relative molar percentage of lanthanide content (Table 2 and Table 3). Note that these values provide information for the overall composition of the nanoparticle, not the composition within a specific shell. Table 2: ICP results for the Er core and High Yb samples: FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 Table 4: ICP results for the core-shell samples: FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 Optical Measurements UCNP Emission Spectra The spectra for the different shell thickness UCNPs are shown in FIG.11B. Emission peaks were assigned to transitions between states in FIG.12. Green emission centered at 520 nm is attributed to the transitions from the2H11 / 2excited state to the4I15 / 2ground state, and emission centered at 541 nm from the4S3 / 24I15 / 2and4F9 / 24I13 / 2transitions. The red emission centered at 654 nm corresponds to the transition from the4F9 / 2excited state to the ground state, as well as from states reached higher-order photon processes,2G7 / 2,2K15 / 2, and4G9 / 2to the excited state4I9 / 2. The single-particle emission was measured as a function of shell thickness between incident power densities of 1 and 30 kW / cm2. The emission curves for different shell thicknesses are shown in FIG. 2H. Across the entire range of power densities, the single- particle brightness increases with shell thickness. At where the core-only UCNPs FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 emit only ~ 2 pps, UCNPs with 3.85 nm shell emit ~40 kpps, increasing by a factor of 20,000. A slope of 2 for the emission curve in the log-log plot indicates a two-photon process. Notably, the slope decreases from 1.8 for the core-only UCNPs to 0.6 for the UCNPs with 3.85 nm shell(Table 4), indicating that the intensity for the saturation of UCNP brightness ( ) shifts to lowerintensities with increasing shell thickness. This is consistent with previous reports that have shown that the energetically inert shell separates the excited lanthanide ions from the solvent, keeping the excitations trapped within the nanoparticle. For the most intense power density, the emission is shown as a function of shell thickness in FIG.2I. At , the single-particle brightness increases from ~80 pps forthe core-only UCNPs to ~ pps for the UCNPs with 3.85 nm shell. The brightnessdistribution as a function of shell thickness is shown in FIG. 14. Comparing the sigma-over- mu ratio of the brightness distribution to that of the size distribution from TEM reveals that there was only an increase of 5% in the variability. This shows homogeneous ion distribution of the dopant within particles of the same sample. Notably, the emission saturates with a shell thickness of about 2 nm, in agreement with previously reports. This shell thickness was selected to further investigate the effect of Er3+doping on emission. Emission spectra were obtained with a Thorlabs CCD spectrometer (P.N. CCS175) at a power density of 3.7 kW / cm2. Samples were taken from stock, placed in a 300 cuvette and irradiated along a 0.2 cm path length by the 976 nm laser redirected from the widefield microscope path. The fiber leading to the CCD spectrometer observed the transverse direction to the excitation beam path and excitation light was blocked by a 950 nm shortpass filter. FIG. 11 shows representative spectra for UCNPs synthesized in this work. Widefield Microscope Setup To demonstrate multicolor imaging, a mixture of these three types of UCNPs was first imaged on a coverslip (FIG. 5D – FIG.5F). The luminescence of the 10% Er3+UCNPs only appear in the Green channel (FIG. 5D). While the signals from the two Tm3+doped UCNPs are imaged in both the Blue (FIG. 5D) and NIR (FIG. 5F) channels, their distinct ratios of Blue to NIR emission make them easily distinguishable. FIG.5G – FIG.5H are the registered image with the three channels blended. The three colors are well separated and present distinct signals. Notably, multicolor single-particle imaging can be achieved with only one laser excitation as all three probes are excited by the same 976 nm laser. Details on the registration between channels are shown in FIG.17. The widefield microscope carried two diode lasers, a Thorlabs 976 nm fiber coupled laser (P.N. BL976-PAG900) and a Cobolt 638 nm free-space -30- FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 laser (P.N. 06-MLD). The 976 nm laser was reflected by a 950 nm dichroic into an upright Nikon 60X oil objective (NA 1.49) atop which the sample was mounted. Samples were able to be irradiated with power densities between 1 and 30 kW / cm2. The emission was captured by the objective and passed through the 950 nm dichroic into the emission pathway. The image was focused by a 400 mm tube lens, through a 950 nm shortpass filter. The emission passed through an iris and then into a dark box where it was successively split into three channels by two dichroics at 510 nm and at 750 nm. FIG. 11A shows the spectral bins overlaid on the spectra of an 8% Er-doped UCNP. The individual channel emissions were steered by independent mirrors towards another