Spatiotemporally mapping temperature dynamics of lysosomes and mitochondria using cascade organelle-targeting upconversion nanoparticles.

The intracellular metabolism of organelles, like lysosomes and mitochondria, is highly coordinated spatiotemporally and functionally. The activities of lysosomal enzymes significantly rely on the cytoplasmic temperature, and heat is constantly released by mitochondria as the byproduct of adenosine triphosphate (ATP) generation during active metabolism. Here, we developed temperature-sensitive LysoDots and MitoDots to monitor the in situ thermal dynamics of lysosomes and mitochondria. The design is based on upconversion nanoparticles (UCNPs) with high-density surface modifications to achieve the exceptionally high sensitivity of 2.7% K-1 and low uncertainty of 0.8 K for nanothermometry to be used in living cells. We show the measurement is independent of the ion concentrations and pH values. With Ca2+ ion shock, the temperatures of both lysosomes and mitochondria increased by ∼2 to 4 °C. Intriguingly, with chloroquine (CQ) treatment, the lysosomal temperature was observed to decrease by up to ∼3 °C, while mitochondria remained relatively stable. Lastly, with oxidative phosphorylation inhibitor treatment, we observed an ∼3 to 7 °C temperature increase and a thermal transition from mitochondria to lysosomes. These observations indicate different metabolic pathways and thermal transitions between lysosomes and mitochondria inside HeLa cells. The nanothermometry probes provide a powerful tool for multimodality functional imaging of subcellular organelles and interactions with high spatial, temporal, and thermal dynamics resolutions.

Metasurface-Controlled Photonic Rashba Effect for Upconversion Photoluminescence.

We demonstrate the effect of spin-momentum locking of upconversion photoluminescence emitted from rare-earth doped nanocrystals coupled to a phase-gradient dielectric metasurface. We observe different directionalities for left and right circular polarized light and associate this experimental observation with the photonic Rashba effect realized for upconverted photoluminescence that is manifested in the spin-dependent splitting of emitted light in the momentum space.

Precise Defect Engineering on Graphitic Carbon Nitrides for Boosted Solar H2 Production.

Defect engineering has been regarded as an "all-in-one strategy" to alleviate the insufficient solar utilization in g-C3 N4 . However, without appropriate modification, the defect benefits will be partly offset due to the formation of deep localized defect states and deteriorated surface states, lowering the photocarrier separation efficiency. To this end, the defective g-C3 N4 is designed with both S dopants and N vacancies via a dual-solvent-assisted synthetic approach. The precise defect control is realized by the addition of ethylene glycol (EG) into precursor formation and molten sulfur into the pyrolysis process, which simultaneously induced g-C3N4. with shallow defect states. These shallow defect energy levels can act as a temporary electron reservoir, which are critical to evoke the migrated electrons from CB with a moderate trapping ability, thus suppressing the bulky photocarrier recombination. Additionally, the optimized surface states of DCN-ES are also demonstrated by the highest electron-trapping resistance (Rtrapping ) of 9.56 × 103 Ω cm2 and the slowest decay kinetics of surface carriers (0.057 s-1 ), which guaranteed the smooth surface charge transfer rather than being the recombination sites. As a result, DCN-ES exhibited a superior H2 evolution rate of 4219.9 µmol g-1 h-1 , which is 29.1-fold higher than unmodified g-C3 N4 .

High Spatial and Temporal Resolution NIR-IIb Gastrointestinal Imaging in Mice.

Conventional biomedical imaging modalities, including endoscopy, X-rays, and magnetic resonance, are invasive and insufficient in spatial and temporal resolutions for gastrointestinal (GI) tract imaging to guide prognosis and therapy. Here we report a noninvasive method based on lanthanide-doped nanocrystals with ∼1530 nm fluorescence in the near-infrared-IIb window (NIR-IIb, 1500-1700 nm). The rational design of nanocrystals have led to an absolute quantum yield (QY) up to 48.6%. Further benefiting from the minimized scattering through the NIR-IIb window, we enhanced the spatial resolution to ∼1 mm in GI tract imaging, which is ∼3 times higher compared with the near-infrared-IIa (NIR-IIa, 1000-1500 nm) method. The approach also realized a high temporal resolution of 8 frames per second; thus the moment of mice intestinal peristalsis can be captured. Furthermore, with a light-sheet imaging system, we demonstrated a three-dimensional (3D) imaging on the GI tract. Moreover, we successfully translated these advances to diagnose inflammatory bowel disease.

