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.

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.

Rapid, Simple, and Inexpensive Spatial Patterning of Wettability in Microfluidic Devices for Double Emulsion Generation.

Water-in-oil-in-water (w/o/w) double emulsion (DE) encapsulation has been widely used as a promising platform technology for various applications in the fields of food, cosmetics, pharmacy, chemical engineering, materials science, and synthetic biology. Unfortunately, DEs formed by conventional emulsion generation approaches in most cases are highly polydisperse, making them less desirable for quantitative assays, controlled biomaterial synthesis, and entrapped ingredient release. Microfluidic devices can generate monodisperse DEs with controllable size, morphology, and production rate, but these generally require multistep fabrication processes and use of different solvents or bulky external instrumentation to pattern channel wettability. To overcome these limitations, we propose a rapid, simple, and inexpensive method to spatially pattern wettability in microfluidic devices for the continuous generation of monodisperse DEs. This is achieved by applying corona-plasma treatment to a select zone of the microchannel surface aided by a custom-designed corona resistance microchannel to strictly confine the plasma-treatment zone in a single polydimethylsiloxane (PDMS) microfluidic device. The properties of PDMS channel surfaces and key microchannel regions for DE generation are characterized under different levels of treatment. The size, shell thickness, and number of inner cores of generated DEs are shown to be highly controllable by tuning the phase flow rate ratios. Using DEs as templates, we successfully achieve a one-step generation and collection of gelatin microgels. Additionally, we demonstrate the biological capability of generated DEs by flow cytometric screening of the encapsulation and growth of yeast cells within DEs. We expect that the proposed approach will be widely used to create microfluidic devices with more complex wettability patterns.

An Analysis of Formal Patient Complaints and Malpractice Events Involving Hand and Upper Extremity Surgeons.

INTRODUCTION: Our purpose was to define and categorize patient complaints within a hand surgery practice over a 10-year period. In addition, we aimed to define surgeon and patient factors associated with formal complaints. METHODS: All patients who filed a complaint with our institution's patient advocacy service against six hand surgeons in an academic practice over a 10-year period were recorded and categorized using the Patient Complaint Analysis System. A control group consisting of all patients seen by the surgeons during the study period was created. Demographic differences between the complaint and control groups were analyzed, as were complaint rates between surgeons. We obtained the number of malpractice events involving each of the surgeons. RESULTS: During the 10-year study period, 73 of 36,010 unique patients seen (0.20%) filed a complaint. Care and treatment category comprised the highest percentage of complaint designations (30%), followed by access and availability (23%). Forty-three patients (59%) who filed complaints were treated surgically. Patients with a complaint had a significantly higher percentage of mental, behavioral, or neurodevelopmental disorders compared with controls (55% versus 42%, P = 0.03). The complaint rate (total complaints/total new patients seen) ranged between 0.09% and 0.29% for the six surgeons, and these results were not statistically significant. DISCUSSION: Within an academic hand and upper extremity surgery practice, the rate of patient complaints is 0.20% or approximately one complaint for every 500 new patients seen. Most patient complaints are categorized within the care and treatment domain. Underlying mental health conditions are associated with more frequent complaints. Communication issues appear to represent a modifiable area that hand surgeons can improve to help mitigate potential complaints. Understanding both the frequency and types of patient complaints may allow hand surgeons to recognize areas for improvement and avoid potential exposure to malpractice litigation. LEVEL OF EVIDENCE: Prognostic level III (case-control).

Formal Patient Complaints and Malpractice Events Against Pediatric Orthopaedic Surgeons.

BACKGROUND: Formal patient complaints are associated with increased malpractice litigation and can have adverse occupational consequences for surgeons. Our purpose was to define and categorize patient complaints within an academic pediatric orthopaedic surgery practice over a 10-year period. We further aimed to define risk factors associated with patient complaints. METHODS: We reviewed all complaints within our institution's patient advocacy service filed on behalf of a patient against 4 pediatric orthopaedic surgeons over a 10-year period. Complaints were categorized using the Patient Complaint Analysis System. A control group of all patients seen by the surgeons during the study period was created. We compared baseline demographics between the patients with a complaint and the control group and compared complaint rates between the surgeons. Any malpractice events (lawsuits and claims) associated with the surgeons were obtained. We queried our institutional MIDAS reporting system (which allows for anonymous reporting of potential patient-safety or "near-miss" events), to assess whether patients with a complaint had a reported event. RESULTS: The 4 pediatric orthopaedic surgeons saw a total of 25,747 unique patients during the study period. Forty-one patients had a formal complaint, resulting in a complaint rate of 0.15%. Complaint rates varied from 0.08% to 0.30% between surgeons. Humanness was the most frequent complaint designation category (32%) followed by Care and Treatment (19%). Of the 41 patients with a complaint, 18 (44%) underwent surgical treatment. Only 1 patient with a complaint also had an entry within our institutional patient-safety reporting system. CONCLUSIONS: The rate of patient complaints within an academic pediatric orthopaedic surgery practice over a decade was 0.15%, or ~1 complaint for every 670 new patients seen. The majority of patient complaints involved communication; a potentially modifiable area that can be targeted for improvement. While complaint rates among surgeons can vary, patient demographic factors are not associated with increased complaints. Understanding patient complaints rates and types may allow surgeons to target areas for improvement and decrease exposure to malpractice litigation. LEVEL OF EVIDENCE: Level II-prognostic.

