Unveiling the Relationship between the Perovskite Precursor Solution and the Resulting Device Performance.

For the fabrication of perovskite solar cells (PSCs) using a solution process, it is essential to understand the characteristics of the perovskite precursor solution to achieve high performance and reproducibility. The colloids (iodoplumbates) in the perovskite precursors under various conditions were investigated by UV-visible absorption, dynamic light scattering, photoluminescence, and total internal reflection fluorescence microscopy techniques. Their local structure was examined by in situ X-ray absorption fine structure studies. Perovskite thin films on a substrate with precursor solutions were characterized by transmission electron microscopy, X-ray diffraction analysis, space-charge-limited current, and Kelvin probe force microscopy. The colloidal properties of the perovskite precursor solutions were found to be directly correlated with the defect concentration and crystallinity of the perovskite film. This work provides guidelines for controlling perovskite films by varying the precursor solution, making it possible to use colloid-engineered lead halide perovskite layers to fabricate efficient PSCs.

Direct-laser writing for subnanometer focusing and single-molecule imaging.

Two-photon direct laser writing is an additive fabrication process that utilizes two-photon absorption of tightly focused femtosecond laser pulses to implement spatially controlled polymerization of a liquid-phase photoresist. Two-photon direct laser writing is capable of nanofabricating arbitrary three-dimensional structures with nanometer accuracy. Here, we explore direct laser writing for high-resolution optical microscopy by fabricating unique 3D optical fiducials for single-molecule tracking and 3D single-molecule localization microscopy. By having control over the position and three-dimensional architecture of the fiducials, we improve axial discrimination and demonstrate isotropic subnanometer 3D focusing (<0.8 nm) over tens of micrometers using a standard inverted microscope. We perform 3D single-molecule acquisitions over cellular volumes, unsupervised data acquisition and live-cell single-particle tracking with nanometer accuracy.

Magnetic field induced aggregation of nanoparticles for sensitive molecular detection.

A molecular detection method utilizing the magnetically induced aggregation of silver nanoparticle (NP)-embedded silica NPs for SERS activation is described. Here, silver embedded magnetic NPs (Ag-M-dots) composed of a magnetic core and silica shells, on whose surface silver NPs were formed, were used. Because the magnetic field induced aggregated Ag-M-dots exhibit a strong SERS signal compared to the dispersed Ag-M-dots, the system allows for the detection of adsorbed Raman label compound even at the 100 fM level. Adenine was tested as a model biocompound and its Raman spectrum could be observed at concentrations as low as 1 pM. The experimental results were supported by the theoretical calculations.

Building a Total Internal Reflection Microscope (TIRF) with Active Stabilization (Feedback SMLM).

The data quality of high-resolution imaging can be markedly improved with active stabilization, which is based on feedback loops within the microscope that maintain the sample in the same location throughout the experiment. The purpose is to provide a highly accurate focus lock, therefore eliminating drift and improving localization precision. Here, we describe a step-by-step protocol for building a total internal reflection microscope combined with the feedback loops necessary for sample and detection stabilization, which we routinely use in single-molecule localization microscopy (SMLM). The performance of the final microscope with feedback loops, called feedback SMLM, has previously been described. We demonstrate how to build a replica of our system and include a list of the necessary optical components, tips, and an alignment strategy.

3D active stabilization for single-molecule imaging.

A key part of any super-resolution technique involves accurately correcting for mechanical motion of the sample and setup during acquisition. If left uncorrected, drift degrades the resolution of the final reconstructed image and can introduce unwanted artifacts. Here, we describe how to implement active stabilization, thereby reducing drift to ~1 nm across all three dimensions. In this protocol, we show how to implement our method on custom and standard microscopy hardware. We detail the construction of a separate illumination and detection path, dedicated exclusively to acquiring the diffraction pattern of fiducials deposited on the imaging slide. We also show how to focus lock and adjust the focus in arbitrary nanometer step size increments. Our real-time focus locking is based on kHz calculations performed using the graphics processing unit. The fast calculations allow for rapid repositioning of the sample, which reduces drift below the photon-limited localization precision. Our approach allows for a single-molecule and/or super-resolution image acquisition free from movement artifacts and eliminates the need for complex algorithms or hardware installations. The method is also useful for long acquisitions which span over hours or days, such as multicolor super resolution. Installation does not require specialist knowledge and can be implemented in standard biological laboratories. The full protocol can be implemented within ~2 weeks.

Multifunctional silver-embedded magnetic nanoparticles as SERS nanoprobes and their applications.

