An antigen-strengthened dye-modified fully-human-nanobody-based immunoprobe for second near infrared bioimaging of metastatic tumors.

Conventional immunoprobes have absorption capabilities across the visible to near infrared (NIR-I, 650-900 nm) region. Recently, second near infrared (NIR-II, 1000-1700 nm) window have gained much attention due to their deeper penetration depth and improved visualization. Here, we describe the design and synthesis of a fully human nanobody-based fluorescent immunoprobe (ICGM-n501) for NIR-II bioimaging with strengthened fluorescent emission by antigen for the first time. By site-directed conjugation of an FDA-approved dye analogue, indocyanine green decorated with maleimide (ICGM), into a tumor-specific n501, ICGM-n501 provides real-time monitoring of abdominal transportation pathway of antibody-based bioagents with high resolution (0.21 mm), presents better accuracy and lower dosage (0.21 µmol kg-1) in bioimaging of peritoneal metastatic tumors than bioluminescence agent D-luciferin. In this work, ICGM-n501 demonstrates its potential in clinical surgery guidance, provide an expanded category of available NIR-II fluorophores and a template for next-generation immunoassay bioagents.

Er-Based Luminescent Nanothermometer to Explore the Real-Time Temperature of Cells under External Stimuli.

Temperature as a typical parameter, which influences the status of living creatures, is essential to life activities and indicates the initial cellular activities. In recent years, the rapid development of nanotechnology provides a new tool for studying temperature variation at the micro- or nano-scales. In this study, an important phenomenon is observed at the cell level using luminescent probes to explore intracellular temperature changes, based on Yb-Er doping nanoparticles with special upconversion readout mode and intensity ratio signals (I525 and I545 ). Further optimization of this four-layer core-shell ratio nanothermometer endows it with remarkable characteristics: super photostability, sensitivity, and protection owing to the shell. Thus this kind of thermal probe has the property of anti-interference to the complex chemical environment, responding exclusively to temperature, when it is used in liquid and cells to reflect external temperature changes at the nanoscale. The intracellular temperature of living RAW and CAOV3 cells are observed to have a resistance mechanism to external stimuli and approach a more favorable temperature, especially for CAOV3 cells with good heat resistance, with the intracellular temperature 4.8 °C higher than incubated medium under 5 °C environment, and 4.4 °C lower than the medium under 60 °C environment.

Measurement of Temperature Distribution at the Nanoscale with Luminescent Probes Based on Lanthanide Nanoparticles and Quantum Dots.

It is very challenging to probe the temperature in a nanoscale because of the lack of detection technique. Temperature-sensitive luminescent probes at a nanoscale provide the possibility to solve this problem. Herein, we fabricated a model, which combined two kinds of temperature sensitive nanoprobes and gold nanoparticle heater within mesoporous silica nanoparticles. Upconverting nanoparticles and quantum dots located at different positions inside 110 nm nanoparticles reported different temperatures when the gold nanoparticles generated heat by 532 nm laser irradiation. The temperature difference between two probes with an average distance of 55 nm can reach about 30 °C. Our results prove that the temperature distribution at a nanoscale can be measured, and it will be noteworthy if a nano-heater is applied.

Highly Doped Upconversion Nanoparticles for In Vivo Applications Under Mild Excitation Power.

One of the major challenges in using upconversion nanoparticles (UCNPs) is to improve their brightness. This is particularly true for in vivo studies, as the low power excitation is required to prevent the potential photo toxicity to live cells and tissues. Here, we report that the typical NaYF4:Yb0.2,Er0.02 nanoparticles can be highly doped, and the formula of NaYF4:Yb0.8,Er0.06 can gain orders of magnitude more brightness, which is applicable to a range of mild 980 nm excitation power densities, from 0.005 W/cm2 to 0.5 W/cm2. Our results reveal that the concentration of Yb3+ sensitizer ions plays an essential role, while increasing the doping concentration of Er3+ activator ions to 6 mol % only has incremental effect. We further demonstrated a type of bright UCNPs 12 nm in total diameter for in vivo tumor imaging at a power density as low as 0.0027 W/cm2, bringing down the excitation power requirement by 42 times. This work redefines the doping concentrations to fight for the issue of concentration quenching, so that ultrasmall and bright nanoparticles can be used to further improve the performance of upconversion nanotechnology in photodynamic therapy, light-triggered drug release, optogenetics, and night vision enhancement.

Quantitative Mapping of Liver Hypoxia in Living Mice Using Time-Resolved Wide-Field Phosphorescence Lifetime Imaging.

Hypoxia has been identified to contribute the pathogenesis of a wide range of liver diseases, and therefore, quantitative mapping of liver hypoxia is important for providing critical information in the diagnosis and treatment of hepatic diseases. However, the existing imaging methods are unsuitable to quantitatively assess liver hypoxia due to the need of liver-specific contrast agents and be easily affected by other imaging factors. Here, a time-resolved lifetime-based imaging method is established for quantitative mapping of the distribution of hypoxia in the livers of mice by combining a wide-field luminescence lifetime imaging system with an oxygen-sensitive nanoprobe. It is shown that the method is suitable for real-time quantification of the change of oxygen pressure in the process of hepatic ischemia-reperfusion of the mouse. Moreover, the developed lifetime imaging methodology is used to quantitatively map liver hypoxia regions in the mouse model of orthotopic liver tumor, where the average oxygen pressure in tumorous liver is far below the normal liver.

