In-section Click-iT detection and super-resolution CLEM analysis of nucleolar ultrastructure and replication in plants.

Correlative light and electron microscopy (CLEM) is an important tool for the localisation of target molecule(s) and their spatial correlation with the ultrastructural map of subcellular features at the nanometre scale. Adoption of these advanced imaging methods has been limited in plant biology, due to challenges with plant tissue permeability, fluorescence labelling efficiency, indexing of features of interest throughout the complex 3D volume and their re-localization on micrographs of ultrathin cross-sections. Here, we demonstrate an imaging approach based on tissue processing and embedding into methacrylate resin followed by imaging of sections by both, single-molecule localization microscopy and transmission electron microscopy using consecutive CLEM and same-section CLEM correlative workflow. Importantly, we demonstrate that the use of a particular type of embedding resin is not only compatible with single-molecule localization microscopy but shows improvements in the fluorophore blinking behavior relative to the whole-mount approaches. Here, we use a commercially available Click-iT ethynyl-deoxyuridine cell proliferation kit to visualize the DNA replication sites of wild-type Arabidopsis thaliana seedlings, as well as fasciata1 and nucleolin1 plants and apply our in-section CLEM imaging workflow for the analysis of S-phase progression and nucleolar organization in mutant plants with aberrant nucleolar phenotypes.

Open-source microscope add-on for structured illumination microscopy.

Super-resolution techniques expand the abilities of researchers who have the knowledge and resources to either build or purchase a system. This excludes the part of the research community without these capabilities. Here we introduce the openSIM add-on to upgrade existing optical microscopes to Structured Illumination super-resolution Microscopes (SIM). The openSIM is an open-hardware system, designed and documented to be easily duplicated by other laboratories, making super-resolution modality accessible to facilitate innovative research. The add-on approach gives a performance improvement for pre-existing lab equipment without the need to build a completely new system.

Geometric Control of Cell Behavior by Biomolecule Nanodistribution.

Many dynamic interactions within the cell microenvironment modulate cell behavior and cell fate. However, the pathways and mechanisms behind cell-cell or cell-extracellular matrix interactions remain understudied, as they occur at a nanoscale level. Recent progress in nanotechnology allows for mimicking of the microenvironment at nanoscale in vitro; electron-beam lithography (EBL) is currently the most promising technique. Although this nanopatterning technique can generate nanostructures of good quality and resolution, it has resulted, thus far, in the production of only simple shapes (e.g., rectangles) over a relatively small area (100 × 100 µm), leaving its potential in biological applications unfulfilled. Here, we used EBL for cell-interaction studies by coating cell-culture-relevant material with electron-conductive indium tin oxide, which formed nanopatterns of complex nanohexagonal structures over a large area (500 × 500 µm). We confirmed the potential of EBL for use in cell-interaction studies by analyzing specific cell responses toward differentially distributed nanohexagons spaced at 1000, 500, and 250 nm. We found that our optimized technique of EBL with HaloTags enabled the investigation of broad changes to a cell-culture-relevant surface and can provide an understanding of cellular signaling mechanisms at a single-molecule level.

Highly compact and cost-effective 2-beam super-resolution structured illumination microscope based on all-fiber optic components.

Current super-resolution structured illumination microscopes (SR-SIM) utilize relatively expensive electro-optic components and free-space optics, resulting in large setups. Moreover, high power laser sources are required to compensate for the losses associated with generating the illumination pattern by diffractive optics. Here, we present a highly compact and flexible 2D SR-SIM microscope based on all-fiber optic components (fiberSIM). Fiber-splitters deliver the laser light to the sample resulting in the interference illumination pattern. A microelectromechanical systems (MEMS) based fiber switch performs rapid pattern rotation. The pattern phase shift is achieved by the spatial displacement of one arm of the fiber interferometer using a piezoelectric crystal. Compared with existing methods, fiberSIM is highly compact and significantly reduces the SR-SIM component cost while achieving comparable results, thus providing a route to making SR-SIM technology accessible to even more laboratories in the life sciences.

