bluuebunny/crossref_metadata_2025 huggingface dataset:
Second harmonic imaging of collagen organization in connective tissues
Authors: SCHANNE‐KLEIN Marie‐Claire
Published: December 2016

Type I collagen is a major structural protein in mammals. This biopolymer is synthesized as a triple helix, which self‐assembles into fibrils (diameter: 10‐300 nm) and further forms various 3D organizations specific to each tissue. In recent years Second Harmonic Generation (SHG) microscopy has emerged as a powerful technique for the
in situ
investigation of the fibrillar collagen structures in matrices or tissues [1]. However, as an optical technique with typically 300 nm lateral resolution, SHG microscopy cannot resolve most of the collagen fibrils. Moreover, in contrast to incoherent fluorescence signals that scale linearly with the chromophore concentration, SHG is a coherent multiphoton signal that scales quadratically with the density of collagen triple helices aligned with the same polarity in the focal volume. Consequently, quantitative SHG measurements have been limited so far to averaged phenomenological parameters [1].

In this study, we correlated SHG and transmission electron microscopies to determine the sensitivity of SHG microscopy and calibrate SHG signals as a function of the diameter of the collagen fibril [2]. To that end, we synthesized in vitro isolated fibrils with various diameters and successfully imaged the very same fibrils with both techniques, down to 30 nm diameter (see figure 1). We observed that SHG signals scale as the fourth power of the fibril diameter, as expected from analytical and numerical calculations. It validated our quantitative bottom‐up approach used to calculate the non‐linear response at the fibrillar scale and demonstrated that the high sensitivity of SHG microscopy originates from the parallel alignment of triple helices within the fibrils and the subsequent constructive interference of SHG radiations. This calibration was then applied to intact rat corneas, where we successfully recovered the diameter of hyperglycemia‐induced fibrils in the Descemet's membrane without having to resolve them [2,3].

Importantly, this calibration only applies to isolated fibrils. Nevertheless, complementary techniques can probe the sub‐micrometer structure of dense distributions of collagen fibrils. In particular, we have shown that polarization‐resolved SHG microscopy can probe the main orientation of collagen fibrils and their orientation disorder within the focal volume [4]. Combination of this modality with traction assays then provides a new method to measure the reorganization of the collagen network upon stretching and to correlate this microscopic response to the biomechanical response at macroscopic scale [4, 5].

In conclusion, our data represent a major step towards quantitative SHG imaging of collagen organization in biomaterials or connective tissues.
The effects of Noni on the
TNF
‐α,
iNOS
levels and plasma cell numbers in the spleen of
PNMC
administered rats
Authors: İlkay Ikitimur‐Armutak Elif , Daglioglu Suzan , Yigit Funda , Behzat Burak Esener Osman , Kivilcim Sonmez , Gurel‐Gurevin Ebru , Aktas Abit , Nadim Yilmazer
Published: December 2016

Noni is a native fruit which grows abundantly from Southeast Asia to Australia and is cultivated in Polynesia, India, the Caribbean, Central and northern South America. Morinda citrifolia L. (Noni), family Rubiaceae, has been used in Polynesia for over 2000 years in traditional medicine. Noni, has been reported to show a wide range of therapeutic effects including anticancer, anti inflammatory and antioxidant effects. The 3‐methyl‐4‐nitrophenol (PNMC) isolated from diesel exhaust particles and also a degradation product of the insecticide fenitrothion. In this study we aimed to evaluate the effects on TNF‐α and iNOS immunohistochemically and investigate the effects of Noni on plasma cell distribution in the spleen of rats exposed to PNMC. In the study, fifty‐six mature male rats were randomly divided into eight groups (n: 7). Control group received phosphate‐buffered saline+0.05% Tween‐80 (vehicle). First experimental group received only Noni and other experimental groups received 1, 10 and 100 mg/kg PNMC (S.C) alone or in combination with Noni (2 ml per rat by gavage) respectively for 5 days. At the sixth day animals were sacrificed and spleen tissues were removed and weighted. After routine histological process, paraffin sections were stained with methyl green‐pyronin for identification of plasma cells and TNF‐α and iNOS antibodies were applied immunohistochemically and evaluated under light microscope. Immunohistochemical staining of TNF‐α and iNOS were eveluated with histological scoring system (H‐scores). Spleen indices were close to control in Noni administered groups. Plasma cell numbers were decreased in 1, 10 and 100 mg/kg PNMC recevied groups (P<0.001) while plasma cell numbers were close to control in Noni recevied groups. According to TNF‐α and iNOS H‐score results, the values of Noni recevied groups were similar to control. TNF‐α and iNOS H‐Score levels were increased in all PNMC recevied groups. Noni administration showed no effect on TNF‐α H‐Score levels, however in Noni+1 mg/kg PNMC combination group iNOS H‐Score levels were decreased obviously with the effect of Noni (P<0.001). According to these results, it has been shown that oral administration of Noni has positive effects on plasma cell numbers in the spleen of rats which exposed to various doses of PNMC and on iNOS levels in the spleen of 1 mg/kg PNMC administered rats.
Electron interferometry techniques for strain analysis using a multi‐biprism microscope
Authors: Denneulin Thibaud , Röder Falk , Houdellier Florent , Gatel Christophe , Snoeck Etienne , Hÿtch Martin
Published: December 2016

Electron interferometric techniques have progressed in the last years thanks to the development of multiple biprisms microscopes. Here, we will discuss some recent developments in the field of strain measurement carried out with the I2TEM microscope (In‐situ Interferometry Transmission Electron Microscope) installed in Toulouse in 2012. The I2TEM is a Hitachi HF‐3300 equipped with one pre‐specimen electrostatic biprism, three post‐specimen biprisms, an image corrector (CEOS B‐COR for correcting off‐axial aberrations) and two stages (objective stage and Lorentz stage above the objective lens).