In this study, surface-enhanced Raman spectroscopy (SERS)-encoded magnetic nanoparticles (NPs) are prepared and utilized as a multifunctional tagging material for cancer-cell targeting and separation. First, silver-embedded magnetic NPs are prepared, composed of an 18-nm magnetic core and a 16-nm-thick silica shell with silver NPs formed on the surface. After simple aromatic compounds are adsorbed on the silver-embedded magnetic NPs, they are coated with silica to provide them with chemical and physical stability. The resulting silica-encapsulated magnetic NPs (M-SERS dots) produce strong SERS signals and have magnetic properties. In a model application as a tagging material, the M-SERS dots are successfully utilized for targeting breast-cancer cells (SKBR3) and floating leukemia cells (SP2/O). The targeted cancer cells can be easily separated from the untargeted cells using an external magnetic field. The separated targeted cancer cells exhibit a Raman signal originating from the M-SERS dots. This system proves to be an efficient tool for separating targeted cells. Additionally, the magnetic-field-induced hot spots, which can provide a 1000-times-stronger SERS intensity due to aggregation of the NPs, are studied.

Toxicity and clearance of intratracheally administered multiwalled carbon nanotubes from murine lung.

Carbon nanotubes (CNT) are known to have widespread industrial applications; however, several reports indicated that these compounds may be associated with adverse effects in humans. In this study, multiwalled carbon nanotubes were administered to murine lungs intratracheally to determine whether acute and chronic pulmonary toxicity occurred. In particular, pristine multiwalled carbon nanotubes (PMWCNT) and acid-treated multiwalled carbon nanotubes (TMWCNT) were used in this study. In broncheoalveolar lavage fluid (BALF) cell analysis, PMWCNT induced more severe acute inflammatory cell recruitment than TMWCNT. Histopathologically, both PMWCNT and TMWCNT induced multifocal inflammatory granulomas in a dose-dependent manner. The observed granulomas were reversible, with TMWCNT-induced granulomas diminishing faster than PMWCNT-induced granulomas. Although the area of granuloma reduced with time, hyperplasia and dysplastic characteristics such as mitotic figures, anisokaryosis, and anisocytosis were still observed. These findings demonstrate that MWCNT induces granulomatous inflammation, and the duration and pattern of inflammation seem to vary depending upon the types of MWCNT to which mice are exposed. Therefore, toxicity studies on various types of CNT are needed as the responsiveness to these compounds differs.

A 3D Bioprinter Specifically Designed for the High-Throughput Production of Matrix-Embedded Multicellular Spheroids.

3D in vitro cancer models are important therapeutic and biological discovery tools, yet formation of matrix-embedded multicellular spheroids prepared in high-throughput (HTP), and in a highly controlled manner, remains challenging. This is important to achieve robust and statistically relevant data. Here, we developed an enabling technology consisting of a bespoke drop-on-demand 3D bioprinter capable of HTP printing of 96-well plates of spheroids. 3D multicellular spheroids are embedded inside a hydrogel matrix with precise control over size and cell number, with the intra-experiment variability of embedded spheroid diameter coefficient of variation being between 4.2% and 8.7%. Application of 3D bioprinting HTP drug screening was demonstrated with doxorubicin. Measurements of IC50 values showed sensitivity to spheroid size, embedding, and how spheroids conform to the embedding, revealing parameters shaping biological responses in these models. Our study demonstrates the potential of 3D bioprinting as a robust HTP platform to screen biological and therapeutic parameters.

Ultraprecise single-molecule localization microscopy enables in situ distance measurements in intact cells.

Single-molecule localization microscopy (SMLM) has the potential to quantify the diversity in spatial arrangements of molecules in intact cells. However, this requires that the single-molecule emitters are localized with ultrahigh precision irrespective of the sample format and the length of the data acquisition. We advance SMLM to enable direct distance measurements between molecules in intact cells on the scale between 1 and 20 nm. Our actively stabilized microscope combines three-dimensional real-time drift corrections and achieves a stabilization of <1 nm and localization precision of ~1 nm. To demonstrate the biological applicability of the new microscope, we show a 4- to 7-nm difference in spatial separations between signaling T cell receptors and phosphatases (CD45) in active and resting T cells. In summary, by overcoming the major bottlenecks in SMLM imaging, it is possible to generate molecular images with nanometer accuracy and conduct distance measurements on the biological relevant length scales.

Multiplex immunoassay using fluorescent-surface enhanced Raman spectroscopic dots for the detection of bronchioalveolar stem cells in murine lung.