Super-resolution fluorescence microscopy by line-scanning with an unmodified two-photon microscope.

Fluorescence-based microscopy as one of the standard tools in biomedical research benefits more and more from super-resolution methods, which offer enhanced spatial resolution allowing insights into new biological processes. A typical drawback of using these methods is the need for new, complex optical set-ups. This becomes even more significant when using two-photon fluorescence excitation, which offers deep tissue imaging and excellent z-sectioning. We show that the generation of striped-illumination patterns in two-photon laser scanning microscopy can readily be exploited for achieving optical super-resolution and contrast enhancement using open-source image reconstruction software. The special appeal of this approach is that even in the case of a commercial two-photon laser scanning microscope no optomechanical modifications are required to achieve this modality. Modifying the scanning software with a custom-written macro to address the scanning mirrors in combination with rapid intensity switching by an electro-optic modulator is sufficient to accomplish the acquisition of two-photon striped-illumination patterns on an sCMOS camera. We demonstrate and analyse the resulting resolution improvement by applying different recently published image resolution evaluation procedures to the reconstructed filtered widefield and super-resolved images. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'.

Differentiation of neural rosettes from human pluripotent stem cells in vitro is sequentially regulated on a molecular level and accomplished by the mechanism reminiscent of secondary neurulation.

Development of neural tube has been extensively modeled in vitro using human pluripotent stem cells (hPSCs) that are able to form radially organized cellular structures called neural rosettes. While a great amount of research has been done using neural rosettes, studies have only inadequately addressed how rosettes are formed and what the molecular mechanisms and pathways involved in their formation are. Here we address this question by detailed analysis of the expression of pluripotency and differentiation-associated proteins during the early onset of differentiation of hPSCs towards neural rosettes. Additionally, we show that the BMP signaling is likely contributing to the formation of the complex cluster of neural rosettes and its inhibition leads to the altered expression of PAX6, SOX2 and SOX1 proteins and the rosette morphology. Finally, we provide evidence that the mechanism of neural rosettes formation in vitro is reminiscent of the process of secondary neurulation rather than that of primary neurulation in vivo. Since secondary neurulation is a largely unexplored process, its understanding will ultimately assist the development of methods to prevent caudal neural tube defects in humans.

Imaging tissues and cells beyond the diffraction limit with structured illumination microscopy and Bayesian image reconstruction.

Background: Structured illumination microscopy (SIM) is a family of methods in optical fluorescence microscopy that can achieve both optical sectioning and super-resolution effects. SIM is a valuable method for high-resolution imaging of fixed cells or tissues labeled with conventional fluorophores, as well as for imaging the dynamics of live cells expressing fluorescent protein constructs. In SIM, one acquires a set of images with shifting illumination patterns. This set of images is subsequently treated with image analysis algorithms to produce an image with reduced out-of-focus light (optical sectioning) and/or with improved resolution (super-resolution). Findings: Five complete, freely available SIM datasets are presented including raw and analyzed data. We report methods for image acquisition and analysis using open-source software along with examples of the resulting images when processed with different methods. We processed the data using established optical sectioning SIM and super-resolution SIM methods and with newer Bayesian restoration approaches that we are developing. Conclusions: Various methods for SIM data acquisition and processing are actively being developed, but complete raw data from SIM experiments are not typically published. Publically available, high-quality raw data with examples of processed results will aid researchers when developing new methods in SIM. Biologists will also find interest in the high-resolution images of animal tissues and cells we acquired. All of the data were processed with SIMToolbox, an open-source and freely available software solution for SIM.

Quantitative super-resolution single molecule microscopy dataset of YFP-tagged growth factor receptors.