symmetric pair of dichroics which re-joined them. In each individual channel was a 255 mm collimating lens. Before hitting the camera, the emission was focused by a 300 mm lens for an overall image with each pixel corresponding to 100 nm. Each emission was steered to occupy one corner of the camera detector, and the iris was sized to ensure no bleed over between quadrants. The 638 nm laser was steered into a micromirror assembly below the backport of the objective which did not significantly occlude the emission image. It was angled such that it totally internally reflected on the sample and was collected by the objective. The outgoing beam was focused onto a quadrant photodetector and allowed for the correction of stage z-drift. This allowed for focus to be maintained at the same level during long imaging sessions. UCNP Emission Saturation Curve Measurement Procedures for measuring UCNP emission saturation curves have been described previously. Approximately 400 ng / mL UCNPs in hexane were drop cast onto a clean and dry 1.5 cover glass pre-coated with 1% (w / v) poly-L-lysine. Hexane was used to rinse off excess nanoparticles. For rigid support, the cover glass was attached to a standard microscope slide using double-sided tape after particles were left to dry for 5 minutes. Single particle optical characterization was performed using the home-built widefield microscope described. Custom Python-based code was used to identify single point-spread functions for each particle and perform a 2D gaussian fit to determine the particle emission in units of photons per second (pps). Only point-spread functions located in the center 128 pixel x 128 pixel region of the camera field-of-view were registered and analyzed to account for the gaussian falloff of the beam near the edges of the field-of-view. The slopes of the log-log saturation curves for Tm- doped UCNPs can be found in Table 5. FIG.15 shows the volume-normalized ( ) emission of all doping series as wellas the 10 nm probes, at the largest power density of . Figure 14 shows FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 representative histograms of particle brightness distribution at the largest power density of for Er-doped UCNPs of increasing shell thickness. Table S4: Pre-saturation slopes of UCNP saturation curves: FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 Correlative SEM and Widefield Fluorescence Imaging Nanoparticles were prepared in the same way as for optical measurements, with the addition of a V-shaped glass scratch made by a diamond scribe before dropcasting the sample onto the treated coverslip. The sample was imaged by the widefield microscope and nanoparticle locations were registered in relation to the tip of the scratch. The sample was then sputtered with an Au-Pd alloy to prepare for SEM imaging. Nanoparticles were imaged using a Zeiss Gemini 450 Scanning Electron Microscope and inLens secondary electron detection. Imaged particles were correlated to optical images using relative positions to the tip of the scratch on the coverslip. With a map between particles and optical point-spread-functions, a zoom-in was performed to verify the oligomeric state and the size of each individual nanoparticle. FIG. 13A shows an SEM field-of-view for an 8% Er-doped CS UCNP sample with 8 nm core and 3.84 nm shell. FIG. 13B shows an optical image with the same field-of- view of FIG.13A highlighted by the purple rectangle. FIG.13C – FIG.13F show SEM zoom- ins on the particles of FIG.13A. Image Registration Image registration between the three different channels was done with the 6% Tm- doped UCNP imaged at long exposure. Separate channels were coarse-aligned on the camera with independent steering mirrors. Fine registration was done by selecting and resolving point- spread functions present in all three channels to produce transformation matrices between channels (e.g. the blue probe’s signal in the NIR and Green Channels), which was then applied to every image and movie. Registration errors were found to be in the range between 5 nm and FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 20 nm. FIG. 16A shows the raw image from the camera, with the 3-channel split. FIG. 16B shows the three channels overlaid with colors assigned but misaligned. FIG. 16C shows the final aligned result. FIG.16D shows a 3D rendering of the un-registered image, and FIG.16E shows a 3D rendering of the registered image. For this figure, coarse-alignment of the channels through steering mirrors was not performed to highlight the effect of the registration. The coarse-alignment procedure produces an image much closer to the final registered image even before registration. Live-cell Imaging Experiments As a proof of concept, membrane proteins labeled with the three colors of probes on live HeLa cells are tracked for 10 minutes. Such nanoparticles may be used to study a variety of biological processes that benefit from photostable, small, and multiplexed probes such as transcription factors, receptor dimerization, protein condensates and more. All UCNPs were synthesized