Upconversion nanoparticle-assisted single-molecule assay for detecting circulating antigens of aggressive prostate cancer.

Sensitive and quantitative detection of molecular biomarkers is crucial for the early diagnosis of diseases like metabolic syndrome and cancer. Here we present a single-molecule sandwich immunoassay by imaging the number of single nanoparticles to diagnose aggressive prostate cancer. Our assay employed the photo-stable upconversion nanoparticles (UCNPs) as labels to detect the four types of circulating antigens in blood circulation, including glypican-1 (GPC-1), leptin, osteopontin (OPN), and vascular endothelial growth factor (VEGF), as their serum concentrations indicate aggressive prostate cancer. Under a wide-field microscope, a single UCNP doped with thousands of lanthanide ions can emit sufficiently bright anti-Stokes' luminescence to become quantitatively detectable. By counting every single streptavidin-functionalized UCNP which specifically labeled on each sandwich immune complex across multiple fields of views, we achieved the Limit of Detection (LOD) of 0.0123 ng/ml, 0.2711 ng/ml, 0.1238 ng/ml, and 0.0158 ng/ml for GPC-1, leptin, OPN and VEGF, respectively. The serum circulating level of GPC-1, leptin, OPN, and VEGF in a mixture of 10 healthy normal human serum was 25.17 ng/ml, 18.04 ng/ml, 11.34 ng/ml, and 1.55 ng/ml, which was within the assay dynamic detection range for each analyte. Moreover, a 20% increase of GPC-1 and OPN was observed by spiking the normal human serum with recombinant antigens to confirm the accuracy of the assay. We observed no cross-reactivity among the four biomarker analytes, which eliminates the false positives and enhances the detection accuracy. The developed single upconversion nanoparticle-assisted single-molecule assay suggests its potential in clinical usage for prostate cancer detection by monitoring tiny concentration differences in a panel of serum biomarkers.

Controlling the Biological Behaviors of Polymer-Coated Upconverting Nanoparticles by Adjusting the Linker Length of Estrone Ligands.

The estrone ligand is used for modifying nanoparticle surfaces to improve their targeting effect on cancer cell lines. However, to date, there is no common agreement on the ideal linker length to be used for the optimum targeting performance. In this study, we aimed to investigate the impact of poly(poly ethylene glycol methyl ether methacrylate) (PPEGMEMA) linker length on the cellular uptake behavior of polymer-coated upconverting nanoparticles (UCNPs). Different triblock terpolymers, poly(poly (ethylene glycol) methyl ether methacrylate)-block-polymethacrylic acid-block-polyethylene glycol methacrylate phosphate (PPEGMEMAx-b-PMAAy-b-PEGMP3: x = 7, 15, 33, and 80; y = 16, 20, 18, and 18), were synthesized with different polymer linker chain lengths between the surface and the targeting ligand by reversible addition-fragmentation chain transfer polymerization. The estrone ligand was attached to the polymer via specific terminal conjugation. The cellular association of polymer-coated UCNPs with linker chain lengths was evaluated in MCF-7 cells by flow cytometry. Our results showed that the bioactivity of ligand modification is dependent on the length of the polymer linker. The shortest polymer PPEGMEMA7-b-PMAA16-b-PEGMP3 with estrone at the end of the polymer chain was found to have the best cellular association behavior in the estrogen receptor (ER)α-positive expression cell line MCF-7. Additionally, the anticancer drug doxorubicin•HCl was encapsulated in the nanocarrier to evaluate the 2D and 3D cytotoxicity. The results showed that estrone modification could efficiently improve the cellular uptake in ERα-positive expression cell lines and in 3D spheroid models.

Preselectable Optical Fingerprints of Heterogeneous Upconversion Nanoparticles.