Background: Super-resolution single molecule localization microscopy (SMLM) is a method for achieving resolution beyond the classical limit in optical microscopes (approx. 200 nm laterally). Yellow fluorescent protein (YFP) has been used for super-resolution single molecule localization microscopy, but less frequently than other fluorescent probes. Working with YFP in SMLM is a challenge because a lower number of photons are emitted per molecule compared with organic dyes, which are more commonly used. Publically available experimental data can facilitate development of new data analysis algorithms. Findings: Four complete, freely available single molecule super-resolution microscopy datasets on YFP-tagged growth factor receptors expressed in a human cell line are presented, including both raw and analyzed data. We report methods for sample preparation, for data acquisition, and for data analysis, as well as examples of the acquired images. We also analyzed the SMLM datasets using a different method: super-resolution optical fluctuation imaging (SOFI). The 2 modes of analysis offer complementary information about the sample. A fifth single molecule super-resolution microscopy dataset acquired with the dye Alexa 532 is included for comparison purposes. Conclusions: This dataset has potential for extensive reuse. Complete raw data from SMLM experiments have typically not been published. The YFP data exhibit low signal-to-noise ratios, making data analysis a challenge. These datasets will be useful to investigators developing their own algorithms for SMLM, SOFI, and related methods. The data will also be useful for researchers investigating growth factor receptors such as ErbB3.

A Comprehensive In Vitro Comparison of Preparation Techniques for Fat Grafting.

BACKGROUND: Lipomodeling is a technique that uses the patient's own fat for tissue regeneration and augmentation. The extent of regenerative effect is reported to be determined by the numbers of adipose-derived stem cells and the viability of cells in processed adipose tissue which, together with other factors, influence the degree of graft retention. This study addresses whether differences exist in properties of fat graft obtained by three commonly used techniques. METHODS: Adipose tissue harvested from the hypogastric regions of 14 patients was processed by decantation, centrifugation, and membrane-based tissue filtration. The morphology of each preparation was assessed by electron microscopy and overall cell viability was assessed by live/dead assay. The number of adipose-derived stem cells was determined and their stem cell character was assessed by the presence of cell surface molecules (i.e., CD105, CD90, CD31, and CD45) and by their capacity to differentiate into adipogenic and osteogenic lineages. RESULTS: First, morphologies of processed fat samples obtained by individual procedures differed, but no preparation caused obvious damage to cellular or acellular components. Second, although the highest numbers of adipose-derived stem cells were contained in the upper fraction of centrifuged lipoaspirates, the difference between preparations was marginal. Third, the maximal concentration of adipose fraction (removal of watery component) of lipoaspirate was achieved by membrane-based tissue filtration. Finally, no significant differences in overall viability were detected. CONCLUSIONS: Properties of processed lipoaspirate were influenced by the preparation procedure. However, the differences were not dramatic; both centrifugation and membrane-based filtration are methods of choice whose selection depends on other criteria (e.g., practicality) for individual surgical settings.

Toward structured macroporous hydrogel composites: electron beam-initiated polymerization of layered cryogels.

The ability to tailor mechanical properties and architecture is crucial in creating macroporous hydrogel scaffolds for tissue engineering. In the present work, a technique for the modification of the pore size and stiffness of acrylamide-based cryogels is demonstrated via the regulation of an electron beam irradiation dose. The samples were characterized by equilibrium swelling measurements, light and scanning electron microscopy, mercury porosimetry, Brunauer-Emmett-Teller surface area analysis, and stiffness measurements. Their properties were compared to cryogels prepared by a standard redox-initiated radical polymerization. A (125)I radiolabeled azidopentanoyl-GGGRGDSGGGY-NH2 peptide was bound to the surface to determine the concentration of the adhesive sites available for biomimetic modification. The functionality of the prepared substrates was evaluated by in vitro cultivation of adipose-derived stem cells. Moreover, the feasibility of preparing layered cryogels was demonstrated. This may be the key to the future preparation of complex hydrogel-based scaffolds to mimic the extracellular microenvironment in a wide range of applications.