In the dark‐field off‐axis scheme [1], an electron beam diffracted by an epitaxially grown region is interfered with a beam diffracted by the substrate thanks to a post‐specimen biprism (Fig. 1(a)).  Fig. 1(b‐d) is an example obtained on a
p
‐MOSFET like transistor with SiGe source/drain. The deformation is recorded as a frequency modulation (FM) in the hologram (Fig. 1(c)) and it can be calculated from the gradient of the reconstructed phase image (Fig 1(d)).

In a recently proposed variant called differential phase contrast dark‐field holography (DPC‐DFEH) [2], a pre‐specimen biprism is used to create two overlapping waves on the sample (Fig. 1(e)). The interference of beams diffracted by slightly distant regions is acquired in a defocused plane. The deformation is recorded as a phase modulation (PM) in the hologram (Fig. 1(f)) and the DPC phase is directly proportional to the deformation (Fig. 1(g)).

Another option is the 4‐wave dark‐field setup where two biprisms oriented perpendicularly are used to interfere three reference waves and one object wave (Fig. 1(h‐j)). The holographic fringes are modulated in amplitude (AM) and each amplitude contour corresponds to a given displacement of the lattice planes [3].  It can provide live information if a sufficient fringe contrast is achieved.

In all cases, strain measurement requires a reference wave diffracted by a region of known lattice parameter (usually the substrate). One solution is the “tilted reference wave” (TRW) where a pre‐specimen biprism and the condenser system are used to create an object‐independent reference wave with an adjustable tilt angle [4]. Fig. 2(a,b) is an example acquired in the vacuum and Fig. 2(c,d) shows the dark‐field configuration for strain measurement.

Finally, a pre‐specimen biprism can also be useful for electron diffraction techniques. For instance, one can create two parallel half cones on the specimen (splitting convergent beam electron diffraction, SCBED) with a controllable distance (Fig. 3(a)) [5]. Each spot in the diffraction pattern contains two lobes related to the regions crossed by the two probes. Fig. 3(b) shows an example of SCBED pattern series where the left and right lobes are related to unstrained and increasingly strained regions respectively.
Recent investigations of small‐scale plasticity mechanisms in 3D and small‐sized systems using advanced in‐situ
TEM
nanomechanical testing
Authors: Idrissi Hosni
Published: December 2016

Recently, the development of a new generation of advanced instruments for in‐situ TEM nanomechanical testing has allowed establishing a one‐to‐one relationship between load‐displacement characteristics and stress‐induced microstructure evolution in the transmission electron microscope (TEM). In the present work, it will be demonstrated that a step forward in the investigation of structural defects and small‐scale plasticity mechanisms can be made by combining commercial [1] and in‐house developed lab‐on‐chip [2] nanomechanical testing techniques with advanced TEM techniques.

High resolution aberration corrected TEM and STEM as well as automated crystallographic orientation and phase mapping in TEM (ACOM‐TEM) have been used to reveal the nanoscale plasticity mechanisms controlling the mechanical responses of nanocrystalline (nc) metallic Al and Pd freestanding thin films. Special attention was paid to the strain rate sensitivity of nanostructured metallic materials involving thermally activated plasticity mechanisms [3] (Figure 1). Furthermore, the microstructure of nc Pd thin films subjected to hydriding cycles has been investigated in order to unravel the interaction mechanisms of hydrogen with dislocations and interfaces [4] as well as the influence of hydrogen cycling on the mechanical properties of the Pd films. An original method combining the measurement of dislocation mobility using in‐situ TEM nanomechanical testing and dislocation dynamic (DD) simulations has also been used to investigate the plasticity of olivine small‐sized crystals at low temperature. It demonstrates for the first time the possibility of characterizing the mechanical properties of specimens, which could be available in the form of micron‐sized samples only [5].

