Qiuqiang Zhan

Using 915 nm Laser Excited Tm3+/Er3+/Ho3+-Doped NaYbF4 Upconversion Nanoparticles for in Vitro and Deeper in Vivo Bioimaging without Overheating Irradiation

Successful further development of superhigh-constrast upconversion (UC) bioimaging requires addressing the existing paradox: 980 nm laser light is used to excite upconversion nanoparticles (UCNPs), while 980 nm light has strong optical absorption of water and biological specimens. The overheating caused by 980 nm excitation laser light in UC bioimaging is computationally and experimentally investigated for the first time. A new promising excitation approach for better near-infrared to near-infrared (NIR-to-NIR) UC photoluminescence in vitro or in vivo imaging is proposed employing a cost-effective 915 nm laser. This novel laser excitation method provides drastically less heating of the biological specimen and larger imaging depth in the animals or tissues due to quite low water absorption. Experimentally obtained thermal-graphic maps of the mouse in response to the laser heating are investigated to demonstrate the less heating advantage of the 915 nm laser. Our tissue phantom experiments and simulations verified that the 915 nm laser is superior to the 980 nm laser for deep tissue imaging. A novel and facile strategy for surface functionalization is utilized to render UCNPs hydrophilic, stable, and cell targeting. These as-prepared UCNPs were characterized by TEM, emission spectroscopy, XRD, FTIR, and zeta potential. Specifically targeting UCNPs excited with a 915 nm laser have shown very high contrast UC bioimaging. Highly stable DSPE-mPEG-5000-encapsulated UCNPs were injected into mice to perform in vivo imaging. Imaging and spectroscopy analysis of UC photoluminescence demonstrated that a 915 nm laser can serve as a new promising excitation light for UC animal imaging.

Photosensitizer encapsulated organically modified silica nanoparticles for direct two-photon photodynamic therapy and In Vivo functional imaging

Nanoparticle-assisted two-photon imaging and near infrared (NIR) imaging are two important technologies in biophotonics research. In the present paper, organically modified silica (ORMOSIL) nanoparticles encapsulated with either PpIX (protoporphyrin IX) photosensitizers or IR-820 NIR fluorophores were synthesized and optically characterized. Using the former ORMOSIL nanoparticles, we showed: (i) direct excitation of the fluorescence of PpIX through its efficient two-photon absorption in the intracellular environment of tumor cells, and (ii) cytotoxicity towards tumor cells by PpIX under two-photon irradiation. The latter ORMOSIL nanoparticles can be used as efficient NIR fluorescent contrast agents for various types in vivo animal imaging. We applied IR-820 doped ORMOSIL nanoparticles in in vivo brain imaging of mice. We also demonstrated the applications of them to sentinel lymph node (SLN) mapping of mice. Finally, we showed that the nanoprobes could target the subcutaneously xenografted tumor of a mouse for long time observations. ORMOSIL nanoparticles have great potentials for disease diagnosis and clinical therapies.

A study of mesoporous silica-encapsulated gold nanorods as enhanced light scattering probes for cancer cell imaging

Mesoporous encapsulation of gold nanorods (GNRs) in a silica shell of controllable thickness (4.5-25.5 nm) was realized through a single-step coating method without any intermediary coating. The dependence of localized surface plasmon resonance (LSPR) extinction spectra of the coated GNRs on the thickness of the silica shell was investigated with both simulation and experiments, which agreed well with each other. It was found that cetyltrimethyl ammonium bromide (CTAB) molecules, which act as surfactants for the GNRs and dissociate in the solution, greatly affect the silica coating. Mesoporous silica-encapsulated GNRs were also shown to be highly biocompatible and stable in bio-environments. Based on LSPR enhanced scattering, mesoporous silica-encapsulated GNRs were utilized for dark field scattering imaging of cancer cells. Biomolecule-conjugated mesoporous silica-encapsulated GNRs were specifically taken up by cancer cells in vitro, justifying their use as effective optical probes for early cancer diagnosis. Mesoporous silica can also be modified with functional groups and conjugated with certain biomolecules for specific labeling on mammalian cells as well as carrying drugs or biomolecules into biological cells.