Microdroplet enabled cultivation of single yeast cells correlates with bulk growth and reveals subpopulation phenomena.

Yeast has been engineered for cost-effective organic acid production through metabolic engineering and synthetic biology techniques. However, cell growth assays in these processes were performed in bulk at the population level, thus obscuring the dynamics of rare single cells exhibiting beneficial traits. Here, we introduce the use of monodisperse picolitre droplets as bioreactors to cultivate yeast at the single-cell level. We investigated the effect of acid stress on growth and the effect of potassium ions on propionic acid tolerance for single yeast cells of different species, genotypes, and phenotypes. The results showed that the average growth of single yeast cells in microdroplets experiences the same trend to those of yeast populations grown in bulk, and microdroplet compartments do not significantly affect cell viability. This approach offers the prospect of detecting cell-to-cell variations in growth and physiology and is expected to be applied for the engineering of yeast to produce value-added bioproducts.

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.

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.

Sensitive and Direct DNA Mutation Detection by Surface-Enhanced Raman Spectroscopy Using Rational Designed and Tunable Plasmonic Nanostructures.

Efficient DNA mutation detection methods are required for diagnosis, personalized therapy development, and prognosis assessment for diseases such as cancer. To address this issue, we proposed a straightforward approach by combining active plasmonic nanostructures, surface-enhanced Raman spectroscopy (SERS), and polymerase chain reaction (PCR) with a statistical tool to identify and classify BRAF wild type (WT) and V600E mutant genes. The nanostructures provide enhanced sensitivity, while PCR offers high specificity toward target DNA. A series of positively charged plasmonic nanostructures including gold/silver nanospheres, nanoshells, nanoflowers, and nanostars were synthesized with a one-pot strategy and characterized. By changing the shape of nanostructures, we are able to vary the surface plasmon resonance from 551 to 693 nm. The gold/silver nanostar showed the highest SERS activity, which was employed for DNA mutation detection. We reproducibly analyzed as few as 100 copies of target DNA sequences using gold/silver nanostars, thus demonstrating the high sensitivity of the direct SERS detection. By means of statistical analysis (principal component analysis-linear discriminant analysis), this method was successfully applied to differentiate the WT and V600E mutant both from whole genome DNA lysed from cell line and from cell-free DNA collected from cell culture media. We further proved that this assay is capable of specifically amplifying and accurately classifying a real plasma sample. Thus, this direct SERS strategy combined with the active plasmonic nanostructures has the potential for wide applications as an alternative tool for sensitively monitoring and evaluating important clinical nucleotide biomarkers.

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.

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.

Sensitive and Multiplexed SERS Nanotags for the Detection of Cytokines Secreted by Lymphoma.

The sensitive and simultaneous detection of cytokines will provide new insights into the physiological process and disease pathways due to the complex nature of cytokine networks. However, the key challenge is the lack of probes that can simultaneously detect multiple cytokines in a single sample. In this contribution, we proposed an alternative approach for sensitive cytokine detection in a multiplex manner by the use of a new set of surface-enhanced Raman spectroscopy (SERS) nanotags. Typically, the newly designed SERS nanotags are composed of gold nanoparticles as the core, tuneable Raman molecules as the reporters, and a thin silver layer as the shell. As demonstrated through rigorous numerical simulations, enhanced Raman signal is achieved due to a strong localization of light in the 0.2 nm thin, optically deep-subwavelength region between the Au core and the Ag shell. Sensitive detection of cytokines is realized by forming a sandwich immunoassay. The detection limit is down to 4.5 pg mL-1 (S/N = 3). The specificity of the assay is proved as negligible signals were detected for the false targets. Furthermore, multiple cytokines are simultaneously detected in a single assay from the secretion of B-lymphocyte cell line (Raji) after concanavalin A (Con A) stimulation. The results indicate that our method holds a significant potential for sensitive and multiplexed detection of cytokines and offers the opportunity for future applications in clinical settings.

Time-Gated Luminescent In Situ Hybridization (LISH): Highly Sensitive Detection of Pathogenic Staphylococcus aureus.