More recently, quantitative nanobeam electron diffraction (NBED) was used to investigate the relationship between the local atomic order and the activation of shear transformation zones (STZs) in nanostructured ZrNi metallic glasses freestanding thin films. These films exhibit outstanding mechanical properties involving very large homogenous plastic deformation and giant ductility without the observation of mature shear bands until fracture. The basic principle of NBED is shown in Figure 2, consisting of a coherent electron beam with diameter of around 0.4 nm in order to produce two‐dimensional diffraction patterns from atomic clusters with comparable size. Furthermore, high resolution HAADF‐STEM and EELS revealed a heterogeneous microstructure with Ni‐rich and Zr‐rich regions exhibiting different atomic densities with characteristic length of 2‐3 nm. The role of such behaviour in the absence of shear bands and the delay of fracture in the ZrNi thin films is discussed.
Next Gen CLEM: super‐accurate correlation and intelligent image acquisition
Authors: Collinson Lucy
Published: December 2016

Correlative light and electron microscopy (CLEM) combines the benefits of fluorescence and electron imaging, revealing protein localisation against the backdrop of cellular architecture. CLEM is usually performed by growing cells on gridded coverslips, imaging the cells (live or fixed) using confocal microscopy, preparing the cells for electron microscopy, relocating the cell position and plane for electron imaging of the fluorescent structure, selecting and modeling the 3D data in both modalities, and overlaying the two datasets to identify the structure of interest. This process usually requires collaboration with expert electron microscopists, and has sufficient steps and complexity to deter many researchers. Nevertheless, for those who commit to the process, there are rich rewards in the understanding of biological processes. Our recent work has focused on improving the speed, accuracy and accessibility of CLEM.

During this development work, it became clear that the technical challenges associated with CLEM are exaggerated when working in 3D. To increase protein localisation precision, we developed an ‘In‐Resin Fluorescence’ (IRF) protocol that preserves the activity of GFP and related fluorophores in resin‐embedded cells and tissues. The sample preparation is relatively fast, and also introduces electron contrast so that cell structure can be visualised in the electron microscope. Once the resin blocks have been cut into ultrathin sections, out‐of‐plane fluorescence is removed resulting in physical ‘super‐resolution’ light microscopy in the axial direction, which increases the accuracy of the LM‐EM overlays. Localisation precision is further increased by imaging the IRF sections
in vacuo
in the next generation of commercial integrated light and electron microscopes (ILEM). We were able to further improve accuracy by developing integrated super‐resolution light and electron microscopy, using the novel and remarkable blinking property of GFP and YFP in‐resin
in vacuo
, and implementing automated 3D imaging for 3D functional and structural analysis of whole cells and tissues at the nanoscale.

With the advent of dual fluorescence‐electron samples comes the challenge of locating and following fluorescent cells during sample preparation and automated 3D EM image acquisition. We present a new locator tool – the miniature light microscope (miniLM), designed to integrate with the ultramicrotome to locate cells during trimming and sectioning, and an even smaller version that fits into the extremely tight space of the 3D SEM vacuum chamber for on‐the‐fly tracking of fluorescent cells during long automated imaging runs.
Quantitative Nanoplasmonics in the
TEM
Authors: Bosman Michel
Published: December 2016

Quantitative Nanoplasmonics in the TEM

Michel Bosman
1

1
Institute of Materials Research and Engineering, A*STAR (Agency for Science, Technology and Research), 2 Fusionopolis Way, #08‐03 Innovis, Singapore 138634
.
michel.bosman@gmail.com

Scanning TEM (STEM)‐based surface plasmon characterization will be discussed in this paper, and it will be argued that some plasmon properties can be measured quantitatively in the STEM, by carefully analyzing the local spectral response. Examples will be given that demonstrate the unique capability of STEM‐based plasmon analysis in comparison with other experimental techniques.

Some very impressive experimental results have recently been published in which plasmons on subwavelength metal nanostructures were mapped with 1‐100 fs time resolution. These include time‐domain techniques based on photoelectron emission microscopy (PEEM) [1, 2], time‐resolved scanning near‐field optical microscopy (SNOM) [3], ultrafast TEM [4] and even plasmon mapping with free electron lasers [5].

STEM‐based electron energy‐loss spectroscopy (EELS) is performed in the energy/frequency domain, and it has a long history as an experimental technique for surface plasmon characterization [6‐17]. However, since surface plasmon resonances are damped harmonic oscillators, it is possible to interpret the EELS plasmon spectra as Fourier‐transforms of oscillations in the time‐domain [18].

This paper will explore the implications of this approach, and will apply it to quantify local dynamic materials properties [19].
Correlative immuno Light Electron Microscopy (
CLEM
) of subcellular compartments
Authors: Klumperman Judith
Published: December 2016

Correlative light and electron microscopy (CLEM) methods integrate light and electron microscopy on a single sample, literally bridging the gap between these two microscopy techniques. Most methods use fluorescent microscopy of thin or semi‐thin sections to define a region‐of‐interest, which then is traced back in the EM to provide subcellular context information (e.g. membrane organization, non‐labeled surroundings of the fluorescent structure). The most powerful application of CLEM is correlative live cell imaging‐EM, by which dynamic information is inferred to structures seen in static EM pictures. By merging the strengths of the two techniques a novel and integrated type of image is created that combines parameters that cannot ‐ or not easily ‐ be obtained when using separate images of related events. However, because imaging requirements are intrinsically different between light and electron microscopy, creating conditions that are ideal for both modalities is a challenging process. Moreover, correlating fluorescent labeling to the cellular architecture seen in the EM is not always straightforward. The most pressing challenges in CLEM are currently therefore 1. development of sample preparation methods that are appropriate for both LM and EM imaging. 2. development of bi‐modal probes that are visible in both LM and EM and 3. development of software that allows for rapid and accurate correlation of LM and EM images.

