Demonstration of intracellular real-time molecular quantification via FRET-enhanced optical microcavity.

Single cell analysis is crucial for elucidating cellular diversity and heterogeneity as well as for medical diagnostics operating at the ultimate detection limit. Although superbly sensitive biosensors have been developed using the strongly enhanced evanescent fields provided by optical microcavities, real-time quantification of intracellular molecules remains challenging due to the extreme low quantity and limitations of the current techniques. Here, we introduce an active-mode optical microcavity sensing stage with enhanced sensitivity that operates via Förster resonant energy transferring (FRET) mechanism. The mutual effects of optical microcavity and FRET greatly enhances the sensing performance by four orders of magnitude compared to pure Whispering gallery mode (WGM) microcavity sensing system. We demonstrate distinct sensing mechanism of FRET-WGM from pure WGM. Predicted lasing wavelengths of both donor and acceptor by theoretical calculations are in perfect agreement with the experimental data. The proposed sensor enables quantitative molecular analysis at single cell resolution, and real-time monitoring of intracellular molecules over extended periods while maintaining the cell viability. By achieving high sensitivity at single cell level, our approach provides a path toward FRET-enhanced real-time quantitative analysis of intracellular molecules.

Photoluminescence Lifetime of Black Phosphorus Nanoparticles and Their Applications in Live Cell Imaging.

Black phosphorus (BP) has attracted much attention as a new member of 2D materials due to its unique electronic and optical properties and a wide range of promising applications. Here, for the first time, we report the photoluminescence lifetime of BP nanomaterial and its applications as an efficient agent for live cell imaging. With a lateral size of ∼35 nm and a thickness of ∼6 nm, the fabricated BP nanoparticles (BPNPs) exhibited a unique photoluminescent (PL) emission at ∼690 nm. The photoluminescence lifetime (PLT) of BPNPs was determined to be 110.5 ps. Coating a layer of mesoporous silica on the surface of BPNPs (BPNPs@mSiO2) extended the lifetime to 267 ps, suggesting a change in the microenvironment. The lifetime was also influenced by ionic strength and intracellular microenvironment, which implies BPNPs as valuable probes for sensing variations in the microenvironment. Live cell imaging was achieved via directly probing the photoluminescence intensity or the photoluminescence lifetime. Our findings are significant, implying that BPNPs can be of large value in sensing variations of the cellular microenvironment and in probing cells with distinct cytosolic contents. This research leads to promising prospects for BPNPs in multiple biomedical applications.

Rituxan nanoconjugation prolongs drug/cell interaction and enables simultaneous depletion and enhanced Raman detection of lymphoma cells.

Antibody therapy is a type of immunotherapy that uses monoclonal antibodies (mAbs) to specifically bind to target cells to fight cancer. These mAbs have been conjugated with a number of nanocarriers to realize the targeted delivery of chemotherapeutics. Previous data indicate that the conjugation of a mAb to a nanocarrier promoted the faster internalization (endocytosis) of the nanoconjugates. In this study, we report that the crosslinking of Rituxan, an anti-lymphoma mAb, with silver nanoparticles prevented Rituxan from entering cells and prolonged drug/cell interaction. This finding was confirmed by both imaging flow cytometric and laser scanning confocal microscopic analyses of the fluorescently labeled nanoconjugates and cells. Furthermore, nanoconjugated Rituxan resulted in the enhanced capping of CD20 molecules on the cell membrane. We were able to show that the prolonged drug/cell interaction and enhanced capping directly contributed towards improved therapeutic efficiency. This represents a significant finding indicating that the nanocarrier served not only as a simple platform for targeted delivery, but also markedly altered the antibody performance at the molecular level and mediated key biological functions. The nanoconjugates also served as a Raman probe to specifically detect single live lymphoma cells with high sensitivity via surface-enhanced Raman scattering (SERS). Via integrating the single-cell level detection sensitivity of SERS with targeted and enhanced depletion ability, this nanoconjugate can be applied as a promising tool in lymphoma theranostics.

Reintroduction of rare arable plants by seed transfer. What are the optimal sowing rates?

During the past decades, agro-biodiversity has markedly declined and some species are close to extinction in large parts of Europe. Reintroduction of rare arable plant species in suitable habitats could counteract this negative trend. The study investigates optimal sowing rates of three endangered species (Legousia speculum-veneris (L.) Chaix, Consolida regalis Gray, and Lithospermum arvense L.), in terms of establishment success, seed production, and crop yield losses.A field experiment with partial additive design was performed in an organically managed winter rye stand with study species added in ten sowing rates of 5-10,000 seeds m(-2). They were sown as a single species or as a three-species mixture (pure vs. mixed sowing) and with vs. without removal of spontaneous weeds. Winter rye was sown at a fixed rate of 350 grains m(-2). Performance of the study species was assessed as plant establishment and seed production. Crop response was determined as grain yield.Plant numbers and seed production were significantly affected by the sowing rate, but not by sowing type (pure vs. mixed sowing of the three study species), and weed removal. All rare arable plant species established and reproduced at sowing rates >25 seeds m(-2), with best performance of L. speculum-veneris. Negative density effects occurred to some extent for plant establishment and more markedly for seed production.The impact of the three study species on crop yield followed sigmoidal functions. Depending on the species, a yield loss of 10% occurred at >100 seeds m(-2). Synthesis and applications: The study shows that reintroduction of rare arable plants by seed transfer is a suitable method to establish them on extensively managed fields, for example, in organic farms with low nutrient level and without mechanical weed control. Sowing rates of 100 seeds m(-2) for C. regalis and L. arvense, and 50 seeds m(-2) for L. speculum-veneris are recommended, to achieve successful establishment with negligible crop yield losses.