via an optimized thermal coprecipitation method varied according to appropriate doping concentrations. Nanoparticles consist of an optically active -NaYb1-xLnxF4core (Ln: Er, Tm), and an optically inert -NaY0.8Gd0.2F4shell. A diagram of the core-shell (CS) structure is shown in Figure 1a. To perform live-cell single-particle tracking using these three 10 nm probes, the UCNPs were functionalized with silica coating followed by PEGylation and biotinylation (FIG. 18). HeLa cells were incubated with biotinylated-wheat germ agglutinin (WGA) which nonspecifically targets glycosylated membrane proteins. The cells were then sequentially incubated with streptavidin and biotinylated UCNPs. UCNPs of all three colors were observed to label the membrane proteins at similar density and could be visually distinguished (FIG. 6A). The three probes in the focal plane corresponding to top of the cell membrane were imaged for 10 minutes at a power density of 21 kW / cm2. FIG.6A shows the last frame from a 10-minute video. We tracked the motion of these particles in the three separate channels and registered the trajectories on the merged image (FIG. 6B). Some proteins were more mobile while others were more static. Within the mobile proteins, various modes of diffusion were observed at room temperature (FIG. 19), with diffusion coefficients ranging from 10-5to 10-2μm2 / s. No correlation between probe color and diffusion coefficients was observed. FIG. 6C – FIG. 6D displays the interaction dynamics for one pair of membrane proteins labeled by green and NIR probes respectively. At t = 0 min, the Green- and NIR- FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 labeled membrane proteins were far apart from each other (> ) and began to move closer. At t = 2 min, as the Green-NIR pair got closer, a blue-labeled protein diffused rapidly by them. At t = 4 min and 30 s, the Green-labeled protein approached the NIR-labeled protein and they co-localized for about a minute (FIG.20). At t = 10 minutes, the Green-labeled protein moved away from the NIR-labeled protein and had since been diffusing on the top-left corner of the field-of-view. Time-colored trajectory of the green-labeled protein shown in FIG.6E exhibits the highly dynamic behavior of membrane proteins on live cells over the course of 10 minutes. Such complex single-molecule behavior over a long time would have been impossible to measure with traditional probes that photobleach within tens of seconds. All three types of UCNPs remain photostable without any signal decay after 10 minutes (FIG.15) and thus even longer single-molecule imaging over hours can be achieved. HeLa Cell Culture The Human Hela cells were purchased from ATCC (CCL-2). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies) containing 10% fetal bovine serum (FBS) (Gibco) and 1× penicillin / streptomycin (Life Technologies). HeLa cells were plated on 18-well IBIDI chambers (Ibidi 81816) and cultured in the humidified incubatorat 37 °C with 5% CO2.Silica-coating of Nanoparticles The reverse microemulsion method was used to grow a 4-nm shell of silicon dioxide on the 10-nm CS nanoparticles. 2.5 g IGEPAL CO-520 were added to a 20 mL scintillation vial along with 10 mL hexane and sonicated for 10 minutes. The solution was then stirred for 15 minutes at room temperature.100 μL of stock CS nanoparticles were added dropwise. After 15 minutes of stirring, 200 μL NH4OH were added dropwise. After an additional 15 minutes, 10 μL TEOS diluted in 200 μL hexane were added dropwise. The solution was stirred for 3 hours. After 3 hours, 10 mL of ethanol were added to the vial and the solution was poured into a 50 mL centrifuge tube.20 mL of ethanol were used to rinse the vial and then poured into the 50 mL centrifuge tube. Nanoparticles were then centrifuged at 25,000 g for 30 minutes. The supernatant was carefully discarded and the nanoparticles were dispersed in 4 mL ethanol. Surface Functionalization of Silica-coated UCNPs Biotinylation of silica coated UCNPs was carried out by silane chemistry. 20 mg of 2- [Methoxy-(polyethyleneoxy)propyl] trimethoxysilane (silane-mPEG, molar mass ~500 g / mol) and 7 mg of silane-PEG(3.4k)-biotin were separately dissolved in 0.45 mL of ethanol. They were added to 1 mL of silica-coated UCNPs in ethanol dropwise while stirring. Then, 100 μL FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 of water and 10 μL of ammonia (28%) were added to the mixture. The mixture was heated to 50oC and stirred overnight. The mixture was then cooled to room temperature. The biotinylated UCNPs were collected by passing the whole mixture through a 100 kDa membrane filter at 3000 g for 5 min with a centrifuge, followed by with 1x PBS buffer supplemented with 1 mM NaF. The final particles were stored in 1x PBS buffer supplemented with 1 mM NaF. Cell Labeling and Imaging To perform UCNP binding to live cells, 10 μL of 1 μM of biotin-WGA (Sigma L5142) was added to each well having 40 μL of cell culture media. After 10 min incubation, cells were washed with 100 μL of cell culture media for 3 times. Then, 10 μL of 1 