Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy.

Lanthanide-doped glasses and crystals are attractive for laser applications because the metastable energy levels of the trivalent lanthanide ions facilitate the establishment of population inversion and amplified stimulated emission at relatively low pump power. At the nanometre scale, lanthanide-doped upconversion nanoparticles (UCNPs) can now be made with precisely controlled phase, dimension and doping level. When excited in the near-infrared, these UCNPs emit stable, bright visible luminescence at a variety of selectable wavelengths, with single-nanoparticle sensitivity, which makes them suitable for advanced luminescence microscopy applications. Here we show that UCNPs doped with high concentrations of thulium ions (Tm3+), excited at a wavelength of 980 nanometres, can readily establish a population inversion on their intermediate metastable 3H4 level: the reduced inter-emitter distance at high Tm3+ doping concentration leads to intense cross-relaxation, inducing a photon-avalanche-like effect that rapidly populates the metastable 3H4 level, resulting in population inversion relative to the 3H6 ground level within a single nanoparticle. As a result, illumination by a laser at 808 nanometres, matching the upconversion band of the 3H4 → 3H6 transition, can trigger amplified stimulated emission to discharge the 3H4 intermediate level, so that the upconversion pathway to generate blue luminescence can be optically inhibited. We harness these properties to realize low-power super-resolution stimulated emission depletion (STED) microscopy and achieve nanometre-scale optical resolution (nanoscopy), imaging single UCNPs; the resolution is 28 nanometres, that is, 1/36th of the wavelength. These engineered nanocrystals offer saturation intensity two orders of magnitude lower than those of fluorescent probes currently employed in stimulated emission depletion microscopy, suggesting a new way of alleviating the square-root law that typically limits the resolution that can be practically achieved by such techniques.

Light-Emitting Diode Excitation for Upconversion Microscopy: A Quantitative Assessment.

Lanthanide-based upconversion nanoparticles (UCNPs) generally require high power laser excitation. Here, we report wide-field upconversion microscopy at single-nanoparticle sensitivity using incoherent excitation of a 970 nm light-emitting diode (LED). We show that due to its broad emission spectrum, LED excitation is about 3 times less effective for UCNPs and generates high background compared to laser illumination. To counter this, we use time-gated luminescence detection to eliminate the residual background from the LED source, so that individual UCNPs with high sensitizer (Yb3+) doping and inert shell protection become clearly identified under LED excitation at 1.18 W cm-2, as confirmed by correlated electron microscopy images. Hydrophilic UCNPs are obtained by polysaccharide coating via a facile ligand exchange protocol to demonstrate imaging of cellular uptake using LED excitation. These results suggest a viable approach to bypassing the limitations associated with high-power lasers when applying UCNPs and upconversion microscopy to life science research.

Simultaneous super-linear excitation-emission and emission depletion allows imaging of upconversion nanoparticles with higher sub-diffraction resolution.

Upconversion nanoparticles (UCNPs) are becoming increasingly popular as biological markers as they offer photo-stable imaging in the near-infrared (NIR) biological transparency window. Imaging at NIR wavelengths benefits from low auto-fluorescence background and minimal photo-damage. However, as the diffraction limit increases with the wavelength, the imaging resolution deteriorates. To address this limitation, recently two independent approaches have been proposed for imaging UCNPs with sub-diffraction resolution, namely stimulated emission-depletion (STED) microscopy and super linear excitation-emission (uSEE) microscopy. Both methods are very sensitive to the UCNP composition and the imaging conditions, i.e. to the excitation and depletion power. Here, we demonstrate that the imaging conditions can be chosen in a way that activates both super-resolution regimes simultaneously when imaging NaYF4:Yb,Tm UCNPs. The combined uSEE-STED mode benefits from the advantages of both techniques, allowing for imaging with lateral resolution about six times better than the diffraction limit due to STED and simultaneous improvement of the axial resolution about twice over the diffraction limit due to uSEE. Conveniently, at certain imaging conditions, the uSEE-STED modality can achieve better resolution at four times lower laser power compared to STED mode, making the method appealing for biological applications. We illustrate this by imaging UCNPs functionalized by colominic acid in fixed neuronal phenotype cells.

High-Contrast Visualization of Upconversion Luminescence in Mice Using Time-Gating Approach.