The focus of our research is to develop new probes and CLEM pipelines that allow us to efficiently and with high accuracy define the three‐dimensional (3D) ultrastructural context of fluorescently‐tagged proteins previously localized in fixed or living cells. We apply our technologies to study the cellular pathways and mechanisms that control the cell's digestive system – i.e. the endo‐lysosomal system ‐ in health and disease conditions. The main CLEM technology that we use in the lab is based on the use of immunogold labeling of ultrathin cryosections (the Tokuyasu technique) [1]. Most CLEM approaches, however, are restricted in their EM approach by the lack of 3D structural information. To overcome this limitation, we apply Focused Ion Beam Scanning Electron Microscopy (FIB‐SEM) as 3D‐EM approach in a live cell‐CLEM set up. To visualize endo‐lysosomes in live cells we combine fluorescent tagged endo‐lysosomal proteins (such as LAMP1‐mGFP) with endocytic tracers (such as fluorescently labeled dextran). This approach enables live‐cell tracking of specific endo‐lysosomal compartments, after which the samples are fixed, stained and resin‐embedded for FIB‐SEM imaging. Figure 1 presents an example of Dextran‐Alexa646 and/or LAMP1‐mGFP labeled endo‐lysosomal compartments in live cells (A) and in 3D‐EM (D), providing the cellular context at ultrastructural resolution. In my presentation I will show various examples of both immunoEM and FIB.SEM‐based CLEM approaches, which are designed to optimally image individual, membrane‐bounded compartments.
In situ
TEM
for understanding electrical and thermal transport properties on nano and atomic scales
Authors: Olsson Eva
Published: December 2016

In situ electron microscopy allows studies of transport of charges and matter in complex structures as well as thermal properties. We can study mechanically and thermally induced changes of charge transport properties using holders designed to enable different stimuli allowing the direct observation and correlation between material structure and properties. The direct correlation between structure and properties on the small scale involving individual interfaces, defects and atoms provides access to new information about which microstructural constituents that are active in determining the material properties on the macro, micro, nano and atomic scale. This talk addresses examples of in situ electrical, mechanical and thermal studies. A few examples are briefly described below.

The nanoscale dimensions of semiconducting nanowires (NWs) provide extended strain relaxation capability between lattice‐mismatched materials and enable the fabrication of single‐NW p‐i‐n junction solar cells on low‐cost substrates. Due to its sub‐wavelength dimension, semiconductor NWs can function as optical antennas and exhibit a “self‐concentrating” effect that enhances optical absorption. The strain relaxation capability and the enhanced absorption cross section make NWs potential candidates as highly efficient and low‐cost solar cells. Due to the effects of  elastic strain applied on the electronic band structure the strain can be used to achieve NW‐based photovoltaic devices with new functionality. We have studied the effect of mechanical strain on the electrical resistance of nanowires. Electron energy loss spectroscopy was used to study the effect of strain on the electronic structure with emphasis on the low energy loss interval of 0 to 50 eV. Electron beam induced current measurements were also performed to study the effect of strain on the diffusion length of the charge carriers [1].

Heating of a transmission electron microscopy (TEM) specimen can be performed in several parallel modes and this talk will address three types of heating modes and show experimental results from nanostructured materials. One mode is by resistive heating of a ring shaped support in contact with the circumference of the entire TEM sample. An additional mode is by use of a heating wire patterned on the TEM sample where the wire is contacted by leads fed through the TEM sample holder. The third mode is by active Joule heating of the nanostructure of study, such as carbon nanotubes, graphene, or metal nanowires. The purpose of having several parallel modes of heating is to enable the separation of temperature dependence, effects of self‐Joule heating, effects of radiative heating and thermal transport. It is also important to be able to extract the three dimensional information about the geometry of the investigated structures [2].
Scanning Probe Microscopy goes Live: seeing dynamic phenomena with
STM
Authors: Frenken Joost
Published: December 2016

Scanning Tunneling Microscopy (STM) and other forms of Scanning Probe Microscopy (SPM), are traditionally applied mainly to static structures that are investigated mainly under relatively artificial conditions, such as ultrahigh vacuum (UHV). This is surprising in the light of the relative insensitivity of the operation mechanism of STM and that of most other types of SPM to such aspects as temperature or gas pressure or even the presence of liquids.

In this talk, I will demonstrate that it is possible to apply SPM techniques without compromising their atomic resolution even under harsh conditions [1‐4]. Extra attention is required to construct the SPM instrumentation such that it avoids the complications that are introduced by these conditions, such as excessive thermal drift or damage to delicate components. This is, in principle, a straightforward engineering task, which leads typically to dedicated designs for specific classes of imaging conditions [1‐4].

The examples provided in this talk are all
live
STM observations of relevant dynamic surface phenomena. They range from model catalysts under the conditions of high temperatures and high pressures at which they are being used in the chemical industry, to the chemical vapor deposition of graphene on metal substrates and the atom‐by‐atom deposition or erosion of surfaces under the influence of atom and ion beams.
Metal Contaminated Environments And Interactions With Biota
Authors: Isaure Marie‐Pierre
Published: December 2016

Contamination of the environment with metals has dramatically increased during the last century due to intense anthropogenic activities and increasing metal need. Living organisms present in soils and aquatic systems are affected by these metals, and some of them are able to develop some specific strategies to cope with metal toxicity. To decipher the impact of metals on these organisms, and particularly their trafficking and storage, the determination of their localization at the tissue and cell level as well as their chemical forms (speciation) are crucial.