μM of streptavidin was added to each well having 40 μL of cell culture media for 5 minutes. Excess streptavidin was removed by washing with 100 μL of cell culture media for 5 times. After washing, 1 nM of biotinylated UCNPs (diluted in 1x PBS) were added and cells were incubated at 37oC with 5% CO2for 5 min. Before imaging, free UCNPs were washed away with 50 μL of cell culture media. Living cells were imaged for 10 minutes at 300 ms time resolution at room temperature (22 °C) and a power density of . The focal plane was set to the top of the cell membrane and kept there for the duration of the experiment. Single-particle Tracking For single particle tracking, individual point-spread-functions were localized and their time-trajectories were resolved using custom python and Matlab scripts which performed 2D Gaussian fitting with multiple-target tracing (MTT) method. Self-labeling Protein Tag Experiments Nanoparticle Functionalization To functionalize the nanoparticles, 1 mL of as-prepared core-shell nanoparticles was dried under vacuum at room temperature followed by addition of 150 mg PMAO dissolved in 7 mL chloroform. The mixed solution was stirred at room temperature for 1.5 h and then chloroform was evaporated under vacuum at room temperature. The residue was sonicated and re-dispersed in 10 mL of 0.05M NaF solution containing 150 mg DMAP. The whole solution was centrifuged at 32,000 rcf for 2 hours and washed once with 0.05M NaF solution to remove excess PMAO and DMAP. The PMAO-coated nanoparticles were dispersed in 3 ml 1xPBS supplemented with 0.05M NaF. For ligand conjugation, 1.5 mL of PMAO-coated nanoparticles was mixed with 6 μL of 350 mM Methyl-PEG-Amine and 15 μL of 8mM X-PEG-Amine (X = Halotag ligand, SNAPtag ligand or CLIPtag ligand) under magnetic stirring. Then 30 mg FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 EDC dissolved in 100 μL water was added and the mixture was stirred for 2 h. To ensure higher conjugation level of PEG, 30 mg EDC and 6 μL of 350 mM Methyl-PEG-Amine was added again and reacted for another 2 h. The solution was centrifuged at 20,000 rcf for 2 h. The supernatant containing mono-disperse ligand-conjugated nanoparticles was concentrated and washed with centrifugal filter (100kDa). The Halotag ligand-PEG-Amine was obtained by a two-step reaction. First, 150 μL of 10 mM Halotag-NH2in anhydrous DMF was mixed with 15 μL of 100 mM SMCC in DMF and 1 μL Triethylamine. The mixture was reacted overnight at 37oC followed by addition of 10 μL of 150 mM SH-PEG-NH2in anhydrous DMF. The mixture was kept at room temperature for 10 h and stored at -20oC. The SNAPtag ligand-PEG-Amine was obtained by mixing equal amount of BG-maleimide and SH-PEG-NH2in anhydrous DMF. Cell Culture U2OS cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1× Penicillin / Streptomycin (Life Technologies). U2OS (HTB-96), HEK293T (CRL-3216), and NIH-3T3 (CRL-1658) cell lines were purchased from the American Type Culture Collection (ATCC). A431 cells were maintained in RPMI 1640 medium (Corning) supplemented with 10% FBS and 1× Penicillin / Streptomycin. A431 cells (CRL-1555) were also purchased from ATCC. All cells were cultured at 37 °C with 5% CO2. Cells were dissociated using 0.05% Trypsin-EDTA (Life Technologies) at a split ratio of 1:3 to 1:6 every 3-6 days. Mycoplasma contamination was routinely monitored to ensure cell culture quality. Plasmid Construction and Lentivirus Preparation Primers were synthesized by IDT. For transient expression plasmids, HaloTag, SNAP- tag, or CLIP-tag fragments, along with the targeted genes, were cloned into the pcDNA3.1 vector. For lentiviral plasmids, HaloTag and EGFR fragments were cloned into pSin vectors. All constructs were verified by Sanger sequencing. To prepare lentiviruses, plasmids were transfected into HEK293T cells using Lipo3000 (Life Technologies) in Opti-MEM medium (Life Technologies). After 6 hours, the medium was replaced with regular cell culture medium. Lentiviruses were harvested and filtered at 48 and 72 hours post-transfection. For lentiviral infection, U2OS cells at 60-70% confluency were incubated with the viruses for 4 hours, followed by selection with 1 μg / mL puromycin (Life Technologies) for 3-5 days. UCNP Labeling and Live-cell Imaging FH12722918.2 Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 Plasmids were transfected into U2OS cells using Lipo3000 (Life Technologies) in Opti- MEM medium (Life Technologies). After 6 hours, the medium was replaced with regular cell culture medium. To perform UCNP binding to live cells, cells were blocked in 1% casein (Sigma) in medium for 5 minutes. Subsequently, 1 nM of UCNPs (diluted in 1× PBS) wereadded, and cells were incubated at 37 °C with 5% CO for 5 minutes. Before imaging, unboundUCNPs were removed by washing with 1% casein medium. Results for the UCNP labeling and live-cell imaging experiments are depicted in FIG.21. INCORPORATION BY REFERENCE All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. EQUIVALENTS While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations. FH12722918.2

