Dendritic upconverting nanoparticles enable in vivo multiphoton microscopy with low-power continuous wave sources

Abstract

We report a group of optical imaging probes, comprising upconverting lanthanide nanoparticles (UCNPs) and polyanionic dendrimers. Dendrimers with rigid cores and multiple carboxylate groups at the periphery are able to tightly bind to surfaces of UCNPs pretreated with NOBF(4), yielding stable, water-soluble, biocompatible nanomaterials. Unlike conventional linear polymers, dendrimers adhere to UCNPs by donating only a fraction of their peripheral groups to the UCNP-surface interactions. The remaining termini make up an interface between the nanoparticle and the aqueous phase, enhancing solubility and offering multiple possibilities for subsequent modification. Using optical probes as dendrimer cores makes it possible to couple the UCNPs signal to analyte-sensitive detection via UCNP-to-chromophore excitation energy transfer (EET). As an example, we demonstrate that UCNPs modified with porphyrin-dendrimers can operate as upconverting ratiometric pH nanosensors. Dendritic UCNPs possess excellent photostability, solubility, and biocompatibility, which make them directly suitable for in vivo imaging. Polyglutamic dendritic UCNPs injected in the blood of a mouse allowed mapping of the cortical vasculature down to 400 μm under the tissue surface, thus demonstrating feasibility of in vivo high-resolution two-photon microscopy with continuous wave (CW) excitation sources. Dendrimerization as a method of solubilization of UCNPs opens up numerous possibilities for use of these unique agents in biological imaging and sensing.

Fig. 1.

(A–C) Structures of cores P (eight anchor groups), C1 (eight anchor groups), and C2 (three anchor groups) used for construction of dendrimers. Dihedral angles show orientation of the planes containing anchor groups relative to the core. (D–F) Polyglutamic dendrimers P-Glu⁴ (128 carboxyls), C1-Glu³ (64 carboxyls), C1-Glu⁴ (128 carboxyls), and C2-Glu⁴ (48 carboxyls). (G) Polyglutamic dendrons Glu³ and Glu⁴. (H) Size distributions of UCNP/dendrimers in aqueous solutions by DLS: UCNP/C1-Glu³ (blue), UCNP/C1-Glu⁴ (cyan), UCNP/C2-Glu⁴ (red), UCNP/PAA (green), and UCNP/UCNP-BF₄⁻ in DMF (black). (I) Vials with aqueous colloidal solutions of UCNP/PAA (Left) and UCNP/C1-Glu⁴ (Right) containing equal amounts of the inorganic material (20 mg/mL). (J) Gel formed upon centrifugation of UCNP/P-Glu⁴. A hand-held laser pointer (980 nm) generates a green luminescent trace (near the bottom of the tube). (K) A laser beam (980 nm) passing through a solution of UCNP/C1-Glu⁴ is able to excite luminescence of UCNP/C2-Glu⁴ in the vial behind. (L) The order of vials is changed. The beam now is strongly scattered by UCNP/C2-Glu⁴, and no luminescence of UCNP/C1-Glu⁴ can be seen. (M) TEM image of UCNP/PAA. (N) TEM image of UCNP/C1-Glu⁴.

Fig. 2.

(A) Emission of UCNP-BF₄⁻ in DMF and UCNP/C1-Glu⁴ (in H₂O) induced by CW excitation at 980 nm. The samples contain equal amounts (by weight) of inorganic UCNPs. (B) Dependence of emission intensity at 540 and 650 nm for UCNP/C1-Glu⁴ on the incident power (log–log plot). A nonfocused beam (1.5 mm in diameter) was used in this experiment (see SI Appendix for details). (C–E) Mouse brain imaging. (C) Maximal intensity projection (MIP) image of a 200-μm-thick image stack, from the surface down, acquired using FITC–dextran and a mode-locked Ti:sapphire laser (100 fs; 80-MHz repetition rate; 800-nm excitation wavelength). (D) MIP image of the same stack obtained with UCNP/C1-Glu⁴ and the same laser operating in CW mode at 980 nm. (E) MIP images acquired at different depth. Stacks extend 20 μm down from the level marked above. (Scale bars: 100 μm.)

Fig. 3.

(A and B) Changes in the absorption spectra of porphyrin–dendrimer P-Glu⁴ with change in pH. (C) Q bands of free-base porphyrin H₂P (red) and porphyrin dication H₄P²⁺ (blue) overlap with emission bands of UCNP. (D) Cartoon illustrating pH sensing by UCNP/P-Glu⁴ via upconversion and EET. (E) pH-titration curve obtained by rationing integrated intensities of UCNP/P-Glu⁴ transitions at 520–540 nm and 660 nm (λ_ex = 980 nm). (F) Time-resolved emission traces of UCNP/P-Glu⁴ at 660 nm recorded upon pulsed excitation at 980 nm at three different pH levels. (G) Steady-state emission spectra for the same three samples.