The control in optical uniformity of single nanoparticles and tuning their diversity in multiple dimensions, dot to dot, holds the key to unlocking nanoscale applications. Here we report that the entire lifetime profile of the single upconversion nanoparticle (τ2 profile) can be resolved by confocal, wide-field, and super-resolution microscopy techniques. The advances in both spatial and temporal resolutions push the limit of optical multiplexing from microscale to nanoscale. We further demonstrate that the time-domain optical fingerprints can be created by utilizing nanophotonic upconversion schemes, including interfacial energy migration, concentration dependency, energy transfer, and isolation of surface quenchers. We exemplify that three multiple dimensions, including the excitation wavelength, emission color, and τ2 profile, can be built into the nanoscale derivative τ2-dots. Creating a vast library of individually preselectable nanotags opens up a new horizon for diverse applications, spanning from sub-diffraction-limit data storage to high-throughput single-molecule digital assays and super-resolution imaging.

Upconversion Nonlinear Structured Illumination Microscopy.

Video-rate super-resolution imaging through biological tissue can visualize and track biomolecule interplays and transportations inside cellular organisms. Structured illumination microscopy allows for wide-field super resolution observation of biological samples but is limited by the strong extinction of light by biological tissues, which restricts the imaging depth and degrades its imaging resolution. Here we report a photon upconversion scheme using lanthanide-doped nanoparticles for wide-field super-resolution imaging through the biological transparent window, featured by near-infrared and low-irradiance nonlinear structured illumination. We demonstrate that the 976 nm excitation and 800 nm upconverted emission can mitigate the aberration. We found that the nonlinear response of upconversion emissions from single nanoparticles can effectively generate the required high spatial frequency components in the Fourier domain. These strategies lead to a new modality in microscopy with a resolution below 131 nm, 1/7th of the excitation wavelength, and an imaging rate of 1 Hz.

Quantitative Lateral Flow Strip Sensor Using Highly Doped Upconversion Nanoparticles.

Paper-based lateral flow assays, though being low-cost and widely used for rapid in vitro diagnostics, are indicative and do not provide sufficient sensitivity for the detection and quantification of low abundant biomarkers for early stage cancer diagnosis. Here, we design a compact device to create a focused illumination spot with high irradiance, which activates a range of highly doped 50 nm upconversion nanoparticles (UCNPs) to produce orders of magnitude brighter emissions. The device employs a very low-cost laser diode, simplified excitation, and collection optics and permits a mobile phone camera to record the results. Using highly erbium ion (Er3+)-doped and thulium ion (Tm3+)-doped UCNPs as two independent reporters on two-color lateral flow strips, new records of limit of detection (LOD), 89 and 400 pg/mL, have been achieved for the ultrasensitive detection of prostate specific antigen (PSA) and ephrin type-A receptor 2 (EphA2) biomarkers, respectively, without crosstalk. The technique and device presented in this work suggests a broad scope of low-cost, rapid, and quantitative lateral flow assays in early detection of bioanalytes.

Enhancement of near-infrared to near-infrared upconversion emission in the CeO2: Er³âº, Tm³âº, Yb³âº inverse opals.

In this Letter, CeO2: Er³âº, Tm³âº, Yb³âº inverse opal with near-infrared to near-infrared upconversion emission was prepared by polystyrene colloidal crystal templates, and the influence of photonic bandgap on the upconversion emission was investigated. Comparing with the reference sample, suppression of the blue or red upconversion luminescence was observed in the inverse opals. It is interesting that the near-infrared upconversion emission located at about 803 nm was enhanced due to the inhibition of visible upconversion emission in the inverse opals. Additionally, the variety of upconversion emission mechanisms was observed and discussed in the CeO2: Er³âº, Tm³âº, Yb³âº inverse opals.

[Preparation and up-conversion luminescence properties of Yb3+/Tm3+ co-doped Sb2O4 powder].