Claims

Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 We claim:

1. An upconverting nanoparticle, comprising: a) a core comprising one or more activator ions and one or more emitter ions; wherein, upon excitation with radiation, the one or more activator ions emit radiation of a wavelength capable of exciting the one or more emitter ions; and b) an optically inert shell enveloping the core.

2. The upconverting nanoparticle of claim 1, wherein the upconverting nanoparticle has a diameter of about 5-20 nm.

3. The upconverting nanoparticle of claim 2, wherein the upconverting nanoparticle has a diameter of about 10 nm.

4. The upconverting nanoparticle of any one of claims 1-3, wherein the one or more activator ions are lanthanide ions.

5. The upconverting nanoparticle of claim 4, wherein the one or more activator ions are selected from Lu3+, Yb3+, Ho3+, Pr3+, Nd3+, Eu3+, Tb3+, Dy3+, Ce3+, Sm3+, and La3+, or a combination thereof.

6. The upconverting nanoparticle of claim 4 or 5, wherein the one or more activator ions are Yb3+.

7. The upconverting nanoparticle of any one of claims 1-6, wherein the one or more emitter ions are lanthanide ions.

8. The upconverting nanoparticle of claim 7, wherein the one or more emitter ions are selected from Lu3+, Er3+, Tm3+, Ho3+, Pr3+, Nd3+, Eu3+, Tb3+, Dy3+, Ce3+, Sm3+, and La3+, or a combination thereof.

