A Phosphorescent Iridium(III) Complex-Modified Nanoprobe for Hypoxia Bioimaging Via Time-Resolved Luminescence Microscopy

Abstract

Oxygen plays a crucial role in many biological processes. Accurate monitoring of oxygen level is important for diagnosis and treatment of diseases. Autofluorescence is an unavoidable interference in luminescent bioimaging, so that an amount of research work has been devoted to reducing background autofluorescence. Herein, a phosphorescent iridium(III) complex-modified nanoprobe is developed, which can monitor oxygen concentration and also reduce autofluorescence under both downconversion and upconversion channels. The nanoprobe is designed based on the mesoporous silica coated lanthanide-doped upconversion nanoparticles, which contains oxygen-sensitive iridium(III) complex in the outer silica shell. To image intracellular hypoxia without the interferences of autofluorescence, time-resolved luminescent imaging technology and near-infrared light excitation, both of which can reduce autofluorescence effectively, are adopted in this work. Moreover, gradient O₂ concentration can be detected clearly through confocal microscopy luminescence intensity imaging, phosphorescence lifetime imaging microscopy, and time-gated imaging, which is meaningful to oxygen sensing in tissues with nonuniform oxygen distribution.

Keywords: biosensors; energy transfer; oxygen nanoprobes; phosphorescence; time‐resolved luminescent imaging.

Figure 1

Schematic illustration of a) oxygen‐sensitive mechanism with downconversion channel and upconversion channel and b) synthesis of core–shell UCNPs@mSiO₂‐Ir.

Figure 2

TEM and HR‐TEM images of a,d) core UCNPs, b,e) core–shell UCNPs, and c,f) core–shell UCNPs@mSiO₂‐Ir.

Figure 3

a) X‐ray diffraction pattern of core–shell UCNPs (NaYF₄: 20 mol% Yb/0.2 mol% Tm@ NaYF₄) and the standard pattern of β‐NaYF₄ (JCPDS 00‐016‐0334). b) Dynamic light scattering measurement of core–shell UCNPs@mSiO₂‐Ir in ethanol. c) Zeta potentials of core–shell UCNPs@mSiO₂‐Ir and core–shell UCNPs@mSiO₂ in ethanol. d) Fourier transform infrared spectra of core–shell UCNPs, complex Ir, core–shell UCNPs@mSiO₂, and core–shell UCNPs@mSiO₂‐Ir. The measurements were conducted at 25 °C.

Figure 4

Normalized UV/visible absorption (black solid line) and phosphorescence spectra (red, orange, and green lines) of complex Ir under different oxygen concentrations in toluene and upconversion luminescence spectra of NaYF₄: 20 mol% Yb/0.2 mol% Tm@NaYF₄ core–shell UCNPs (blue solid line). The spectra were measured at 25 °C.

Figure 5

a) Upconversion luminescence spectra of core–shell UCNPs@mSiO₂‐Ir (1.0 mg UCNPs mL⁻¹) under different oxygen concentrations in ethanol and b) the corresponding Stem–Volmer plots (K _SV = 0.08%⁻¹) of the quenching by oxygen. Inset: a) Emission spectra ranging from 560 to 630 nm. λ _ex = 980 nm, P = 2.5 W. The spectra were measured at 25 °C.

Figure 6

a) Luminescence spectra of core–shell UCNPs@mSiO₂‐Ir (1.0 mg UCNPs mL⁻¹) under different oxygen concentrations in ethanol and b) the corresponding Stem–Volmer plots of the quenching by oxygen (black points stand for the values of I ₀/I and corresponding K _SV = 0.17%⁻¹, red points stand for the values of τ ₀/τ and corresponding K _SV = 0.18%⁻¹). λ _ex = 450 nm. Inset: a) the photos showing luminescence change under different oxygen concentrations when excited at 365 nm. The spectra were measured at 25 °C.

Figure 7

In vitro cell viability of Hela cells incubated with core–shell UCNPs@mSiO₂‐Ir at different concentrations at 37 °C for 48 h.

Figure 8

a,b,f,g) Confocal luminescent images, c,h) PLIM images, and d,e,i,j) TGLI images (delayed time = 200 or 500 ns) of living Hela cells incubated with core–shell UCNPs@mSiO₂‐Ir (200 μg mL⁻¹) at 37 °C for 2 h and then incubated at 37 °C and under 2.5% and 21% O₂ for another 1 h by 405 nm excitation. All the images share the same scale bar of 30 μm. Images were taken at 25 °C.

Figure 9

a) Schematic diagram of uniform and gradient O₂ distribution generated by placing the slide A) under and B) on top of the cells at 37 °C for 3 h after the cells were incubated with core–shell UCNPs@mSiO₂‐Ir (200 μg mL⁻¹) at 37 °C for 2 h, b,c) confocal, d) PLIM, and e) TGLI images of living Hela cells incubated with core–shell UCNPs@mSiO₂‐Ir (200 μg mL⁻¹) upon excitation at 405 nm underuniform O₂ distribution (21% O₂, 10% O₂, and 2.5% O₂) and gradient O₂ distribution. All the images share the same scale bar of 500 μm. Images were taken at 25 °C.