The Sb2O4:Yb3+, Tm3+ up-conversion luminescence powder with excellent physical, chemical stability and relative low phonon energy was synthesized by the high temperature solid-state reaction and its up-conversion luminescence property was investigated. Under the 980 nm excitation, infrared and blue up-conversion emissions centered at 800 and 480 nm were observed, which were assigned to the 1G4-->3H6 and 3H4-->3 He transitions of Tm2+, respectively. The influence of Yb3+ and Tm3+ concentration on the up-conversion emission property was also obtained. The up-conversion luminescence increases with increasing of Yb3+ and Tm3+ concentration. Additionally, the up-conversion luminescence mechanism was discussed based on the dependence of Tm3+ up-conversion luminescence on pump power. It is interesting that two photon excitation processes for blue and infrared emission were observed in the Sb2O04: Yb3+, Tm3+ powder under a 980 nm excitation. Based on the energy level diagram of Tma3 and Yb2+ ions, we think that two photons blue emission is contributed to the cooperation energy transfer between Tm"+ and Yb3+ ions. We believe that the Sbz04 : Yb3 , Tm2+ up-conversion luminescence powder will have potential application for new optical devices in up-conversion color displays, sensors, detection of infrared radiation, and lasers.

Small Extracellular Vesicle-Derived Circular RNA hsa_circ_0007386 as a Biomarker for the Diagnosis of Pleural Mesothelioma.

Pleural mesothelioma (PM) is a highly aggressive tumor that is caused by asbestos exposure and lacks effective therapeutic regimens. Current procedures for PM diagnosis are invasive and can take a long time to reach a definitive result. Small extracellular vesicles (sEVs) have been identified as important communicators between tumor cells and their microenvironment via their cargo including circular RNAs (circRNAs). CircRNAs are thermodynamically stable, highly conserved, and have been found to be dysregulated in cancer. This study aimed to identify potential biomarkers for PM diagnosis by investigating the expression of specific circRNA gene pattern (hsa_circ_0007386) in cells and sEVs using digital polymerase chain reaction (dPCR). For this reason, 5 PM, 14 non-PM, and one normal mesothelial cell line were cultured. The sEV was isolated from the cells using the gold standard ultracentrifuge method. The RNA was extracted from both cells and sEVs, cDNA was synthesized, and dPCR was run. Results showed that hsa_circ_0007386 was significantly overexpressed in PM cell lines and sEVs compared to non-PM and normal mesothelial cell lines (p < 0.0001). The upregulation of hsa_circ_0007386 in PM highlights its potential as a diagnostic biomarker. This study underscores the importance and potential of circRNAs and sEVs as cancer diagnostic tools.

Optical Nonlinearity Enabled Super-Resolved Multiplexing Microscopy.

Optical multiplexing for nanoscale object recognition is of great significance within the intricate domains of biology, medicine, anti-counterfeiting, and microscopic imaging. Traditionally, the multiplexing dimensions of nanoscopy are limited to emission intensity, color, lifetime, and polarization. Here, a novel dimension, optical nonlinearity, is proposed for super-resolved multiplexing microscopy. This optical nonlinearity is attributable to the energy transitions between multiple energy levels of the doped lanthanide ions in upconversion nanoparticles (UCNPs), resulting in unique optical fingerprints for UCNPs with different compositions. A vortex beam is applied to transport the optical nonlinearity onto the imaging point-spread function (PSF), creating a robust super-resolved multiplexing imaging strategy for differentiating UCNPs with distinctive optical nonlinearities. The composition information of the nanoparticles can be retrieved with variations of the corresponding PSF in the obtained image. Four channels multiplexing super-resolved imaging with a single scanning, applying emission color and nonlinearity of two orthogonal imaging dimensions with a spatial resolution higher than 150 nm (1/6.5λ), are demonstrated. This work provides a new and orthogonal dimension - optical nonlinearity - to existing multiplexing dimensions, which shows great potential in bioimaging, anti-counterfeiting, microarray assays, deep tissue multiplexing detection, and high-density data storage.

Investigation of upconversion and near infrared emission properties in CeO2: Er³âº, Yb³âº inverse opals.