9. The upconverting nanoparticle of claim 7 or 8, wherein the one or more emitter ions are Er3+, Tm3+, or a combination thereof. FH12722918.2Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 10. The upconverting nanoparticle of any one of claims 1-9, wherein the core has an average diameter of about 5-10 nm.

11. The upconverting nanoparticle of claim 10, wherein the core has an average diameter of about 8 nm.

12. The upconverting nanoparticle of any one of claims 1-11, wherein the optically inert shell has an average thickness of about 0.5-8 nm.

13. The upconverting nanoparticle of claim 12, wherein the optically inert shell has an average thickness of about 1 nm.

14. The upconverting nanoparticle of any one of claims 1-13, wherein the optically inert shell comprises -NaY0.8Gd0.2F4.

15. The upconverting nanoparticle of any one of claims 1-14, wherein the core comprises -NaYb1-xLnxF4, wherein Ln is selected from Yb3+, Er3+, and Tm3+, and x is from 0 to 1.

16. The upconverting nanoparticle of any one of claims 1-15, wherein the core comprises NaY(1-x-y)Yb(y)Tm(x)F4, and (x+y) = 1.

17. The upconverting nanoparticle of any one of claims 1-15, wherein the core comprises NaY(1-x-y)Yb(y)Er(x)F4, and (x+y) = 1.

18. The upconverting nanoparticle of any one of claims 15-17, wherein x is from 0 to 1.

19. The upconverting nanoparticle of any one of claims 16-18, wherein y is from 0 to 1.

20. The upconverting nanoparticle of any one of claims 15-19, wherein x is from 0 to 0.5, and y is from 0.5 to 1.

21. The upconverting nanoparticle of any one of claims 16-20, wherein x is from 0 to 0.25, and y is from 0.75 to 1. FH12722918.2Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 22. The upconverting nanoparticle of claim 15, wherein the core comprises NaYb0.9Er0.1F4.

23. The upconverting nanoparticle of claim 15, wherein the core comprises NaYb0.85Tm0.15F4.

24. The upconverting nanoparticle of claim 16, wherein the core comprises NaY0.69Yb0.30Tm0.01F4.

25. The upconverting nanoparticle of any one of claims 1-24, wherein the core does not comprise a seed crystal that does not contain activator ions or emitter ions.

26. The upconverting nanoparticle of any one of claims 1-25, wherein the upconverting nanoparticle emits radiation of a second wavelength when excited with infrared radiation of a first wavelength.

27. The upconverting nanoparticle of claim 26, wherein the first wavelength ranges from about 900 nm to about 1400 nm.

28. The upconverting nanoparticle of claim 27, wherein the first wavelength is about 976 nm.

29. The upconverting nanoparticle of any one of claims 26-28, wherein the second wavelength ranges from about 350 nm to about 770 nm.

30. The upconverting nanoparticle of any one of claims 26-28, wherein the second wavelength ranges from about 780 nm to about 1500 nm.

31. The upconverting nanoparticle of any one of claims 1-30, wherein the upconverting nanoparticle continues to emit radiation at a substantially constant intensity after about 15 hours of exposure to radiation at a wavelength of 976 nm and a power density of about 5-30 kW / cm2. FH12722918.2Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 32. The upconverting nanoparticle of any one of claims 1-31, wherein the upconverting nanoparticle emits substantially continuous radiation during exposure to radiation at a wavelength of 976 nm and a power density of about 5-30 kW / cm2.

33. The upconverting nanoparticle of any one of claims 1-32, wherein the upconverting nanoparticle further comprises a reactive shell comprising SiO2enveloping the optically inert shell.

34. The upconverting nanoparticle of claim 33, wherein the reactive shell has a thickness of about 1-6 nm.

35. The upconverting nanoparticle of claim 34, wherein the reactive shell has a thickness of about 2 nm.

36. The upconverting nanoparticle of any one of claims 33-35, wherein the reactive shell is functionalized with an organic compound (e.g., a fluorophore, a peptide, a nucleic acid, an affinity tag, such as biotin, an enzyme, or a drug).