We describe simple direct conjugation of a single TEGylated Europium chelate to DNA that binds to intracellular rRNA and is then detected using a homogeneous luminescent in situ hybridisation (LISH) technique. As a proof-of-principle, Staphylococcus aureus (S. aureus) was selected as a model for our study to show the ability of this probe to bind to intracellular 16S ribosomal rRNA. A highly purified Europium chelate conjugated oligonucleotide probe complementary to an rRNA sequence-specific S. aureus was prepared and found to be soluble and stable in aqueous solution. The probe was able to bind specifically to S. aureus via in situ hybridisation to differentiate S. aureus from a closely related but less pathogenic Staphylococcus species (S. epidermidis). A time-gated luminescent (TGL) microscope system was used to generate the high signal-to-noise ratio (SNR) images of the S. aureus. After excitation (365 nm, Chelate λmax = 335 nm), the long-lived (Eu3+) luminescent emission from the probe was detected without interference from natural background autofluorescence typically seen in biological samples. The luminescent images were found to have 6 times higher SNR or sensitivity compared to the fluorescent images using conventional fluorophore Alexa Fluor 488. The TEGylated Europium chelate -oligo probe stained S. aureus with mean signal intensity 3.5 times higher than the threshold level of signal from S. epidermidis (with SNR 8 times higher). A positive control probe (EUB338-BHHTEGST-Eu3+) has mean signal intensity for S. aureus and S. epidermidis equally 3.2 times higher than the threshold of signal for a negative NON-EUB338 control probe. The direct conjugation of a single Europium chelate to DNA provides simplicity and improvement over existing bovine serum albumin (BSA)/streptavidin/biotinylated DNA platforms for multi-attachment of Europium chelate per DNA and more importantly makes it feasible for hybridisation to intracellular RNA targets. This probe has great potential for highly sensitive homogeneous in situ hybridisation detection of the vast range of intracellular DNA targets.

Use of Mammalian Target of Rapamycin Inhibitors for Pancreas Transplant Immunosuppression Is Associated With Improved Allograft Survival and Improved Early Patient Survival.

OBJECTIVES: Mammalian targets of rapamycin inhibitors (mTORi) are considered second-line immunosuppression agents because of associated increases in rejection and impaired wound healing. Recent reports indicate mTORi have been linked to improved survival, decreased inflammatory response in pancreatitis, and antiproliferative and antiangiogenic activity. Mammalian targets of rapamycin inhibitors have not been extensively analyzed in pancreas transplant recipients. METHODS: Adults with pancreas and kidney-pancreas transplants from 1987 to 2016 in the United Network for Organ Sharing database were analyzed (N = 25,837). Subjects were stratified into 2 groups: use of mTORi (n = 4174) and use of non-mTORi-based immunosuppression (n = 21,663). The log-rank test compared survival rates. Univariate and multivariate Cox regression analyses assessed patient and graft survival. RESULTS: Mammalian targets of rapamycin inhibitors were associated with a 7% risk reduction in allograft failure (hazard ratio, 0.931; P = 0.006). Allograft survival rates were significantly different between mTORi versus non-mTORi (P < 0.0001).The mTORi group showed a significantly higher patient survival rate 1, 3, 5, and 10 years posttransplant compared. Patient survival at 15 years was not significantly different. CONCLUSIONS: The use of mTORi for immunosuppression in pancreas transplant is associated with improved allograft survival and early patient survival posttransplant (up to 10 years).

A comparison of portal venous versus systemic venous drainage in pancreas transplantation.

BACKGROUND: The decision to utilize portal or systemic venous drainage in pancreas transplantation is surgeon- and center-dependent. Information regarding the superior method is based on single-center reports and animal models. METHODS: UNOS data on adults receiving pancreas and kidney-pancreas transplants from 1987 to 2016 were analyzed (n = 29 078). The groups analyzed were: systemic venous pancreas graft drainage (SVD, n = 24 512) or portal venous pancreas graft drainage (PVD, n = 4566). A Cox proportional hazard model compared patient and allograft survival between groups. RESULTS: No statistically significant differences were observed for patient and allograft survival at 1, 3, 5, 10, or 15 years post-transplant at each time interval and cumulatively (patient - HR:1.041; 95% CI:0.989-1.095; allograft - HR:0.951; 95% CI:0.881-1.027). PVD reduced the risk of death by 22.0% (P = 0.017) compared to SVD for patients undergoing pancreas after kidney transplant (PAK); no statistically significant difference was found for patients undergoing other types of transplants. CONCLUSION: There is no significant clinical difference in patient or allograft survival between PVD and SVD in pancreas transplantation for the majority of patients. For the subgroup of PAK, PVD was associated with decreased mortality. For individual surgeons, center and patient scenarios should dictate which technique is performed.

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.

Follicular dendritic cell sarcoma of the porta hepatis.

Follicular dendritic cell (FDC) sarcoma is a very rare neoplasm which commonly involves the lymph nodes and less commonly involves extranodal organs such as the liver. Most cases of FDC sarcoma are idiopathic, however some cases are associated with other disease states. Management of FDC sarcoma is primarily focused on surgical resection of the mass, and secondarily focused on radiotherapy, chemotherapy and/or biologic pharmacotherapy. We report the case of a patient who was found to have FDC sarcoma presenting as an obstructing mass of the porta hepatis, a manifestation which does not appear to be reported in the literature.

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.