Highly Stretchable,Biocompatible, Striated Substrate Made from Fugitive Glue

We developed a novel substrate made from fugitive glue (styrenic block copolymer) that can be used to analyze the effects of large strains on biological samples. The substrate has the following attributes: (1) It is easy to make from inexpensive components; (2) It is transparent and can be used in optical microscopy; (3) It is extremely stretchable as it can be stretched up to 700% strain; (4) It can be micro-molded, for example we created micro-ridges that are 6 μm high and 13 μm wide; (5) It is adhesive to biological fibers (we tested fibrin fibers), and can be used to uniformly stretch those fibers; (6) It is non-toxic to cells (we tested human mammary epithelial cells); (7) It can tolerate various salt concentrations up to 5 M NaCl and low (pH 0) and high (pH 14) pH values. Stretching of this extraordinary stretchable substrate is relatively uniform and thus, can be used to test multiple cells or fibers in parallel under the same conditions.     

Electron microscopy of whole cells in liquid with nanometer resolution

Single gold-tagged epidermal growth factor (EGF) molecules bound to cellular EGF receptors of fixed fibroblast cells were imaged in liquid with a scanning transmission electron microscope (STEM). The cells were placed in buffer solution in a microfluidic device with electron transparent windows inside the vacuum of the electron microscope. A spatial resolution of 4 nm and a pixel dwell time of 20 μs were obtained. The liquid layer was sufficiently thick to contain the cells with a thickness of 7 ± 1 μm. The experimental findings are consistent with a theoretical calculation. Liquid STEM is a unique approach for imaging single molecules in whole cells with significantly improved resolution and imaging speed over existing methods.     

Whole-cell,multicolor superresolution imaging using volumetric multifocus microscopy