4Pi Microscopy.

Optical microscopy has become a key technology in the life sciences today. Its noninvasive nature provides access to the interior of intact and even living cells, where specific molecules can be precisely localized by fluorescent tagging. However, the attainable 3D resolution of an optical microscope has long been hampered by a comparatively poor resolution along the optic axis. By coherent focusing through two objective lenses, 4Pi microscopy improves the axial resolution by three- to fivefold. This primer is intended as a starting point for the design and operation of a 4Pi microscope of type A.

Three-dimensional organization of promyelocytic leukemia nuclear bodies.

Promyelocytic leukemia nuclear bodies (PML-NBs) are mobile subnuclear organelles formed by PML and Sp100 protein. They have been reported to have a role in transcription, DNA replication and repair, telomere lengthening, cell cycle control and tumor suppression. We have conducted high-resolution 4Pi fluorescence laser-scanning microscopy studies complemented with correlative electron microscopy and investigations of the accessibility of the PML-NB subcompartment. During interphase PML-NBs adopt a spherical organization characterized by the assembly of PML and Sp100 proteins into patches within a 50- to 100-nm-thick shell. This spherical shell of PML and Sp100 imposes little constraint to the exchange of components between the PML-NB interior and the nucleoplasm. Post-translational SUMO modifications, telomere repeats and heterochromatin protein 1 were found to localize in characteristic patterns with respect to PML and Sp100. From our findings, we derived a model that explains how the three-dimensional organization of PML-NBs serves to concentrate different biological activities while allowing for an efficient exchange of components.

Successful use of transvenous coil electrodes as single element subcutaneous array leads.

For implantable cardioverter defibrillator a 10 J safety margin between the defibrillation threshold (DFT) and the maximum output of the device is intended. In complex cases, the additional placement of a subcutaneous array lead is a common strategy for lowering the DFT. We report the successful use of transvenous coil electrodes as single element subcutaneous array leads in order to lower the DFT.

4Pi microscopy with linear fluorescence excitation.

Practical 4Pi microscopy has so far exclusively relied on multiphoton excitation of fluorescence, because the nonlinear suppression of contributions from higher-order sidelobes was mandatory for unambiguous axial superresolution. We show that novel lenses of 74 degrees semiaperture angle enable biological 4Pi microscopy with regular one-photon fluorescence excitation, thus increasing the signal and reducing system complexity and cost. An axial resolution of 95 nm, corresponding to a more than fourfold improvement over confocal microscopy, is verified in the imaging of microtubules in mammalian cells.

2,2'-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy.

The use of high numerical aperture immersion lenses in optical microscopy is compromised by spherical aberrations induced by the refractive index mismatch between the immersion system and the embedding medium of the sample. Especially when imaging >10 micro m deep into the specimen, the refractive index mismatch results in a noticeable loss of image brightness and resolution. A solution to this problem is to adapt the index of the embedding medium to that of the immersion system. Unfortunately, not many mounting media are known that are both index tunable as well as compatible with fluorescence imaging. Here we introduce a nontoxic embedding medium, 2,2'-thiodiethanol (TDE), which, by being miscible with water at any ratio, allows fine adjustment of the average refractive index of the sample ranging from that of water (1.33) to that of immersion oil (1.52). TDE thus enables high resolution imaging deep inside fixed specimens with objective lenses of the highest available aperture angles and has the potential to render glycerol embedding redundant. The refractive index changes due to larger cellular structures, such as nuclei, are largely compensated. Additionally, as an antioxidant, TDE preserves the fluorescence quantum yield of most of the fluorophores. We present the optical and chemical properties of this new medium as well as its application to a variety of differently stained cells and cellular substructures.

4Pi microscopy of type A with 1-photon excitation in biological fluorescence imaging.

We demonstrate that oil immersion lenses with a semi-aperture angle >/= 74 degrees enable 4Pi confocal fluorescence microscopy of type A with 1 photon excitation. The axial sidelobes amount to < 50 % of the main diffraction maximum, implying that lobe induced artifacts can be removed from the image data. The advancement reported herein enables a relative inexpensive implementation of 4Pi microscopy, providing axially superresolved 3D-imaging in transparent samples. As an example, we show dual-color 4Pi images of double stained Golgi stacks in a mammalian cell with 110 nm axial resolution. The resolution can be further enhanced to values slightly below 100 nm by image deconvolution.