In this context, we combine various imaging techniques including synchrotron micro X‐ray Fluorescence (µXRF), Transmission Electron Microscopy combined with Energy Dispersive X‐ray analysis (TEM‐EDX), and Nano‐ Secondary Ion Mass Spectrometry (nano‐SIMS) to locate metals, and X‐ray Absorption Spectroscopy with focused and non focused beam (µXAS and XAS) to clarify the nature of metal species.

This approach will be illustrated by two environmental studies. The first one is focused on the green micro‐algae
Chlamydomonas reinhardtii
, an eukaryotic photosynthetic model for metal stress.
Chlamydomonas reinhardtii
is tolerant to cadmium but the mechanisms involved in this tolerance are misknown. We will show that intracellular thiol ligands are involved in Cd binding at the intracellular level, and that these sulfur ligands are not the only tolerance mechanism implemented by the cell. The second environmental study is related to mining soils contaminated with Cd and Zn. Among the existing soil treatments, phytoremediation has emerged as an alternative sustainable technique for two decades. We will show here how pioneer plants used in a phytostabilization process interact with metals in the soil.

This presentation illustrates the interest of combining imaging and spectroscopy techniques to get a global view of metals in complex environmental systems.
How do plants read their own shape ?
Authors: Hamant Olivier
Published: December 2016

There is accumulating evidence that single cells in culture can sense mechanical cues to control their division, their growth and their fate. In a multicellular context, this implies that a given cell can use growth‐derived stress and shape‐derived stress to control its behavior. In this scenario, the presence of mechanical forces in tissues may thus be a way for the cell to know its position and control its differentiation accordingly, during development. Whereas this question is difficult to explore in animals, the relatively slow growth and simpler mechanics in plants make them ideal systems to investigate the contribution of “positional mechanical signals” in development. To illustrate this idea, I will present the microtubule response to mechanical stress in Arabidopsis at three different scales: at the single cell level, using the jigsaw puzzle shaped pavement cells; at the tissue scale, using the crease‐shape boundary domain of the shoot apical meristem; at the whole organ scale, using the sepal, which exhibits a very reproducible shape despite high variability at the cell level. Beyond the cytoskeleton, mechanical cues are also controlling gene expression and our recent data suggest that the perception of mechanical stress acts in parallel to major hormones, like auxin, to regulate some of the master regulators of the shoot apical meristem. Altogether, this provides a picture in which mechanical forces add robustness to plant morphogenesis, by channeling the dynamics of cell effectors and molecular pathways.
Real‐time subcellular imaging of cancer cell behavior in living mice
Authors: van Rheenen Jacco
Published: December 2016

Although histological techniques have provided important information on epithelial stem cells and cancer, they draw static images of dynamic processes. To study dynamic processes, we have developed various imaging windows to image intestinal, liver and breast tissue, and visualize the behavior of individual cells at subcellular resolution for several weeks with two‐photon intravital microscropy (IVM). Our IVM experiments illustrate that cellular properties and fate of cells are highly dynamic and change over time. For example, we show in healthy and tumorigenic tissues, that cells can acquire and lose stem cell properties, illustrating that stemness is a state as opposed to an intrinsic property of a cell. Moreover, we show that mammary tumor cells that are surrounded by T cells acquire migration properties. An additional aspect that complicates tumor heterogeneity is that cells may exchange active biomolecules through the release and uptake of extracellular vesicles (EVs). Our data shows, in living mice, that malignant tumor cells, through transfer of EVs, enhance the migratory behavior and metastatic capacity of more benign cells. Taken together, these data exemplify that tumor heterogeneity is far more complex than currently anticipated, which has profound consequences for our ideas on the mechanisms of tumor progression and for designing optimal treatment strategies.
mScarlet, a novel high quantum yield (71%) monomeric red fluorescent protein with enhanced properties for
FRET
‐ and super resolution microscopy
Authors: Haarbosch Lindsay , Bindels Daphne , Postma Marten , Hink Mark , Royant Antoine , Gadella Theodorus
Published: December 2016

mScarlet, a novel red fluorescent protein was generated from a synthetic template based on a consensus amino acid sequence derived from naturally occurring red fluorescent proteins and purple chromoproteins and on consensus monomerization mutations. The encoded synthetic red fluorescent protein was optimized by molecular evolution through site directed and random mutagenesis. Improved variants were selected by quantitative multimode screening for increased fluorescence lifetime, increased photo stability, increased quantum yield and for increased chromophore maturation.