Yb-Based Nanoparticles with the Same Excitation and Emission Wavelength for Sensitive in Vivo Biodetection.

Near-infrared luminescent emission has been widely used as a signal for biological detection with its high spatial resolution and fast response. Rare-earthdoped nanoparticle-dye composites have diverse advantages of a wide operation wavelength and remarkable light stability, while the application is limited by the low luminescence quantum yield of rare-earth nanoparticles. Hence, in this work, we use a singly Yb doped nanoparticle that has strong luminescence emission at 975 nm under excitation at the same wavelength as an energy donor to construct the detection system. An inner filter pair, composed of core-shell nanophosphor NaYF4/20%Yb@NaYF4 (1:2) nanocrystals (csYb) as a luminescent beacon and ClO--responsive cyanine dye Cy890 as a filtering agent, was designed as a model. With a time-gated detection mode, the nanocomposites realize the detection limit at 0.55 ppb as demonstrated in a ClO- detection trial. The csYb&Cy890 nanocomposites can also monitor ClO- by luminescence signals in both living cells and mice models.

Luminescence Lifetime-Based In Vivo Detection with Responsive Rare Earth-Dye Nanocomposite.

For years, luminescence lifetime imaging has served as a quantitative tool in indicating intracellular components and activities. However, very few studies involve the in vivo study of animals, especially in vivo stimuli-responsive activities of animals, as both excitation and emission wavelengths should fall into the near-infrared (NIR) optical transparent window (660-950 and 1000-1500 nm). Herein, this work reports a lifetime-responsive nanocomposite with both excitation and emission in the NIR I window (800 nm) and lifetime in the microsecond region. The incorporation of Tm3+ -doped rare-earth nanocrystals and NIR dye builds an efficient energy transfer pathway that enables a tunable luminescence lifetime range. The NaYF4 :Tm nanocrystal, which absorbs and emits photons at the same energy level, is found to be 33 times brighter than optimized core-shell upconversion nanocrystals, and proved to be an effective donor for NIR luminescence resonance energy transfer (LRET). The anti-interference capability of luminescence lifetime signals is further confirmed by luminescence and lifetime imaging. In vivo studies also verify the lifetime response upon stimulation generated in an arthritis mouse model. This work introduces an intriguing tool for luminescence lifetime-based sensing in the microsecond region.

Time-Gated Ratiometric Detection with the Same Working Wavelength To Minimize the Interferences from Photon Attenuation for Accurate in Vivo Detection.

Luminescence imaging, exhibiting noninvasive, sensitive, rapid, and versatile properties, plays an important role in biomedical applications. It is usually unsuitable for direct biodetection, because the detected luminescence intensity can be influenced by various factors such as the luminescent substance concentration, the depth of the luminescent substance in the organism, etc. Ratiometric imaging may eliminate the interference due to the luminescent substance concentration on the working signal. However, the conventional ratiometric imaging mode has a limited capacity for in vivo signal acquisition and fidelity due to the highly variable and wavelength-dependent scattering and absorption process in biotissue. In this work, we demonstrate a general imaging mode in which two signals with the same working wavelength are used to perform ratiometric sensing ignoring the depth of the luminescent substance in the organism. Dual-channel decoding is achieved by time-gated imaging technology, in which the signals from lanthanide ions and fluorescent dyes are distinguished by their different luminescent lifetimes. The ratiometric signal is proven to be nonsensitive to the detection depth and excitation power densities; thus, we could utilize the working curve measured in vitro to determine the amount of target substance (hypochlorous acid) in vivo.

Eu2+/Eu3+-Based Smart Duplicate Responsive Stimuli and Time-gated Nanohybrid for Optical Recording and Encryption.

With the rapid development of information science, it is urgent that memory devices possessing high security, density, and desirable storage ability should be developed. In this work, a smart duplicate response of stimuli has been developed and a time-gate nanohybrid based on variable valence Eu2+/Eu3+ coencapsulated has been fabricated and acts as active material in the multilevel and multidimensional memory devices. The luminescence lifetime of Eu3+ in this nanohybrid gave a stimuli response due to which the energy level of the coordinated ligand could be modulated. Furthermore, by a simple sintering procedure, Eu3+ was partially in situ reduced to Eu2+ with a short lifetime in the system. And the in situ reduction ensured both Eu3+ and Eu2+ ions' uniform distribution in the nanohybrid and simultaneous response upon light excitation of variable valence Eu ions. Interestingly, Eu3+ revealed a prolonged lifetime because of the presence of an energy-transfer effect of Eu2+ → Eu3+. Such a nanohybrid had abundant luminescent properties, including the short lifetime of Eu2+, the energy transfer from the Eu2+ to Eu3+ ions, and the stimuli response of the Eu3+ lifetimes when exposed to acidic or basic vapor, thus giving birth to interesting recording and encryption performance in spatial-temporal dimensions. We believe that this research will point out a new direction for the future development of multilevel and multidimensional optical recording and encryption materials.