Optical imaging through the near-infrared (NIR) window provides deep penetration of light up to several centimeters into biological tissues. Capable of emitting 800 nm luminescence under 980 nm illumination, the recently developed upconversion nanoparticles (UCNPs) suggest a promising optical contrast agent for in vivo bioimaging. However, presently they require high-power lasers to excite when applied to small animals, leading to significant scattering background that limits the detection sensitivity as well as a detrimental thermal effect. In this work, we show that the time-gating approach implementing pulsed illumination from a NIR diode laser and time-delayed imaging synchronized via an optical chopper offers detection sensitivity more than 1 order of magnitude higher than the conventional approach using optical band-pass filters (S/N, 47321/6353 vs 5339/58), when imaging UCNPs injected into Kunming mice. The pulsed laser illumination (70 µs ON in 200 µs period) also reduces the overall thermal accumulation to 35% of that under the continuous-wave mode. Technical details are given on setting up the time-gating unit comprising an optical chopper, a pinhole, and a microscopy eyepiece. Being generally compatible with any camera, this provides a convenient and low cost solution to NIR animal imaging using UCNPs as well as other luminescent probes.

High-Precision Pinpointing of Luminescent Targets in Encoder-Assisted Scanning Microscopy Allowing High-Speed Quantitative Analysis.

Compared with routine microscopy imaging of a few analytes at a time, rapid scanning through the whole sample area of a microscope slide to locate every single target object offers many advantages in terms of simplicity, speed, throughput, and potential for robust quantitative analysis. Existing techniques that accommodate solid-phase samples incorporating individual micrometer-sized targets generally rely on digital microscopy and image analysis, with intrinsically low throughput and reliability. Here, we report an advanced on-the-fly stage scanning method to achieve high-precision target location across the whole slide. By integrating X- and Y-axis linear encoders to a motorized stage as the virtual "grids" that provide real-time positional references, we demonstrate an orthogonal scanning automated microscopy (OSAM) technique which can search a coverslip area of 50 × 24 mm(2) in just 5.3 min and locate individual 15 µm lanthanide luminescent microspheres with standard deviations of 1.38 and 1.75 µm in X and Y directions. Alongside implementation of an autofocus unit that compensates the tilt of a slide in the Z-axis in real time, we increase the luminescence detection efficiency by 35% with an improved coefficient of variation. We demonstrate the capability of advanced OSAM for robust quantification of luminescence intensities and lifetimes for a variety of micrometer-scale luminescent targets, specifically single down-shifting and upconversion microspheres, crystalline microplates, and color-barcoded microrods, as well as quantitative suspension array assays of biotinylated-DNA functionalized upconversion nanoparticles.

Astrocytic GPCR signaling in the anterior cingulate cortex modulates decision making in rats.

Decision making is a process of selecting a course of action by assessing the worth or value of the potential consequences. Rat Gambling Task (RGT) is a well-established behavioral paradigm that allows for assessment of the decision-making performance of rats. Astrocytes are emerging as key players in modulating cognitive functions. Using repeated RGTs with short intersession time intervals (48 h), the current study demonstrates that Gi pathway activation of astrocytes in the anterior cingulate cortex (ACC) leads to impaired decision-making in consistently good decision-making rats. On the other hand, ACC astrocytic Gq pathway activation improves decision-making in a subset of rats who are not consistently good decision-makers. Furthermore, we show that astrocytic Gq activation is associated with an increase in the L-lactate level in the extracellular fluid of the ACC. Together, these results expand our knowledge of the role of astrocytic GPCR signaling in modulating cognitive functions.

Astrocyte and L-lactate in the anterior cingulate cortex modulate schema memory and neuronal mitochondrial biogenesis.

Astrocyte-derived L-lactate was shown to confer beneficial effects on synaptic plasticity and cognitive functions. However, how astrocytic Gi signaling in the anterior cingulate cortex (ACC) modulates L-lactate levels and schema memory is not clear. Here, using chemogenetic approach and well-established behavioral paradigm, we demonstrate that astrocytic Gi pathway activation in the ACC causes significant impairments in flavor-place paired associates (PAs) learning, schema formation, and PA memory retrieval in rats. It also impairs new PA learning even if a prior associative schema exists. These impairments are mediated by decreased L-lactate in the ACC due to astrocytic Gi activation. Concurrent exogenous L-lactate administration bilaterally into the ACC rescues these impairments. Furthermore, we show that the impaired schema memory formation is associated with a decreased neuronal mitochondrial biogenesis caused by decreased L-lactate level in the ACC upon astrocytic Gi activation. Our study also reveals that L-lactate-mediated mitochondrial biogenesis is dependent on monocarboxylate transporter 2 (MCT2) and NMDA receptor activity - discovering a previously unrecognized signaling role of L-lactate. These findings expand our understanding of the role of astrocytes and L-lactate in the brain functions.