Very bright variants were obtained with high fluorescence lifetimes up to 3.8 ns, quantum yields >70 % and complete maturation. The monomeric status of the variants was confirmed by OSER analysis and with a‐tubulin fusions. The brightness of mScarlet is >2‐ fold increased as compared to bright red fluorescent proteins such as mCherry, mRuby2 and tagRFP‐T as was analyzed with quantitative (single plasmid with viral 2A sequence) coexpression with mTurquoise2 in mammalian cells.

mScarlet can be used as a bright red fluorescent fusion tag for staining various subcellular structures in live cells. Because of their efficient maturation and high quantum yield, mScarlet vastly outperforms existing monomeric red fluorescent proteins in ratiometric FRET‐microscopy applications due to seriously enhanced sensitized emission and lack of photochromism (figure 1).

During evolution mScarlet variants with substantially altered spectroscopic properties were generated including fluorescence lifetime variants, photo labile variants and strongly spectrally shifted variants. Besides looking at generally optimized properties such as increased lifetime, maturation and brightness, some of these variants were rescreened for blinking properties. An mScarlet variant (7Q2BMs‐K) was identified that produced high spontaneous blinking upon illumination at 561 nm. This variant was fused to life‐act and co‐expressed with mVenus‐Lifeact. With 488 and 561 nm excitation, dual life cell single molecule localization microscopy produced perfectly colocalized yellow and red actin structures in living cells (figure 2), demonstrating the usefulness of the novel red fluorescent proteins for life cell super‐resolution microscopy.
Eavesdropping on the molecular signatures of embryonic development
Authors: Fraser Scott
Published: December 2016

The study of embryonic development has been dramatically advanced by the wealth of high‐throughput molecular studies that have defined the genes and proteins involved. This wealth of data now presents the challenge of integrating a working knowledge of how these molecular components, often present at vanishingly small concentrations, generate reliable patterns of cell migration and cell differentiation. In typical cell biology approaches, cultures of isolated cells have been used reveal mechanism. What is needed to understand development is to carry out studies on cells in their normal context interacting with other cells and signals in the intact embryo. Key events of embryonic development take place over dimensions of less than 500um in less than 5 hours, making it tractable for light imaging tools to be used to answer this challenge.

Imaging techniques are challenged by major tradeoffs between spatial resolution, temporal resolution, and the limited photon budget. We are attempting to advance this tradeoff by constructing light sheet microscopes that maintain subcellular resolution in thick and scattering specimens. Our two‐photon light‐sheet microscope, combines the deep penetration of two‐photon microscopy and the speed of light sheet microscopy to generate images with more than ten‐fold improved imaging speed and sensitivity. As with other light sheet technologies, the collection of an entire 2‐D optical section in parallel offers dramatically speeds acquisition rates. By adopting two‐photon SPIM is far less subject to light scattering, permitting subcellular resolution to be maintained far better than conventional light sheet microscopes. The combination of attributes permits cell and molecular imaging with sufficient speed and resolution to generate unambiguous tracing of cells and signals in intact systems, which presents a major challenge in data management, processing and analysis.

Multispectral imaging offers the chance of asking multiple questions of the same embodied cells. Multiplex analyses permit the variance and the “noise” in a system to be exploited by asking about the analytes that co‐vary with a selected gene product. Transforming fluorescent spectra to a point on a 2D‐plot with sinusoidal functions (phasors) provides a powerful tool for the cumbersome tasks of visualizing and analyzing hyper‐spectral imaging data. This technique offers excellent performance in the face of biological and instrumental uncertainties. Our results on live zebrafish embryos document the accuracy of hyper‐spectral phasors and demonstrates their capability to distinguish multiple spectrally overlapping fluorophores in low signal‐to‐noise and fast analysis time.

Combined, these tools offer the multi‐dimensional imaging required to follow key events in embryos as they take place, and allow us to use variance as an experimental tool rather than a limitation.
Characterization of the plasma membrane dynamics during cell signaling processes investigated by spot variation fluorescence correlation spectroscopy (
svFCS
)
Authors: Mailfert Sébastien , Hamon Yannick , He Hai‐Tao , Marguet Didier
Published: December 2016

The plasma membrane organization is highly heterogeneous as a result of the intrinsic molecular Brownian agitation and the vast diversity of membrane components. Selective interactions take place in the formation of local complex multicomponent assemblies of lipids and proteins on different time scales. Still, deciphering this lateral organization on living cells and on the appropriate length and temporal scales has been challenging but is crucial to advance our knowledge on the biological function of the plasma membrane.

Among the methodological developments made during the last decade, the spot variation FCS (svFCS), a fluorescent correlation spectroscopy (FCS)‐based method, has allowed the significant progress in the characterization of cell membrane lateral organization at the sub‐optical level, including to providing compelling evidence for the in vivo existence of lipid‐dependent nanodomains (see for review )
1
.This method provides particular insight to identify possible molecular confinement occurring at the plasma membrane
1, 2
. The svFCS is performed by changing the spot of illumination underfilling the back aperture of the objective
3
. Theoretical models have been developed to predict how geometrical constraints such as the presence of adjacent or isolated domains affect the svFCS observations
4, 5
.