Optimization of Optical Excitation of Upconversion Nanoparticles for Rapid Microscopy and Deeper Tissue Imaging with Higher Quantum Yield

Relatively low quantum yield (QY), time-consuming scanning and strong absorption of light in tissue are some of the issues present in the development of upconversion nanoparticles (UCNPs) for biomedical applications. In this paper we systematically optimize several aspects of optical excitation of UCNPs to improve their applicability in bioimaging and biotherapy. A novel multi-photon evanescent wave (EW) excitation modality is proposed for UCNP-based microscopy. The scanning-free, ultrahigh contrast and high spatiotemporal resolution method could simultaneously track a few particles in a large area with a speed of up to 350 frames per second. The HeLa cancer cell membrane imaging was successfully performed using NaYF4: 20% Yb3+/2% Er3+ targeting nanoparticles. Studies with different tissues were made to illustrate the impact of optical property parameters on the deep imaging ability of 920-nm band excitation. In the experiments a semiconductor laser with a 920 nm wavelength was used to excite UCNPs in tissue phantom at five depths. Our experimental and computational results have shown that in UCNP-based diffusion optical imaging with 920-nm laser excitation could lead to larger imaging depth range compared to traditional 974-nm excitation in a wide dynamic range of tissue species. As the QY is power density dependent, a pulsed laser is proposed to improve the QY of UCNPs. This proposal is promising in drastically increasing the imaging depth and efficiency of photodynamic therapy.

Achieving high-efficiency emission depletion nanoscopy by employing cross relaxation in upconversion nanoparticles

Stimulated emission depletion microscopy provides a powerful sub-diffraction imaging modality for life science studies. Conventionally, stimulated emission depletion requires a relatively high light intensity to obtain an adequate depletion efficiency through only light–matter interaction. Here we show efficient emission depletion for a class of lanthanide-doped upconversion nanoparticles with the assistance of interionic cross relaxation, which significantly lowers the laser intensity requirements of optical depletion. We demonstrate two-color super-resolution imaging using upconversion nanoparticles (resolution ~ 66 nm) with a single pair of excitation/depletion beams. In addition, we show super-resolution imaging of immunostained cytoskeleton structures of fixed cells (resolution ~ 82 nm) using upconversion nanoparticles. These achievements provide a new perspective for the development of photoswitchable luminescent probes and will broaden the applications of lanthanide-doped nanoparticles for sub-diffraction microscopic imaging.Upconversion nanoparticles, which do not suffer from the photophysical artifacts that limit fluorescent molecules, offer an exciting opportunity for biological super-resolution imaging. Here, Zhan et al. develop an efficient STED mechanism using optimized lanthanide upconversion nanoparticles, enabling cytoskeleton nanoscopic imaging.

Using Some Nanoparticles as Contrast Agents for Optical Bioimaging

The introduction of nanometric contrast agents to optical imaging is helpful for the understanding of some biological processes at the molecular level, as well as the development of diagnostic tools and therapies. Optical imaging agents such as gold nanorods (GNRs), quantum dots (QDs), and organically modified silica (ORMOSIL) nanoparticles can overcome many drawbacks of conventional agents, such as poor contrast, photobleaching, and low chemical and optical stability in biological environment. These nanoparticles can also be developed for absorbance, emission, and scattering in the near-IR region, which allows optical approaches for deep-tissue real-time imaging. The synthesis methods and optical properties of GNRs, QDs, and ORMOSIL nanoparticles are briefly introduced, and some of their applications in optical bioimaging are demonstrated. Specific targeting, “green” synthesis methods, and optical signal demodulation are also introduced.

Deep, high contrast microscopic cell imaging using three-photon luminescence of β-(NaYF4:Er(3+)/NaYF4) nanoprobe excited by 1480-nm CW laser of only 1.5-mW.