Assessing the activity of antibodies conjugated to upconversion nanoparticles for immunolabeling.

Surface modification and functionalization is typically required to engineer upconversion nanoparticles (UCNPs) for biosensing and bioimaging applications. Nevertheless, despite various antibody conjugation methods having been applied to UCNPs, no consensus has been reached on the best choice, as the results from individual studies are largely unable to be compared due to inadequate assessment of the properties of the conjugated products. Here, we introduce a systematic approach to quantitatively evaluate the biological activity of antibody-conjugated UCNPs. We determine that the optimal antibody conjugation efficiency to our colominic acid polysaccharide-coated UCNPs via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxy succinimide (EDC/NHS) coupling is approximately 70%, corresponding to 16 antibodies per nanoparticle of 63 nm hydrodynamic diameter, with on average 12 of the 16 antibodies maintaining their affinity to the target antigens. The binding ability of the antibody-conjugated UCNPs to the antigen was well preserved, as verified by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and cellular imaging. This is the first study to quantitate the active antibody binding capacity of polysaccharide coated UCNP nanoparticles, offering a practical guideline for benchmarking functionalised UCNPs in future studies.

Detection of rare prostate cancer cells in human urine offers prospect of non-invasive diagnosis.

Two molecular cytology approaches, (i) time-gated immunoluminescence assay (TGiA) and (ii) Raman-active immunolabeling assay (RiA), have been developed to detect prostate cancer (PCa) cells in urine from five prostate cancer patients. For TGiA, PCa cells stained by a biocompatible europium chelate antibody-conjugated probe were quantitated by automated time-gated microscopy (OSAM). For RiA, PCa cells labeled by antibody-conjugated Raman probe were detected by Raman spectrometer. TGiA and RiA were first optimized by the detection of PCa cultured cells (DU145) spiked into control urine, with TGiA-OSAM showing single-cell PCa detection sensitivity, while RiA had a limit of detection of 4-10 cells/mL. Blinded analysis of each patient urine sample, using MIL-38 antibody specific for PCa cells, was performed using both assays in parallel with control urine. Both assays detected very low abundance PCa cells in patient urine (3-20 PCa cells per mL by TGiA, 4-13 cells/mL by RiA). The normalized mean of the detected PCa cells per 1 ml of urine was plotted against the clinical data including prostate specific antigen (PSA) level and Clinical Risk Assessment for each patient. Both cell detection assays showed correlation with PSA in the high risk patients but aligned with the Clinical Assessment rather than with PSA levels of the low/intermediate risk patients. Despite the limited available urine samples of PCa patients, the data presented in this proof-of-principle work is promising for the development of highly sensitive diagnostic urine tests for PCa.

Near-Infrared-Triggered Nitrogen Fixation over Upconversion Nanoparticles Assembled Carbon Nitride Nanotubes with Nitrogen Vacancies.

Photocatalytic artificial fixation of N2 to NH3 occurs over NaYF4:Yb,Tm (NYF) upconversion nanoparticles (NPs) decorated carbon nitride nanotubes with nitrogen vacancies (NYF/NV-CNNTs) in water under near-infrared (NIR) light irradiation. NYF NPs with a particle size of ca. 20 nm were uniformly distributed on the surface of NV-CNNTs. The NYF/NV-CNNTs with 15 wt % NYF exhibited the highest NH3 production yield of 1.72 mmol L-1 gcat-1, corresponding to an apparent quantum efficiency of 0.50% under NIR light illumination, and about three times higher the activity of the bare CNNTs under UV-filtered solar light. 15N isotope-labeling NMR results confirm that the N source of ammonia originates from the photochemical N2 reduction. The spectroelectrochemical measurements reveal that NVs can greatly facilitate the photogenerated electron transfer without energy loss, while the presence of NYF NPs shifts both the deep trap state and the edge of conduction band toward a lower potential. Moreover, NYF NPs endow the photocatalyst with a NIR light absorption via the fluorescence resonance energy transfer process, and NVs have the ability to enhance the active sites for a stronger adsorption of N2 and decrease the surface quenching effect of NYF NPs, which thus can promote the energy migration within the heterojunctions. This work opens the way toward full solar spectrum photocatalysis for sustainable ammonia synthesis under aqueous system.

Lifetime-Engineered Ruby Nanoparticles (Tau-Rubies) for Multiplexed Imaging of µ-Opioid Receptors.