We will illustrate how the investigations based on svFCS have provided compelling evidence for the
in vivo
existence of lipid‐dependent nanodomains
2, 6, 7
and have allowed significant progress in the characterization of cell membrane lateral organization for different kind of receptors
8‐10
.
New insights into plasma membrane organization from super‐resolution STED microscopy
Authors: Eggeling Christian
Published: December 2016

Plasma membrane interactions such as the transient protein‐protein or protein‐lipid complexes, the formation of lipid nanodomains (often denoted “rafts”), or diffusional restrictions by the cortical cytoskeleton are considered to play a functional part in a whole range of membrane‐associated processes. However, the direct and non‐invasive observation of such structures in living cells is impeded by the resolution limit of >200nm of a conventional far‐field optical microscope. Here we present the use of the combination of super‐resolution STED microscopy with fluorescence correlation spectroscopy (FCS) for the disclosure of complex nanoscopic dynamical processes. By performing FCS measurements in focal spots tuned to a diameter of down to 30 nm, we have obtained new details of molecular membrane dynamics, such as of transient lipid‐protein interactions and of diffusional restrictions by the cortical cytoskeleton. Further insights will be given for molecular dynamics in the plasma membrane of immune cells, specifically during T‐cell activation.
Liquid Cell Electron Microscopy of Dynamic Phenomena at Solid‐Liquid Interfaces
Authors: Zheng Haimei
Published: December 2016

An understanding of solid‐liquid interfaces at the atomic scale is significant for a variety of processes ranging from materials synthesis to material corrosion and energy conversion and storage. My group focuses on the study of precipitation and dissolution at solid‐liquid interfaces primarily by developing and applying liquid cell transmission electron microscopy (TEM). Besides real time TEM imaging of colloidal nanocrystal growth in a liquid solution, we study precipitation/dissolution at electrode‐electrolyte interfaces with an electrochemical liquid cell. The dynamic phenomena including dendritic growth, solid‐electrolyte interface formation and phase transformation of the precipitates during charge cycles have been monitored in situ. With the assistance of theoretical calculation, depth understanding of the dynamic processes can be achieved.
Possibility to Predict Extrusion Die Incidental Fracture by Finite Element Simulation
Authors: Bevacqua Alessandro , Medvedev Alexander E. , Molotnikov Andrey , Axe Richard , Lapovok Rimma
Published: December 2016

Finite element (FE) simulation of the extrusion process can minimize the time and effort in die design and assist with the optimization of technological parameters for obtaining high quality profiles. However, there are cases when extrusion die fails unexpectedly despite the simulations predicting a positive outcome.

In this paper the incidental failure of the die used by Capral Ltd for extrusion of the T‐shape profile from AA6082 aluminium alloy is analysed. FE simulation complemented with a damage modelling is shown to be valuable tool for establishing the reasons for the die failure and for developing the strategy to prevent such an event.
Facile Stem Cell Delivery to Bone Grafts Enabled by Smart Shape Recovery and Stiffening of Degradable Synthetic Periosteal Membranes
Authors: Zhang Ben , Filion Tera M. , Kutikov Artem B. , Song Jie
Published: December 2016

Delivering stem/progenitor cells via a degradable synthetic membrane to devitalized allogenic tissue graft surfaces presents a promising allograft‐mediated tissue regeneration strategy. However, balancing degradability and bioactivity of the synthetic membrane with physical characteristics demanded for successful clinical translation is challenging. Here, well‐integrated composites of hydroxyapatite (HA) and amphiphilic poly(lactide‐co‐glycolide)‐b‐poly(ethylene glycol)‐b‐poly(lactide‐co‐glycolide) (PELGA) with tunable degradation rates are designed that stiffen upon hydration and exhibit excellent shape recovery ability at body temperature for efficiently delivering skeletal progenitor cells around bone grafts. Unlike conventional degradable polymers that weaken upon wetting, these amphiphilic composites stiffen upon hydration as a result of enhanced polyethylene glycol (PEG) crystallization. HA‐PELGA composite membranes support the attachment, proliferation, and osteogenesis of rat periosteum‐derived cells in vitro, as well as the facile transfer of confluent cell sheets of green fluorescent protein‐labeled bone marrow stromal cells. With efficient shape memory behaviors around physiological temperature, the composite membranes can be programmed with a permanent tubular configuration, deformed into a flat temporary shape desired for cell seeding/cell sheet transfer, and triggered to wrap around a femoral bone allograft upon 37 °C saline rinse and subsequently stiffen. These properties combined make electrospun HA‐PELGA promising smart synthetic periosteal membranes for augmenting allograft healing.
Advanced Biowaste‐Based Flexible Photocatalytic Fuel Cell as a Green Wearable Power Generator
Authors: Lui Gregory , Jiang Gaopeng , Lenos Jared , Lin Edric , Fowler Michael , Yu Aiping , Chen Zhongwei
Published: December 2016

This study designs a wearable power generator from a flexible, photocatalytic fuel cell (fPFC) using various biowaste sources (lactic acid, ethanol, methanol, urea, glycerol, and glucose) as fuel. The fPFC uses light irradiation and the decomposition of biowaste to generate electrical power under both flat and bending (r = 3 cm) conditions. When employed as a sweat band, the fPFC generates a maximum power 4.0 mW cm−2 g−1 from human sweat. The wearable fPFC is able to overcome many of the disadvantages of wearable microbial and enzymatic cells while providing comparable if not superior power density.
Lessons Learnt from Spatially Resolved Electro‐ and Photoluminescence Imaging: Interfacial Delamination in CH3NH3PbI3 Planar Perovskite Solar Cells upon Illumination
Authors: Soufiani Arman Mahboubi , Hameiri Ziv , Meyer Steffen , Lim Sean , Tayebjee Murad Jehangir Yusuf , Yun Jae Sung , Ho‐Baillie Anita , Conibeer Gavin J. , Spiccia Leone , Green Martin A.
Published: December 2016