Single molecule-based superresolution imaging has become an essential tool in modern cell biology. Because of the limited depth of field of optical imaging systems, one of the major challenges in superresolution imaging resides in capturing the 3D nanoscale morphology of the whole cell. Despite many previous attempts to extend the application of photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) techniques into three dimensions, effective localization depths do not typically exceed 1.2 µm. Thus, 3D imaging of whole cells (or even large organelles) still demands sequential acquisition at different axial positions and, therefore, suffers from the combined effects of out-of-focus molecule activation (increased background) and bleaching (loss of detections). Here, we present the use of multifocus microscopy for volumetric multicolor superresolution imaging. By simultaneously imaging nine different focal planes, the multifocus microscope instantaneously captures the distribution of single molecules (either fluorescent proteins or synthetic dyes) throughout an ∼4-µm-deep volume, with lateral and axial localization precisions of ∼20 and 50 nm, respectively. The capabilities of multifocus microscopy to rapidly image the 3D organization of intracellular structures are illustrated by superresolution imaging of the mammalian mitochondrial network and yeast microtubules during cell division.Because of its specificity and ability to image live samples, fluorescence microscopy is the most widely used imaging tool for biological studies. In recent years, several methods have been introduced to increase the resolution of fluorescence microscopy beyond the diffraction limit (1, 2). These methods include stimulated emission depletion (3), structured illumination (4), and single-molecule localization microscopy (LM) (5, 6). In the latter approach, precise control over illumination conditions enables sparse activation of individual fluorescent molecules, permitting determination of their positions with an accuracy of a few tens of nanometers. The sequential photoactivation, imaging, and bleaching (or photoswitching) of large numbers of fluorophores then allow the reconstruction of the investigated structure, embodying the principles of photo-activated localization microscopy (PALM) (5), fluorescence photo-activation localization microscopy (FPALM) (7), and stochastic optical reconstruction microscopy (STORM) (6, 8).LM techniques have garnered significant interest in biological studies (9, 10) but are still predominantly implemented using evanescent-wave (or total internal reflection fluorescence) microscopy (11), which limits its application to 2D or thin structures close to the cell membrane. However, in many biological contexts, it is highly desirable to access the 3D intracellular organization of the cell with subdiffraction resolution.For efficient 3D LM in cultured cells, two issues need to be resolved. First, single molecules must be localized with subdiffraction accuracy both laterally and axially. Second, the axial depth over which localizations are made should be comparable with the thickness of the whole cell. Approaches developed to address the former issue usually rely on encrypting axial information into 2D images by engineering the point-spread function (PSF) of the microscope (8, 12–15), single-photon interferometry (16), or biplane imaging (17–19). However, the typical imaging depth of most of these methods does not exceed 1.2 μm and hence, is insufficient for whole-cell imaging. Furthermore, because of the wide-field excitation configuration predominantly used for imaging in 3D LM, the activation and emission of out-of-focus molecules lead to increased background in the fluorescence image and their unnecessary bleaching (Fig. 1 A and B). In other words, information outside the imaging plane is lost, whereas the signal-to-noise ratio in the image is reduced. Selective plane excitation (20) or activation (21, 22) can circumvent this problem but requires sequential scanning to image entire cell volume.Open in a separate windowFig. 1.Comparison between conventional wide-field detection and multifocus detection. (A and B) The whole volume is excited in the wide-field configuration, and fluorescence from out-of-focus molecules constitutes the background of the recorded in-focus signal. (C and D) In the multifocus configuration, the 3D extent of the PSF is used to localize molecules within the volume as information is obtained from multiple focal planes.We report the successful implementation of a volumetric PALM/STORM superresolution method that avoids the aforementioned problems. Our method relies on the recently developed multifocus microscope (MFM) (23), which achieves simultaneous acquisition of nine equally spaced focal planes (Fig. 1 C and D) on a single camera through the combination of a specialized diffractive grating and chromatic correction elements placed in the microscope emission path (Fig. 2). The spacing between consecutive focal planes is ∼440 nm, allowing accurate 3D localization of single fluorescent molecules with 3D Gaussian fitting of resulting PSFs. We show that such volumetric acquisition is compatible with two-color superresolution PALM/STORM imaging of mammalian and yeast cells, with lateral and axial localization precisions of ∼20 and 50 nm, respectively. The imaging depth is ∼4 µm, well beyond the capabilities of other 3D superresolution techniques (review in ref. 24), and notably, it permits complete 3D imaging of many cellular organelles or whole cells (Movies S1 and S2).Open in a separate windowFig. 2.MFM setup. The excitation lasers are combined in a fiber through an acousto-optic tunable filter, collimated, reflected on a dichroic mirror (DM), and focused at the back aperture of a high-N.A. objective to achieve wide-field excitation. The collected emission is transmitted through the DMs and passes through the MFG placed in a plane conjugated to the back pupil plane of the objective. The diffraction orders pass through the chromatic correction module before being separately focused on the detector. To record sample and stage drift, polystyrene beads (4-μm diameter) were immobilized on the cover glass, illuminated by an infrared light-emitting diode (IR LED), and their diffraction pattern was recorded with an IR camera (IR CAM) through an additional beamsplitter inserted into the emission path. The graphs are the recorded positions of the bead along the x and z axes. (Lower Inset) Example of emitting molecules recorded at different z planes corresponding to labeled nucleopores (the raw data are in Movie S1, and a reconstructed image is in Movie S2). (Scale bar: 5 μm.)     

Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy

We demonstrate how a conventional confocal spinning-disk (CSD) microscope can be converted into a doubly resolving image scanning microscopy (ISM) system without changing any part of its optical or mechanical elements. Making use of the intrinsic properties of a CSD microscope, we illuminate stroboscopically, generating an array of excitation foci that are moved across the sample by varying the phase between stroboscopic excitation and rotation of the spinning disk. ISM then generates an image with nearly doubled resolution. Using conventional fluorophores, we have imaged single nuclear pore complexes in the nuclear membrane and aggregates of GFP-conjugated Tau protein in three dimensions. Multicolor ISM was shown on cytoskeletal-associated structural proteins and on 3D four-color images including MitoTracker and Hoechst staining. The simple adaptation of conventional CSD equipment allows superresolution investigations of a broad variety of cell biological questions.Fluorescence microscopy is an extremely powerful research tool in the life sciences. It combines highest sensitivity with molecular specificity and exceptional image contrast. However, as with all light-based microscopy techniques, its resolution is limited by the diffraction of light to a typical lateral resolution of ∼200 nm and an axial resolution of ∼500 nm (for 500-nm wavelength light). Only recently, this diffraction limit was broken by using the quantum, or nonlinear, character of fluorescence excitation and emission. The first of these superresolution methods was stimulated emission depletion (STED) microscopy (1). Later, methods based on single-molecule localization, such as photoactivated localization microscopy (PALM) (2) and stochastic optical reconstruction microscopy (STORM) (3), joined the field. These methods “break” the diffraction limit because they all use principles that operate beyond the diffraction of light.Although still bound to light diffraction, increased spatial resolution can be achieved in a class of advanced resolution methods that exploit a clever combination of excitation and detection modalities (4–7). Although these methods do not reach the resolution achievable with STED, PALM, STORM, and related techniques, they do not require any specialized labels or high excitation intensities, and they may be applied to any fluorescent sample at any excitation/emission wavelength. The most prominent example of this class is structured illumination microscopy (SIM) (5), in which one scans a sample with a structured illumination pattern while taking images with a wide-field imaging system. Meanwhile, several commercial instruments for SIM have become available. The disadvantages of SIM are its technical complexity, reflected in the rather large cost of the commercially available systems, and its sensitivity to optical imperfections and aberrations, which are unavoidable in biological samples.In a theoretical study in 1988, Sheppard (8) pointed out that it is possible to double the resolution of a scanning confocal microscope in a manner closely related to SIM. In SIM, one starts with a conventional wide-field imaging microscope, and by implementing a scanning structured illumination, one subsequently obtains, after appropriate deconvolution of the recorded images, an image with increased resolution. In image-scanning microscopy (ISM), as proposed by Sheppard, one starts with a conventional confocal microscope that uses a diffraction-limited laser focus for scanning a sample but replaces the point detector typically used for recording the excited fluorescence signal with an imaging detector. Also here, an image with enhanced resolution is obtained after applying an appropriate algorithm to the recorded images.We experimentally realized this idea first in 2010 (4), indeed demonstrating a substantial increase in resolution. The major drawback of this implementation was the slowness of the imaging. At each scan position of the laser focus, an image of the excited region had to be recorded, limiting the scan speed by the frame rate of the imaging camera used. For the small scan area of 2 µm × 2 µm shown with the original ISM setup, data acquisition took 25 s. In 2012, York et al. (6) demonstrated that this limitation may be overcome by using a multifocal excitation scheme. They generated an array of multiple excitation foci by implementing a digital micro-mirror device (DMD) into the excitation path of a wide-field microscope. Using this system, ISM images can be obtained with excellent speed, in two excitation/emission wavelengths (two-color imaging) and in three dimensions. However, this approach requires the incorporation of a DMD with all the necessity of perfect optical alignment.Here, we demonstrate that existing imaging detector-based confocal systems can be converted easily into a doubly resolving ISM system. This mainly includes two kinds of microscopes that are widely available in research laboratories: confocal spinning-disk (CSD) microscopes and rapid laser scanning confocal microscopes with an imaging camera as the detector. We present the results obtained with a CSD system.     