To address the growing demand for simultaneous imaging of multiple biomarkers in highly scattering media such as organotypic cell cultures, we introduce a new type of photoluminescent nanomaterial termed "tau-ruby" composed of ruby nanocrystals (Al2O3:Cr3+) with tunable emission lifetime. The lifetime tuning range from 2.4 to 3.2 ms was achieved by varying the Cr3+ dopant concentration from 0.8% to 0.2%, affording facile implementation of background-free detection. We developed inexpensive scalable production of tau-ruby characterized by bright emission, narrow spectrum (693 ± 2 nm), and virtually unlimited photostability upon excitation with affordable excitation/detection sources, noncytotoxic and insensitive to microenvironmental fluctuations. By functionalizing the surface of tau-rubies with targeting antibodies, we obtained different biomarkers suitable for multiplexed lifetime imaging. As a proof of principle, three tau-ruby bioprobes, characterized by three mean lifetimes, were deployed to label three µ-opioid receptor species expressed on transfected cancer cells, each fused to a unique epitope, so that three types of cells were lifetime-encoded. Robust decoding of photoluminescent signals that report on each cell type was achieved by using a home-built lifetime imaging system and resulted in high-contrast multiplexed lifetime imaging of the cells.

Controlling the non-linear emission of upconversion nanoparticles to enhance super-resolution imaging performance.

Upconversion nanoparticles (UCNPs) exhibit unique optical properties such as photo-emission stability, large anti-Stokes shift, and long excited-state lifetimes, allowing significant advances in a broad range of applications from biomedical sensing to super-resolution microscopy. In recent years, progress on nanoparticle synthesis led to the development of many strategies for enhancing their upconversion luminescence, focused in particular on heavy doping of lanthanide ions and core-shell structures. In this article, we investigate the non-linear emission properties of fully Yb-based core-shell UCNPs and their impact on the super-resolution performance of stimulated excitation-depletion (STED) microscopy and super-linear excitation-emission (uSEE) microscopy. Controlling the power-dependent emission curve enables us to relax constraints on the doping concentrations and to reduce the excitation power required for accessing sub-diffraction regimes. We take advantage of this feature to implement multiplexed super-resolution imaging of a two-sample mixture.

Time-resolved microfluidic flow cytometer for decoding luminescence lifetimes in the microsecond region.

Time-resolved luminescence detection using long-lived probes with lifetimes in the microsecond region have shown great potential in ultrasensitive and multiplexed bioanalysis. In flow cytometry, however, the long lifetime poses a significant challenge to measure wherein the detection window is often too short to determine the decay characteristics. Here we report a time-resolved microfluidic flow cytometer (tr-mFCM) incorporating an acoustic-focusing chip, which allows slowing down of the flow while providing the same detection conditions for every target, achieving accurate lifetime measurement free of autofluorescence interference. Through configuration of the flow velocity and detection aperture with respect to the time-gating sequence, a multi-cycle luminescence decay profile is captured for every event under maximum excitation and detection efficiency. A custom fitting algorithm is then developed to resolve europium-stained polymer microspheres as well as leukemia cells against abundant fluorescent particles, achieving counting efficiency approaching 100% and lifetime CVs (coefficient of variation) around 2-6%. We further demonstrate lifetime-multiplexed detection of prostate and bladder cancer cells stained with different europium probes. Our acoustic-focusing tr-mFCM offers a practical technique for rapid screening of biofluidic samples containing multiple cell types, especially in resource-limited environments such as regional and/or underdeveloped areas as well as for point-of-care applications.

Efficient upconverting carbon nitride nanotubes for near-infrared-driven photocatalytic hydrogen production.

We report a facile chemical technique for synthesizing nanotube-based hybrid materials for near-infrared-driven photocatalytic hydrogen (H2) production. Upconversion nanoparticles (UCNPs), NaYF4:Yb,Tm,Gd (NYFG) and NaYF4:Yb,Tm (NYF), were engineered on C3N4 nanotubes (C3N4 NTs) separately to construct heterojunction structures. With a UCNP loading content of 15 wt%, the NYFG/C3N4 NT heterojunction exhibits the highest H2 generation rate of 311.6 µmol g-1 with an apparent quantum efficiency of 0.80 ‰, about 1.4 times higher than that of the NYF/C3N4 NT nanocomposite under 980 nm laser irradiation. Comprehensive characterization reveals that the enhanced photocatalytic performance of the Gd doped nanostructure is attributed to the synergistic effect, stronger interaction, higher emission intensities, and faster charge transfer between the UCNPs and C3N4 NTs. Moreover, the steady-state and dynamic fluorescence spectra indicate that the energy from NYFG NPs was transferred to C3N4 NTs via a fluorescence-resonance energy-transfer process. Our work demonstrates the potential of developing near-infrared-responsive photocatalysts for energy and environmental applications.