The influence of illumination on the long‐term performance of planar structured perovskite solar cells (PSCs) is investigated using fast and spatially resolved luminescence imaging. The authors analyze the effect of illuminated current density–voltage (J–V) and light‐soaking measurements on pristine PSCs by providing visual evidence for the spatial inhomogeneous evolution of device performance. Regions that are exposed to light initially produce stronger electroluminescence signals than surrounding unilluminated regions, mainly due to a lower contact resistance and, possibly, higher charge collection efficiency. Over a period of several days, however, these initially illuminated regions appear to degrade more quickly despite the device being stored in a dark, moisture‐ and oxygen‐free environment. Using transmission electron microscopy, this accelerated degradation is attributed to delamination between the perovskite and the titanium dioxide (TiO2) layer. An ion migration mechanism is proposed for this delamination process, which is in accordance with previous current–voltage hysteresis observations. These results provide evidence for the intrinsic instability of CH3NH3PbI3‐based devices under illumination and have major implications for the design of PSCs from the standpoint of long‐term performance and stability.
Mössbauer spectroscopy evidence of intrinsic non‐stoichiometry in iron telluride single crystals
Authors: Kiiamov Airat G. , Lysogorskiy Yury V. , Vagizov Farit G. , Tagirov Lenar R. , Tayurskii Dmitrii A. , Croitori Dorina , Tsurkan Vladimir , Loidl Alois
Published: December 2016

The FeTe parent compound for iron‐superconductor chalcogenides was studied applying Mössbauer spectroscopy accompanied by ab initio calculations of electric field gradients at the iron nuclei. Room‐temperature (RT) Mössbauer spectra of single crystals have shown asymmetric doublet structure commonly ascribed to contributions of over‐stoichiometric iron or impurity phases. Low‐temperature Mössbauer spectra of the magnetically ordered compound could be well described by four hyperfine‐split sextets, although no other foreign phases different from Fe1.05Te were detected by XRD and microanalysis within the sensitivity limits of the equipment. Density functional ab initio calculations have shown that over‐stoichiometric iron atoms significantly affect electron charge and spin density up to the second coordination sphere of the iron sub‐lattice, and, as a result, four non‐equivalent groups of iron atoms are formed by their local environment. The resulting four‐group model consistently describes the angular dependence of the single crystals Mössbauer spectra as well as intensity asymmetry of the doublet absorption lines in powdered samples at RT. We suppose that our approach could be extended to the entire class of FeSeTex compounds, which contain excess iron atoms.

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Polymerase‐Mediated Site‐Specific Incorporation of a Synthetic Fluorescent Isomorphic G Surrogate into RNA
Authors: Li Yao , Fin Andrea , McCoy Lisa , Tor Yitzhak
Published: December 2016

An enzyme‐mediated approach for the assembly of singly modified RNA constructs in which specific G residues are replaced with thG, an emissive isomorphic G surrogate, is reported. Transcription in the presence of thG and native nucleoside triphosphates enforces initiation with the unnatural analogue, yielding 5′‐end modified transcripts that can be mono‐phosphorylated and ligated to provide longer site‐specifically modified RNA constructs. The scope of this unprecedented enzymatic approach to non‐canonical purine‐containing RNAs is explored via the assembly of several altered hammerhead (HH) ribozymes and a singly modified HH substrate. By strategically modifying key positions, a mechanistic insight into the ribozyme‐mediated cleavage is gained. Additionally, the emissive features of the modified nucleoside and its responsiveness to environmental changes can be used to monitor cleavage in real time by steady state fluorescence spectroscopy.
Co‐immobilized Phosphorylated Cofactors and Enzymes as Self‐Sufficient Heterogeneous Biocatalysts for Chemical Processes
Authors: Velasco‐Lozano Susana , Benítez‐Mateos Ana I. , López‐Gallego Fernando
Published: December 2016

Enzyme cofactors play a major role in biocatalysis, as many enzymes require them to catalyze highly valuable reactions in organic synthesis. However, the cofactor recycling is often a hurdle to implement enzymes at the industrial level. The fabrication of heterogeneous biocatalysts co‐immobilizing phosphorylated cofactors (PLP, FAD+, and NAD+) and enzymes onto the same solid material is reported to perform chemical reactions without exogeneous addition of cofactors in aqueous media. In these self‐sufficient heterogeneous biocatalysts, the immobilized enzymes are catalytically active and the immobilized cofactors catalytically available and retained into the solid phase for several reaction cycles. Finally, we have applied a NAD+‐dependent heterogeneous biocatalyst to continuous flow asymmetric reduction of prochiral ketones, thus demonstrating the robustness of this approach for large scale biotransformations.