Bioimaging with Upconversion Nanoparticles

Abstract

Bioimaging enables the spatiotemporal visualization of biological processes at various scales empowered by a range of different imaging modalities and contrast agents. Upconversion nanoparticles (UCNPs) represent a distinct type of such contrast agents with the potential to transform bioimaging due to their unique optical properties and functional design flexibilities. This review explores and discusses the opportunities, challenges, and limitations that UCNPs exhibit as bioimaging probes and highlights applications with spatial dimensions ranging from the single nanoparticle level to cellular, tissue, and whole animal imaging. We further summarized recent advancements in bioimaging applications enabled by UCNPs, including super-resolution techniques and multimodal imaging methods, and provide a perspective on the future potential of UCNP-based technologies in bioimaging research and clinical translation. This review may provide a valuable resource for researchers interested in exploring and applying UCNP-based bioimaging technologies.

Keywords: Upconversion; bioimaging; in vivo imaging; luminescence; microscopy; multimodal; nanoparticles; super-resolution.

Figure 1:. Simplified schematic representation of photophysical properties of upconversion nanoparticles (UCNPs).

Colloidal dispersions of (a) β-NaYF4(Yb³⁺/Er³⁺) UCNPs and (b) β-NaYF4(Yb³⁺/Tm³⁺) UCNPs in cyclohexane with corresponding upconversion luminescence spectra upon 980 nm continuous wave (CW) laser excitation (10 W/cm²). Simplified energy level diagrams and energy transfer upconversion mechanisms for (c) Yb³⁺/Er³⁺-doped and (d) Yb³⁺/Tm³⁺-doped sensitizer/activator ion systems. The excitation light (980 nm) is absorbed by Yb³⁺ sensitizer ions and sequentially transferred to Er³⁺/Tm³⁺ activator ions. Arrows indicate radiative, nonradiative energy transfer, and multiphonon relaxation processes.^[5] Adapted with permission from Ref. 5. Copyright 2017 American Chemical Society.

Figure 2:. A depiction of different photon upconverting processes in comparison to single-photon absorption.

Asterisks (*) denote photon absorption, tildes (~) denote photon release, straight arrows denote a shift in energy state within an atom/ion and a curved arrow denotes a transfer of energy between ions. Sequential energy transfers are numbered in the order they occur. An incident photon exciting an electron from one energy to a higher, less stable energy level before emitting a red-shifted (longer wavelength) photon is termed single-photon absorption. Multi-photon absorption enables upconversion of the emitted photon through near-simultaneous absorption of two or more photons through an intermediate, virtual energy state. Unlike multi-photon absorption, second harmonic generation requires the emitted photon is double the frequency, or half the wavelength, of the incident photons.^[218] Finally, by using lanthanide ions in UCNPs, higher upconversion efficiencies can be achieved through multi-step absorptions with physical intermediate states, as opposed to the simultaneous absorption through a virtual intermediate state in multi-photon absorption.^[44] Within lanthanide ions, upconversion can occur through multiple processes, including sequential absorption in a single lanthanide ion (excited state absorption), an ion at an intermediate state transferrin its elevated energy to another intermediate state ion (energy-transfer), or excited state ions transferring their elevated energy to form two intermediate energy ions, which are then able, upon excitation to populate the intermediate state of two more ions, continuing the cycle until many high energy electrons fall back to the ground state, or a photon avalanche.^[46] As compared to second-harmonic generation and multi-photon absorption, upconversion using lanthanide ions may be preferrable as it eliminates the need for more intense and coherent excitation sources while still having the advantages of narrow emission peaks, upconverting photons, and extended emission lifetimes.^[45]

Figure 3:. Interactions between light and biological tissue.

(A) The tissue penetration of light is wavelength-dependent. Longer wavelengths typically tend to exhibit deeper penetration depths in biological tissue. The light tissue penetration data in this diagram is based on a report by Ash et al. and indicates the approximate depth at which 1% of incident light energy of a 10-mm-wide laser in a skin model still exists.^[52],[60] Note: The 808-nm and 980-nm lasers are commonly used excitation sources for upconversion nanoparticles. It should be noted the 808-nm and 980-nm laser depths are from a separate study and thus not directly comparable to the visible light penetration depths due to change in experimental conditions.^[52],[60] (B) This wavelength-dependent light penetration depth is a result of longer wavelengths typically exhibiting lower coefficients of absorbance (μ_a) and scattering (μ_s), i.e. lower extinction. Light absorbance occurs when light energy is transferred to the tissue upon irradiation. Light scattering occurs when light reflects off the tissue components, causing a reduction in the intensity of light continuing through the tissue. (C) The near-infrared (NIR)-I optical window of biological tissue is in the wavelength range of ~700–1,000 nm. Biological tissues exhibit a relatively low tissue attenuation within the NIR-I window, enabling improved light-based imaging through NIR-based lasers. Within this optical window, 808-nm lasers typically exhibit deeper tissue penetration than 980-nm lasers due to the locally elevated absorbance of water in the 950–1,050 nm range. On the other hand, wavelengths <700 nm are absorbed efficiently by tissue components, such as hemoglobin.^[219]

Figure 4:. Visualization of individual upconversion nanoparticles (UCNPs) through super-resolution techniques.