It is challenging to achieve deep microscopic imaging for the strong scattering in biotissue. An efficient three-photon luminescence can effectively increase the penetration depth. Here we report that β-NaYF4: Er(3+)/NaYF4 UCNPs were excited by a 1480-nm CW-laser and emitted 543/653-nm light through a three-photon process. With the merit of the hexagonal crystal phase, sub-milliwatt laser power was utilized to excite the UCNP-probed cells to minimize the heating effect. The polymer-coated UCNPs were shown to be harmless to cells. The deep, high contrast in vitro microscopic imaging was implemented through an artificial phantom. Imaging depth of 800 μm was achieved using only 1.5 mW excitation and a 0.7 NA objective. The green/red emission intensities ratio after penetrating the phantom was studied, indicating that longer emission wavelength is preferred for deep multiphoton microscopy. The proposed and demonstrated β-UCNPs would have great potential in three-photon microscopy.

Optically investigating Nd 3+ -Yb 3+ cascade sensitized upconversion nanoparticles for high resolution, rapid scanning, deep and damage-free bio-imaging

The rapid development of upconversion nanoparticles (UCNPs) has been facing with a great challenge: intense emission, fast scanning, and deep imaging require high-power light irradiation with minimized heating effect (the intrinsic 975-nm excitation of Yb(3+)-sensitized UCNPs have overheating problem). By shifting the excitation peak from 975 nm to 795 nm, Nd(3+)-Yb(3+) cascade sensitized upconversion nanoparticles (Nd-UCNPs) with minimized heating effect were reported as the new generation UCNPs. For the first time, within two optically modeled applications in vitro and in vivo, the damage outcomes under long time high power laser excitation were solidly calculated, complementing the damage-free study of Nd-UCNPs. The higher resolution (20% improvement) and five times faster scanning microscopy were successfully performed using Nd-UCNPs under safety laser power level. The computational results showed the Nd(3+)-Yb(3+) energy transfer efficiency would not compromise the deep imaging ability, and the red (650-nm) emission is worth to be enhanced for deep tissue imaging.

Optical depletion mechanism of upconverting luminescence and its potential for multi-photon STED-like microscopy

Simulated emission depletion (STED) microscopy is very powerful, but still suffers from small tissue penetration depth, photobleaching of fluorescent probes and complicated imaging systems. Here, we propose an optical luminescence depletion mechanism employing upconverting nanoparticles (UCNPs) and explore its potential for multi-photon STED-like microscopy. With the addition of Yb³⁺ ions in NaYF₄:Er³⁺ UCNPs, the two-photon green emission of Er³⁺ under 795-nm excitation was successfully depleted by 1140-nm laser through the synergetic effect of the excited state absorption and the interionic energy transfer. This STED-like depletion mechanism was systematically investigated using steady-state rate equations, evidenced by the surprising emerging of 478-nm emission. The green emission depletion efficiency was about 30%, limited by the current laser source. Our work indicates that NaYF₄:Yb³⁺/Er³⁺ UCNPs will be potential probes for multi-photon super-resolution microscopy with many advantages, including long-wavelength-induced large penetration, non-photobleaching and non-photoblinking properties, cost-effective and simplified imaging systems.

Controlling the excitation of upconverting luminescence for biomedical theranostics: neodymium sensitizing

With the rapid development of nanotechnology over the past decade, lanthanide doped upconverting nanoparticles (UCNPs) have been successfully synthesized and have emerged as an important class of nanomaterials. In contrast to the traditional Yb3+-sensitized UCNPs, the emerging Nd3+-sensitized UCNPs have many merits in controlling the excitation of upconverting luminescence, including minimizing overheating effect, increasing the imaging depth and so on. In this review paper, we provide a comprehensive survey of the most recent advances in developing Nd3+-sensitized UCNPs, which include nanocomposition, mechanisms, and some typical nanostructures of Nd3+-sensitized UCNPs. Furthermore, an important emphasis is placed on various applications including downconversion and upconversion photoluminescence for bioimaging, high-resolution, and deep tissue imaging and tumor diagnosis and therapy. Potential challenges and prospective development are also discussed.