The upconversion emission of rare earth ions can be modified in photonic crystals, however, the influence of upconversion emission modification of rare earths on near infrared emission has not been investigated yet in the photonic crystals. In the paper, CeO2: Er³âº, Yb³âº inverse opals with the photonic band gaps at 545, 680 and 450 nm were prepared by polystyrene colloidal crystal templates. The upconversion and the near infrared emission properties of Er³âº ions were systematically investigated in the CeO2: Er³âº, Yb³âº inverse opals. Comparing with the reference sample, significant suppression of both the green and red upconversion luminescence of Er³âº ions were observed in the inverse opals. It is interesting that the infrared emission located at 1,560 nm was enhanced due to inhibition of upconversion emission in the inverse opals. Additionally, mechanism of upconversion emission of the inverse opal was discussed. The photon avalanche upconversion process is observed.

Bone disease imaging through the near-infrared-II window.

Skeletal disorders are commonly diagnosed by X-ray imaging, but the radiation limits its use. Optical imaging through the near-infrared-II window (NIR-II, 1000-1700 nm) can penetrate deep tissues without radiation risk, but the targeting of contrast agent is non-specific. Here, we report that lanthanide-doped nanocrystals can passively target the bone marrow, which can be effective for over two months. We therefore develop the high-resolution NIR-II imaging method for bone disease diagnosis, including the 3D bone imaging instrumentation to show the intravital bone morphology. We demonstrate the monitoring of 1 mm bone defects with spatial resolution comparable to the X-ray imaging result. Moreover, NIR-II imaging can reveal the early onset inflammation as the synovitis in the early stage of rheumatoid arthritis, comparable to micro computed tomography (µCT) in diagnosis of osteoarthritis, including the symptoms of osteophyte and hyperostosis in the knee joint.

Heterochromatic Nonlinear Optical Responses in Upconversion Nanoparticles for Super-Resolution Nanoscopy.

Point spread function (PSF) engineering by an emitter's response can code higher-spatial-frequency information of an image for microscopy to achieve super-resolution. However, complexed excitation optics or repetitive scans are needed, which explains the issues of low speed, poor stability, and operational complexity associated with the current laser scanning microscopy approaches. Here, the diverse emission responses of upconversion nanoparticles (UCNPs) are reported for super-resolution nanoscopy to improve the imaging quality and speed. The method only needs a doughnut-shaped scanning excitation beam at an appropriate power density. By collecting the four-photon emission of single UCNPs, the high-frequency information of a super-resolution image can be resolved through the doughnut-emission PSF. Meanwhile, the two-photon state of the same nanoparticle is oversaturated, so that the complementary lower-frequency information of the super-resolution image can be simultaneously collected by the Gaussian-like emission PSF. This leads to a method of Fourier-domain heterochromatic fusion, which allows the extended capability of the engineered PSFs to cover both low- and high-frequency information to yield optimized image quality. This approach achieves a spatial resolution of 40 nm, 1/24th of the excitation wavelength. This work suggests a new scope for developing nonlinear multi-color emitting probes in super-resolution nanoscopy.

Optical Fingerprint Classification of Single Upconversion Nanoparticles by Deep Learning.

Highly controlled synthesis of upconversion nanoparticles (UCNPs) can be achieved in the heterogeneous design, so that a library of optical properties can be arbitrarily produced by depositing multiple lanthanide ions. Such a control offers the potential in creating nanoscale barcodes carrying high-capacity information. With the increasing creation of optical information, it poses more challenges in decoding them in an accurate, high-throughput, and speedy fashion. Here, we reported that the deep-learning approach can recognize the complexity of the optical fingerprints from different UCNPs. Under a wide-field microscope, the lifetime profiles of hundreds of single nanoparticles can be collected at once, which offers a sufficient amount of data to develop deep-learning algorithms. We demonstrated that high accuracies of over 90% can be achieved in classifying 14 kinds of UCNPs. This work suggests new opportunities in handling the diverse properties of nanoscale optical barcodes toward the establishment of vast luminescent information carriers.

Differential inflammatory and toxic effects in-vitro of wood smoke and traffic-related particulate matter from Sydney, Australia.