37. The upconverting nanoparticle of claim 36, wherein the organic compound is an affinity tag, such as biotin.

38. The upconverting nanoparticle of any one of claims 33-35, wherein the reactive shell is functionalized with a moiety capable of reacting with a self-labeling protein tag (e.g., a Halotag, a SNAP tag, or a CLIP tag).

39. The upconverting nanoparticle of claim 38, wherein the moiety capable of reacting with a self-labeling protein tag is selected fromFH12722918.2Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196represents the point of attachment to the reactive shell.

40. A composition comprising a plurality of upconverting nanoparticles of any one of claims 1-39, wherein the plurality of upconverting nanoparticles is substantially uniform in size, composition, shape, and / or molecular weight.

41. A composition comprising a plurality of upconverting nanoparticles of any one of claims 1-40, wherein the plurality of upconverting nanoparticles is substantially monodisperse.

42. A composition comprising a plurality of upconverting nanoparticles of any one of claims 1-41, wherein the polydispersity index of the plurality of upconverting nanoparticles is between about 1% and about 10%.

43. The composition of claim 42, wherein the polydispersity index of the plurality of upconverting nanoparticles is between about 3% and about 7%.

44. The composition of claim 42 or 43, wherein the polydispersity index of the plurality of upconverting nanoparticles is between 3.8% and 6.2%.

45. A method of performing single-particle tracking, wherein the particle to be tracked is conjugated to an upconverting nanoparticle of any one of claims 33-44.

46. The method of claim 45, wherein the particle to be tracked is a single cell.

47. The method of claim 45 or 46, wherein the particle to be tracked is a living cell.

48. A method of imaging a target protein, the method comprising: FH12722918.2Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 a) expressing a fusion of the target protein and a self-labeling protein tag in a cell; b) contacting the target protein with an upconverting nanoparticle of claim 38 or 39; c) exposing the upconverting nanoparticle to radiation at a wavelength of 976 nm and a power density of about 5-30 kW / cm2, thereby producing emitted radiation; and d) detecting the emitted radiation, thereby imaging the target protein.

49. The method of claim 48, wherein the self-labeling protein tag is selected from a Halotag, a SNAP tag, and a CLIP tag.

50. The method of claim 48, wherein the cell is a living cell.

51. A method of making an upconverting nanoparticle, comprising: a) contacting a salt of one or more activator ions, a salt of one or more emitter ions, and a solvent, thereby forming a reaction mixture; b) contacting the reaction mixture with a halide salt to form a core; and c) contacting the core with a solution comprising a halide source to form a shell around the core.

52. The method of claim 51, wherein the salt of the one or more activator ions is an acetate salt or a chloride salt.

53. The method of claim 51 or 52, wherein the salt of the one or more emitter ions is an acetate salt or a chloride salt.

54. The method of any one of claims 51-53, wherein the reaction mixture further comprises an alkene and an organic acid.

55. The method of claim 54, wherein the alkene is a terminal alkene.

56. The method of claim 55, wherein the terminal alkene is 1-octadecene.

57. The method of any one of claims 54-56, wherein the organic acid is a carboxylic acid. FH12722918.2Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 58. The method of claim 57, wherein the carboxylic acid is oleic acid.

59. The method of any one of claims 51-58, wherein the halide salt is a fluoride salt.

60. The method of claim 59, wherein the fluoride salt is ammonium fluoride.

61. The method of any one of claims 51-60, wherein the halide source is a trifluoroacetate salt (e.g., lithium trifluoroacetate, sodium trifluoroacetate, or potassium trifluoroacetate).

62. The method of claim 61, wherein the trifluoroacetate salt is sodium trifluoroacetate.

63. The method of any one of claims 51-62, wherein step a) further comprises heating the reaction mixture to a first temperature of about 70-140 °C.