3D sub-diffraction imaging in a conventional confocal configuration by exploiting super-linear emitters.

Sub-diffraction microscopy enables bio-imaging with unprecedented clarity. However, most super-resolution methods require complex, costly purpose-built systems, involve image post-processing and struggle with sub-diffraction imaging in 3D. Here, we realize a conceptually different super-resolution approach which circumvents these limitations and enables 3D sub-diffraction imaging on conventional confocal microscopes. We refer to it as super-linear excitation-emission (SEE) microscopy, as it relies on markers with super-linear dependence of the emission on the excitation power. Super-linear markers proposed here are upconversion nanoparticles of NaYF4, doped with 20% Yb and unconventionally high 8% Tm, which are conveniently excited in the near-infrared biological window. We develop a computational framework calculating the 3D resolution for any viable scanning beam shape and excitation-emission probe profile. Imaging of colominic acid-coated upconversion nanoparticles endocytosed by neuronal cells, at resolutions twice better than the diffraction limit both in lateral and axial directions, illustrates the applicability of SEE microscopy for sub-cellular biology.

Lifetime-engineered NIR-II nanoparticles unlock multiplexed in vivo imaging.

Deep tissue imaging in the second near-infrared (NIR-II) window holds great promise for physiological studies and biomedical applications1-6. However, inhomogeneous signal attenuation in biological matter7,8 hampers the application of multiple-wavelength NIR-II probes to multiplexed imaging. Here, we present lanthanide-doped NIR-II nanoparticles with engineered luminescence lifetimes for in vivo quantitative imaging using time-domain multiplexing. To achieve this, we have devised a systematic approach based on controlled energy relay that creates a tunable lifetime range spanning three orders of magnitude with a single emission band. We consistently resolve selected lifetimes from the NIR-II nanoparticle probes at depths of up to 8 mm in biological tissues, where the signal-to-noise ratio derived from intensity measurements drops below 1.5. We demonstrate that robust lifetime coding is independent of tissue penetration depth, and we apply in vivo multiplexing to identify tumour subtypes in living mice. Our results correlate well with standard ex vivo immunohistochemistry assays, suggesting that luminescence lifetime imaging could be used as a minimally invasive approach for disease diagnosis.

Background-free in-vivo Imaging of Vitamin C using Time-gateable Responsive Probe.

Sensitive optical imaging of active biomolecules in the living organism requires both a molecular probe specifically responsive to the target and a high-contrast approach to remove the background interference from autofluorescence and light scatterings. Here, a responsive probe for ascorbic acid (vitamin C) has been developed by conjugating two nitroxide radicals with a long-lived luminescent europium complex. The nitroxide radical withholds the probe on its "off" state (barely luminescent), until the presence of vitamin C will switch on the probe by forming its hydroxylamine derivative. The probe showed a linear response to vitamin C concentration with a detection limit of 9.1 nM, two orders of magnitude lower than that achieved using electrochemical methods. Time-gated luminescence microscopy (TGLM) method has further enabled real-time, specific and background-free monitoring of cellular uptake or endogenous production of vitamin C, and mapping of vitamin C in living Daphnia magna. This work suggests a rational design of lanthanide complexes for background-free small animal imaging of biologically functional molecules.

Practical implementation, characterization and applications of a multi-colour time-gated luminescence microscope.

Time-gated luminescence microscopy using long-lifetime molecular probes can effectively eliminate autofluorescence to enable high contrast imaging. Here we investigate a new strategy of time-gated imaging for simultaneous visualisation of multiple species of microorganisms stained with long-lived complexes under low-background conditions. This is realized by imaging two pathogenic organisms (Giardia lamblia stained with a red europium probe and Cryptosporidium parvum with a green terbium probe) at UV wavelengths (320-400 nm) through synchronization of a flash lamp with high repetition rate (1 kHz) to a robust time-gating detection unit. This approach provides four times enhancement in signal-to-background ratio over non-time-gated imaging, while the average signal intensity also increases six-fold compared with that under UV LED excitation. The high sensitivity is further confirmed by imaging the single europium-doped Y2O2S nanocrystals (150 nm). We report technical details regarding the time-gating detection unit and demonstrate its compatibility with commercial epi-fluorescence microscopes, providing a valuable and convenient addition to standard laboratory equipment.