BACKGROUND: It is well known that PM2.5 generated by traffic or burning wood is pro-inflammatory and induces various adverse health outcomes in humans. In Sydney, New South Wales, Australia, the main anthropogenic contributors to particulate matter (PM) air pollution are wood combustion heaters, on-road vehicles, and coal-fired power stations. However, the relative toxicity of these local sources has not to date been investigated. METHOD: PM2.5 was collected on filters from the same sampling site in Liverpool, one suburb of Sydney. According to the positive matrix factorisation and collection season, filters were representative of either day with high traffic-related air pollution (TRAP), wood smoke, or both TRAP and woodsmoke (mixed air pollution). The elemental composition of the PM was assessed by accelerator-based ion beam analysis techniques (i.e. PIXE & PIGE) and size by Dynamic Light Scattering. Toxicity and inflammation were assessed in-vitro in human bronchial epithelial cells by measuring interleukin-6 (IL-6), interleukin-8 (IL-8) release, and MTT. RESULTS: Mixed air pollution (TRAP/wood smoke) PM had more nanometer (nm) sized PM than the other two groups. Using an in-vitro model of the lungs, the mixed air pollution PM was the most toxic, whereas the PM from woodsmoke induced greater IL-6 release than TRAP PM. There was no difference in the induction of IL-8 between the three sources of PM. CONCLUSION: Marked differences occur in the cellular response to PM from different sources, with differences in both toxicity and inflammation.

Multiplexed structured illumination super-resolution imaging with lifetime-engineered upconversion nanoparticles.

The emerging optical multiplexing within nanoscale shows super-capacity in encoding information by using lifetime fingerprints from luminescent nanoparticles. However, the optical diffraction limit compromises the decoding accuracy and throughput of the nanoparticles during conventional widefield imaging. This, in turn, challenges the quality of nanoparticles to afford the modulated excitation condition and further retain the multiplexed optical fingerprints for super-resolution multiplexing. Here we report a tailor-made multiplexed super-resolution imaging method using the lifetime-engineered upconversion nanoparticles. We demonstrate that the nanoparticles are bright, uniform, and stable under structured illumination, which supports a lateral resolution of 185 nm, less than 1/4th of the excitation wavelength. We further develop a deep learning algorithm to coordinate with super-resolution images for more accurate decoding compared to a numeric algorithm. We demonstrate a three-channel super-resolution imaging based optical multiplexing with decoding accuracies above 93% for each channel and larger than 60% accuracy for potential seven-channel multiplexing. The improved resolution provides high throughput by resolving the particles within the diffraction-limited spots, which enables higher multiplexing capacity in space. This lifetime multiplexing super-resolution method opens a new horizon for handling the growing amount of information content, disease source, and security risk in modern society.

Stratified Disk Microrobots with Dynamic Maneuverability and Proton-Activatable Luminescence for in Vivo Imaging.

Microrobots can expand our abilities to access remote, confined, and enclosed spaces. Their potential applications inside our body are obvious, e.g., to diagnose diseases, deliver medicine, and monitor treatment efficacy. However, critical requirements exist in relation to their operations in gastrointestinal environments, including resistance to strong gastric acid, responsivity to a narrow proton variation window, and locomotion in confined cavities with hierarchical terrains. Here, we report a proton-activatable microrobot to enable real-time, repeated, and site-selective pH sensing and monitoring in physiological relevant environments. This is achieved by stratifying a hydrogel disk to combine a range of functional nanomaterials, including proton-responsive molecular switches, upconversion nanoparticles, and near-infrared (NIR) emitters. By leveraging the 3D magnetic gradient fields and the anisotropic composition, the microrobot can be steered to locomote as a gyrating "Euler's disk", i.e., aslant relative to the surface and along its low-friction outer circumference, exhibiting a high motility of up to 60 body lengths/s. The enhanced magnetomotility can boost the pH-sensing kinetics by 2-fold. The fluorescence of the molecular switch can respond to pH variations with over 600-fold enhancement when the pH decreases from 8 to 1, and the integration of upconversion nanoparticles further allows both the efficient sensitization of NIR light through deep tissue and energy transfer to activate the pH probes. Moreover, the embedded down-shifting NIR emitters provide sufficient contrast for imaging of a single microrobot inside a live mouse. This work suggests great potential in developing multifunctional microrobots to perform generic site-selective tasks in vivo.