64. The method of claim 63, wherein the first temperature is about 110 °C.

65. The method of claim 63 or 64, wherein step a) is performed over a first time of about 0.5-2.5 h.

66. The method of claim 65, wherein the first time is about 1 h.

67. The method of any one of claims 63-66, wherein step a) further comprises applying a vacuum to the reaction mixture after the reaction mixture is heated.

68. The method of any one of claims 51-67, wherein step b) further comprises contacting the reaction mixture with a first base.

69. The method of claim 68, wherein the first base is an amine, or a salt thereof.

70. The method of claim 69, wherein the amine is oleylamine, or a salt thereof.

71. The method of any one of claims 51-70, wherein step b) further comprises contacting the reaction mixture with a carboxylate salt. FH12722918.2Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 72. The method of claim 71, wherein the carboxylate salt is an oleate salt (e.g., lithium oleate, sodium oleate, or potassium oleate).

73. The method of claim 72, wherein the oleate salt is sodium oleate.

74. The method of any one of claims 67-73, wherein step b) further comprises heating the reaction mixture to a second temperature of about 280-340 °C after the vacuum is applied.

75. The method of claim 74, wherein the second temperature is about 310 °C.

76. The method of claim 74 or 75, wherein step b) is performed over a second time of about 0.5-2.5 h.

77. The method of claim 76, wherein the second time is about 0.8 h.

78. The method of any one of claims 74-77, wherein step c) is performed at a third temperature of about 50-90 °C.

79. The method of claim 78, wherein the third temperature is about 70 °C.

80. The method of claim 78 or 79, wherein step c) is performed over a third time of about 0.1-1.5 h.

81. The method of claim 80, wherein the third time is about 0.5 h.

82. The method of claim 80 or 81, wherein step c) is performed at a fourth temperature of about 250-340 °C after the third time is elapsed.

83. The method of claim 82, wherein the fourth temperature is about 290 °C.

84. The method of claim 82 or 83, wherein step c) is performed over a fourth time of about 0.5-3 h.

85. The method of claim 84, wherein the fourth time is about 1.5 h. FH12722918.2Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 86. The method of any one of claims 51-85, wherein the method further comprises epitaxially growing a reactive shell comprising SiO2on the optically inert shell.

87. The method of claim 86, wherein the method further comprises contacting the reactive shell with an activating agent and a base, thereby forming a functionalized reactive shell.

88. The method of claim 87, wherein the activating agent is poly(maleic anhydride-alt-1- octadecene) (PMAO) and the base is 4-(dimethylamino)pyridine (DMAP).

89. The method of claim 87 or 88, wherein the method further comprises contacting the functionalized reactive shell with a coupling reagent (e.g., an amide coupling reagent) and a reagent comprising a moiety capable of reacting with a self-labeling protein tag.

90. The method of claim 89, wherein the coupling reagent is 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC).

91. The method of claim 89, wherein the reagent comprising a moiety capable of reacting with a self-labeling protein tag is selected from methyl-poly(ethylene glycol)n-amine, Halotag ligand-poly(ethylene glycol)n-amine, SNAP tag ligand-poly(ethylene glycol)n-amine, and CLIP tag ligand-poly(ethylene glycol)n-amine; wherein: n is an integer from 1 to 200.

92. The method of claim 91, wherein the reagent comprising a moiety capable of reacting with a self-labeling protein tag is methyl-poly(ethylene glycol)n-amine.

93. The method of claim 91, wherein the reagent comprising a moiety capable of reacting with a self-labeling protein tag is Halotag ligand-poly(ethylene glycol)n-amine.

94. The method of claim 91, wherein the reagent comprising a moiety capable of reacting with a self-labeling protein tag is SNAP tag ligand-poly(ethylene glycol)n-amine. FH12722918.2Attorney Docket No.: BRH-05425 Broad Ref. No.: BI-11196 95. The method of claim 91, wherein the reagent comprising a moiety capable of reacting with a self-labeling protein tag is CLIP tag ligand-poly(ethylene glycol)n-amine.

96. An upconverting nanoparticle produced by the method of any one of claims 51-95. FH12722918.2

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