Two-Dimensional and Three-Dimensional Single Particle Tracking of Upconverting Nanoparticles in Living Cells.

Lanthanide-doped upconversion nanoparticles (UCNPs) are inorganic nanomaterials in which the lanthanide cations embedded in the host matrix can convert incident near-infrared light to visible or ultraviolet light. These particles are often used for long-term and real-time imaging because they are extremely stable even when subjected to continuous irradiation for a long time. It is now possible to image their movement at the single particle level with a scale of a few nanometers and track their trajectories as a function of time with a scale of a few microseconds. Such UCNP-based single-particle tracking (SPT) technology provides information about the intracellular structures and dynamics in living cells. Thus far, most imaging techniques have been built on fluorescence microscopic techniques (epifluorescence, total internal reflection, etc.). However, two-dimensional (2D) images obtained using these techniques are limited in only being able to visualize those on the focal planes of the objective lens. On the contrary, if three-dimensional (3D) structures and dynamics are known, deeper insights into the biology of the thick cells and tissues can be obtained. In this review, we introduce the status of the fluorescence imaging techniques, discuss the mathematical description of SPT, and outline the past few studies using UCNPs as imaging probes or biologically functionalized carriers.

Cellular uptake efficiency of nanoparticles investigated by three-dimensional imaging.

Understanding the interaction of nanoparticles with living cells on the basis of cellular uptake efficiency is a fundamental requisite in biomedical research. Cellular internalization of nanoparticles takes place by mechanisms like ATP hydrolysis-driven endocytosis that deliver nanoparticles to the cytoplasm, organelles and nuclei. Despite its importance in nanomedicine, this uptake procedure is not understood in-depth because of the complexity of the biochemical mechanisms and the lack of available experimental methods for quantitative analysis. The only breakthrough is likely to be the development of imaging techniques that can visualize, monitor and even count the number of nanoparticles inside the cell. To this end, we report here a new, fast and background-free three-dimensional (3-D) imaging technique with quantitative evaluation of the uptake efficiency for NaYF4:Yb3+,Er3+/NaYF4 core/shell upconversion nanoparticles (UCNPs) functionalized with different chemical and biological groups. Furthermore, the multiple 3-D trajectories of the UCNPs have been analyzed to investigate the cellular dynamics. This study reveals the nuclear uptake of UCNPs to be dependent on the specific chemical groups conjugated to the UCNPs. The developed 3-D imaging technique is of great significance for exploring complex biological systems.

Distinct mechanisms for the upconversion of NaYF4:Yb3+,Er3+ nanoparticles revealed by stimulated emission depletion.

Upconversion nanoparticles (UCNPs) have attracted enormous interest over the past few years because of their unique optical properties and potential for use in various applications such as bioimaging probes, biosensors, and light-harvesting materials for photovoltaics. The improvement of imaging resolution is one of the most important goals for UCNPs used in biological applications. Super-resolution imaging techniques that overcome the fundamental diffraction limit of light rely on the photochemistry of organic dyes or fluorescent proteins. Here we report our progress toward super-resolution microscopy with UCNPs. We found that the red emission (655 nm) of core/shell UCNPs with the structure NaYF4:Yb3+,Er3+/NaYF4 could be modulated by emission depletion (ED) of the intermediate state that interacts resonantly with an infrared beam (1540 nm). In contrast, the green emission bands (525 and 545 nm) of the UCNPs were less affected by irradiation with the infrared beam. The origin of such distinct behaviors between the green and red emissions was attributed to their different photophysical pathways.

Correction: Visualization of intercellular cargo transfer using upconverting nanoparticles.

Correction for 'Visualization of intercellular cargo transfer using upconverting nanoparticles' by Yeongchang Goh et al., Nanoscale, 2022, https://doi.org/10.1039/d2nr01999j.

Visualization of intercellular cargo transfer using upconverting nanoparticles.

Cell-cell communication is important for cellular differentiation, organ function, and immune responses. In intercellular communication, the extracellular vesicles (EVs) play a significant role in delivering the cargo molecules such as genes, proteins, and enzymes, to regulate and control the ability of the recipient cells. In this study, the observation of intercellular cargo transfer via dual-colour imaging using upconverting nanoparticles (UCNPs) has been demonstrated. Using this technique, the intercellular transport via contact-dependent and contact-independent signaling in live HeLa cells was clearly visualized with real-time, long-term single-vesicle tracking. Furthermore, it was demonstrated that the endocytosed UCNPs can be transmitted with the encapsulation of EVs labelled with fluorescent proteins.

Fast and background-free three-dimensional (3D) live-cell imaging with lanthanide-doped upconverting nanoparticles.

We report on the development of a three-dimensional (3D) live-cell imaging technique with high spatiotemporal resolution using lanthanide-doped upconverting nanoparticles (UCNPs). It employs the sectioning capability of confocal microscopy except that the two-dimensional (2D) section images are acquired by wide-field epi-fluorescence microscopy. Although epi-fluorescence images are contaminated with the out-of-focus background in general, the near-infrared (NIR) excitation used for the excitation of UCNPs does not generate any autofluorescence, which helps to lower the background. Moreover, the image blurring due to defocusing was naturally eliminated in the image reconstruction process. The 3D images were used to investigate the cellular dynamics such as nuclear uptake and single-particle tracking that require 3D description.