(A) Super-resolution stimulated emission depletion microscopy (STED) images of 40-nm UCNPs doped with NaYF₄ (20% Yb and 8% Tm). The authors reported a lateral resolution limit of 28 nm when using 13-nm UCNPs at an excitation power of 7.5 MWcm⁻² (scale bar = 500 nm).^[43] Reprinted by permission from Springer Nature: Nature, Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy, Liu et al. Copyright 2017. (B) Upconversion super-linear excitation-emission microscopy (uSEE) can resolve NaYF₄ (20%Yb/8%Tm) UCNPs separated by ~200 nm. The authors reported lateral and axial resolutions of 184 nm & 390 nm, respectively, using the uSEE technique (scale bar = 200 nm).^[131] Reprinted with permission from reference with permission. (C) Upconversion nonlinear structured illumination microscopy (U-NSIM) has been used to image NaYF₄ (20%Yb/4%Tm) UCNPs. The authors noted the ability to resolve induvial UCNPs separated by 161 nm (scale bar = 2 μm).^[50] Adapted with permission from Liu et al. Upconversion Nonlinear Structured Illumination Microscopy. Nano Lett. 20, 4775–4781 (2020). Copyright 2020 American Chemical Society. (D) Photon avalanche upconversion nanoparticles imaged at 828 kW/cm² (left) and 72 kW/cm² (right). At 76 kW/cm², Liang et al. achieved a full width half maximum of 62 nm for a single particle. The projection on the right is a 10-point moving average of the relative pixel intensity of the line drawn through the particle.^[94] Reprinted by permission from Springer Nature: Nature Nanotechnology, Migrating photon avalanche in different emitters at the nanoscale enables 46^th-order optical nonlinearity, Liang et al. Copyright 2022.

Figure 5:. Potential approaches for upconversion nanoparticles (UCNPs)-based multiplexing in cellular imaging.

(A) Wang et al. demonstrated upconversion luminescence lifetime engineering to control the UCNPs’ emission lifetimes through nanoparticle design (scale bars = 2 μm).^[25] The engineered UCNPs can be detected and identified by their characteristic upconversion luminescence lifetimes in selected timepoint images, potentially enabling UCNPs emission lifetime multiplexing for cellular imaging. Reproduced from reference with permission from the Royal Society of Chemistry. (B) Excitation power multiplexing of UCNPs exploits changes in lanthanide ion dopant concentrations of various UCNPs to alter the power thresholds necessary for UCNP detection. Combined with the super-linear relationship between excitation power and upconversion emission intensity, lower-doped UCNPs are visible at lower excitation powers and saturate at similar laser powers where the higher-doped UCNPs begin to be visible, enabling the distinction of the two UCNPs populations for potential applications in cellular imaging (scale bar = 1 μm).^[24] Reproduced from reference with permission from the Royal Society of Chemistry. (C) Vosch et al. demonstrated a frequency-encoding method to enable UCNP multiplex imaging (scale bar = 10 μm). Co-excitation with secondary lasers targeted specific energy transitions for enhanced absorption by either holmium or erbium.^[164] Adapted with permission from Lisberg, M. B., Lahtinen, S., Sloth, A. B., Soukka, T. & Vosch, T. Frequency Encoding of Upconversion Nanoparticle Emission for Multiplexed Imaging of Spectrally and Spatially Overlapping Lanthanide Ions. J. Am. Chem. Soc. 143, 19399–19405 (2021). Copyright 2021. American Chemical Society.

Figure 6:. Upconversion nanoparticles (UCNPs) enable multimodal bioimaging and in vivo imaging.

UCNPs can be designed for multimodal imaging, including but not limited to X-ray/computed tomography (CT) imaging, photoacoustic imaging (PAI), single-photon emission computed tomography (SPECT), and magnetic resonance imaging (MRI). The center circle shows the substantial increase in upconversion luminescence signal following local administration of UCNPs in mice.^[51] Reprinted with permission from Zhao, J., Chu, H., Zhao, Y., Lu, Y. & Li, L. A NIR Light Gated DNA Nanodevice for Spatiotemporally Controlled Imaging of MicroRNA in Cells and Animals. J. Am. Chem. Soc. 141, 7056–7062 (2019). Copyright 2019 American Chemical Society. T₁-MRI images of tumor-bearing mice following injection of PEGylated NaYF₄:Yb,Er@NaGdF₄ encapsulated in tumor-targeting antibody-conjugated micelles (Top Right).^[174] Reproduced with permission from reference . CT imaging following subcutaneous injection of BiF₃:Yb,Er UCNPs (Bottom Right).^[70] Reproduced with permission from reference . Photoacoustic in vivo images of gold nanorod dimer-UCNP-chlorin e6 injected via tail injection accumulating in mouse-grafted HeLa cell tumors (Bottom Left).^[220] Reproduced with permission from reference . SPECT imaging monitoring ¹⁵³Sm radiolabeled UCNP accumulation in specific organs following injection in mice (Top Left).^[221] Reproduced with permission from reference .