Immunoassays using nanomaterials have been rapidly developed for the analysis of multiple biomolecules. Highly sensitive and biocompatible surface enhanced Raman spectroscopy-active nanomaterials have been used for biomolecule analysis by many research groups in order to overcome intrinsic problems of conventional immunoassays. We used fluorescent surface-enhanced Raman spectroscopic dots (F-SERS dots) to detect biomolecules in this study. The F-SERS dots are composed of silver nanoparticle-embedded silica nanospheres, organic Raman tagging materials, and fluorescent dyes. The F-SERS dots demonstrated highly sensitive, selective, and multifunctional characteristics for multiplex targeting, tracking, and imaging of cellular and molecular events in the living organism. We successfully applied F-SERS dots for the detection of three cellular proteins, including CD34, Sca-1, and SP-C. These proteins are simultaneously expressed in bronchioalveolar stem cells (BASCs) in the murine lung. We analyzed the relative expression ratios of each protein in BASCs since external standards were used to evaluate SERS intensity in tissue. Quantitative comparisons of multiple protein expression in tissue were first attempted using SERS-encoded nanoprobes. Our results suggested that immunoassays using F-SERS dots offered significant increases in sensitivity and selectivity. Such immunoassays may serve as the primary next-generation labeling technologies for the simultaneous analysis of multiple biomolecules.

Highly pathogenic avian influenza virus (H5N8) in domestic poultry and its relationship with migratory birds in South Korea during 2014.

Highly pathogenic H5N8 avian influenza viruses (HPAIVs) were introduced into South Korea during 2014, thereby caused outbreaks in wild birds and poultry farms. During the 2014 outbreak, H5N8 HPAIVs were isolated from 38 wild birds and 200 poultry farms (up to May 8, 2014). To better understand the introduction of these viruses and their relationships with wild birds and poultry farm, we analyzed the genetic sequences and available epidemiological data related to the viruses. Genetic analysis of 37 viruses isolated from wild birds and poultry farms showed that all of the isolates belonged to clade 2.3.4.6 of the hemagglutinin (HA) gene, but comprised two distinct groups. During the initial stage of the outbreak, identical isolates from each group were found in wild birds and poultry farms near Donglim Reservoir, which is a resting site for migratory birds, thereby indicating that two types of H5N8 HPAIVs were introduced into the lake at the same time. Interestingly, the one group of H5N8 HPAIV predominated around Donglim Reservoir, and the predominant virus was dispersed by wild birds among the migratory bird habitats in the western region of South Korea as time passed, and it was also detected in nearby poultry farms. Furthermore, compared with the results of the annual AIV surveillance of captured wild birds, which has been performed since 2008, more HPAIVs were isolated and H5 sero-prevalence was also detected during the 2014 outbreak. Overall, our results strongly suggest that migratory birds played a key role in the introduction and spread of viruses during the initial stage of the 2014 outbreak.

Nuclear image analysis study of neuroendocrine tumors.

BACKGROUND: There is a subjective disagreement about nuclear chromatin in the field of pathology. Objective values of red, green, and blue (RGB) light intensities for nuclear chromatin can be obtained through a quantitative analysis using digital images. METHODS: We examined 10 cases of well differentiated neuroendocrine tumors of the rectum, small cell lung carcinomas, and moderately differentiated squamous cell lung carcinomas respectively. For each case, we selected 30 representative cells and captured typical microscopic findings. Using an image analyzer, we determined the longest nuclear line profiles and obtained graph files and Excel data on RGB light intensities. We assessed the meaningful differences in graph files and Excel data among the three different tumors. RESULTS: The nucleus of hematoxylin and eosin-stained tumor cells was expressed as a combination of RGB light sources. The highest intensity was from blue, whereas the lowest intensity was from green. According to the graph files, green showed the most noticeable change in the light intensity, which is consistent with the difference in standard deviations. CONCLUSIONS: The change in the light intensity for green has an important implication for differentiating between tumors. Specific features of the nucleus can be expressed in specific values of RGB light intensities.

Preparation of polydiacetylene immobilized optically encoded beads.

Polydiacetylene (PDA), which can change the chromic and fluorescence properties by inducing environmental perturbations, is immobilized on planar solid supports for many biological applications. In this work, we immobilize PDA onto optically encoded spherical beads (PDA-SERS beads). The prepared PDA immobilized beads (36 µm) exhibit a blue color without fluorescence. By inducing stress, their color and fluorescence properties are changed to red with fluorescence. The SERS spectra of the PDA-SERS beads can be recognized over the PDA background. Moreover, our PDA immobilization methods are successfully applied to silica-surface SERS-encoded beads (5 µm) and proven to also be useful in fluorescence encoding systems.