A Novel Micro-Displacement Sensor Based on Double Optical Fiber Probes Made through Photopolymer Materials

In this paper, a novel micro-displacement sensor with double optical fiber probes is proposed and designed, which can realize the highly sensitive sensing of longitudinal or lateral micro-displacements. The optical fiber probes are made through photopolymer formulation, and the effects of reaction time and optical power on the growth length of the probe are illustrated. The relationship between light intensity and longitudinal micro-displacement is a power function in the range of 0–100 μm at room temperature with a correlation coefficient of 98.92%. For lateral micro-displacement, the sensitivity is −2.9697 dBm/μm in the range of 0–6 μm with a linear fit of 99.61%. In addition, the linear correlation coefficient decreases as the initial longitudinal distance increases, and the function of these correlation coefficients is also linear with a linearity of 96.14%. This sensor has a simple manufacturing process, low cost, high sensitivity, and fast response speed. It is suitable for harsh environments such as strong electromagnetic interference and corrosivity, and has a broad application prospect in the field of micro-displacement sensing.     

Lyophilized Polyvinylpyrrolidone Hydrogel for Culture of Human Oral Mucosa Stem Cells

This work shows the synthesis of a polyvinylpyrrolidone (PVP) hydrogel by heat-activated polymerization and explores the production of hydrogels with an open porous network by lyophilisation to allow the three-dimensional culture of human oral mucosa stem cells (hOMSCs). The swollen hydrogel showed a storage modulus similar to oral mucosa and elastic solid rheological behaviour without sol transition. A comprehensive characterization of porosity by scanning electron microscopy, mercury intrusion porosimetry and nano-computed tomography (with spatial resolution below 1 μm) showed that lyophilisation resulted in the heterogeneous incorporation of closed oval-like pores in the hydrogel with broad size distribution (5 to 180 μm, d₅₀ = 65 μm). Human oral mucosa biopsies were used to isolate hOMSCs, expressing typical markers of mesenchymal stem cells in more than 95% of the cell population. Direct contact cytotoxicity assay demonstrated that PVP hydrogel have no negative effect on cell metabolic activity, allowing the culture of hOMSCs with normal fusiform morphology. Pore connectivity should be improved in future to allow cell growth in the bulk of the PVP hydrogel.     

Lensless high-resolution on-chip optofluidic microscopes for Caenorhabditis elegans and cell imaging

Low-cost and high-resolution on-chip microscopes are vital for reducing cost and improving efficiency for modern biomedicine and bioscience. Despite the needs, the conventional microscope design has proven difficult to miniaturize. Here, we report the implementation and application of two high-resolution (≈0.9 μm for the first and ≈0.8 μm for the second), lensless, and fully on-chip microscopes based on the optofluidic microscopy (OFM) method. These systems abandon the conventional microscope design, which requires expensive lenses and large space to magnify images, and instead utilizes microfluidic flow to deliver specimens across array(s) of micrometer-size apertures defined on a metal-coated CMOS sensor to generate direct projection images. The first system utilizes a gravity-driven microfluidic flow for sample scanning and is suited for imaging elongate objects, such as Caenorhabditis elegans; and the second system employs an electrokinetic drive for flow control and is suited for imaging cells and other spherical/ellipsoidal objects. As a demonstration of the OFM for bioscience research, we show that the prototypes can be used to perform automated phenotype characterization of different Caenorhabditis elegans mutant strains, and to image spores and single cellular entities. The optofluidic microscope design, readily fabricable with existing semiconductor and microfluidic technologies, offers low-cost and highly compact imaging solutions. More functionalities, such as on-chip phase and fluorescence imaging, can also be readily adapted into OFM systems. We anticipate that the OFM can significantly address a range of biomedical and bioscience needs, and engender new microscope applications.     

Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue

Multifocal structured illumination microscopy (MSIM) provides a twofold resolution enhancement beyond the diffraction limit at sample depths up to 50 µm, but scattered and out-of-focus light in thick samples degrades MSIM performance. Here we implement MSIM with a microlens array to enable efficient two-photon excitation. Two-photon MSIM gives resolution-doubled images with better sectioning and contrast in thick scattering samples such as Caenorhabditis elegans embryos, Drosophila melanogaster larval salivary glands, and mouse liver tissue.Fluorescence microscopy is an invaluable tool for biologists. Protein distributions in cells have an interesting structure down to the nanometer scale, but features smaller than 200–300 nm are blurred by diffraction in widefield and confocal fluorescence microscopes. Superresolution techniques like photoactivated localization microscopy (1), stochastic optical reconstruction microscopy (2), or stimulated emission depletion (STED) (3) microscopy allow the imaging of details beyond the limit imposed by diffraction, but usually trade acquisition speed or straightforward sample preparation. And although STED can provide resolution down to 40 nm, STED-specific fluorophores are recommended and it often requires light intensities that are orders of magnitude above widefield and confocal microscopy. On the other hand, structured illumination microscopy (SIM) (4) gives twice the resolution of a conventional fluorescence microscope with light intensities on the order of widefield microscopes and can be used with most common fluorophores. SIM uses contributions from both the excitation and emission point spread functions (PSFs) to substantially improve the transverse resolution and is generally performed by illuminating the sample with a set of sharp light patterns and collecting fluorescence on a multipixel detector, followed by image processing to recover superresolution detail from the interaction of the light pattern with the sample. A related technique, image scanning microscopy (ISM), uses a scanned diffraction-limited spot as the light pattern (5, 6). Multifocal SIM (MSIM) parallelizes ISM by using many excitation spots (7), and has been shown to produce optically sectioned images with ∼145-nm lateral and ∼400-nm axial resolution at depths up to ∼50 µm and at ∼1 Hz imaging frequency. In MSIM, images are excited with a multifocal excitation pattern, and the resulting fluorescence in the multiple foci are pinholed, locally scaled, and summed to generate an image [multifocal-excited, pinholed, scaled, and summed (MPSS)] with root 2-improved resolution relative to widefield microscopy, and improved sectioning compared with SIM due to confocal-like pinholing. Deconvolution is applied to recover the final MSIM image which has a full factor of 2 resolution improvement over the diffraction limit.MSIM works well in highly transparent samples (such as zebrafish embryos), but performance degrades in light scattering samples (such as the Caenorhabditis elegans embryo). Imaging in scattering samples can be improved by two-photon microscopy (8) and although the longer excitation wavelength reduces the resolution in nondescanned detection configurations, this can be partially offset by descanned detection and the addition of a confocal pinhole into the emission path. Whereas the nondescanned mode collects the most signal, the addition of a pinhole in the emission path of a point-scanning system can improve resolution when the pinhole is closed (9). In practice this is seldom done for biological specimens because signal-to-noise decays as the pinhole diameter decreases (9–11).SIM is an obvious choice in improving resolution without a dramatic loss in signal-to-noise, but the high photon density needed for efficient two-photon excitation is likely difficult to achieve in the typical widefield SIM configuration. This has led to other methods, such as line scanning (12) to achieve better depth penetration than confocal microscopy and up to twofold improvements in axial resolution (but with only ∼20% gain in lateral resolution). Multiphoton Bessel plane illumination (13) achieved an anisotropic lateral resolution of 180 nm (only in one direction) but requires an instrument design with two objectives in an orthogonal configuration. Cells and embryos can be readily imaged, but the multiaxis design may hinder the intravital imaging of larger specimens. Here, a combination of multiphoton excitation with MSIM is shown to improve both lateral and axial resolutions twofold compared with conventional multiphoton imaging while improving the sectioning and contrast of MSIM in thick, scattering samples.     

Lanthanide near infrared imaging in living cells with Yb3+ nano metal organic frameworks

We have created unique near-infrared (NIR)–emitting nanoscale metal-organic frameworks (nano-MOFs) incorporating a high density of Yb³⁺ lanthanide cations and sensitizers derived from phenylene. We establish here that these nano-MOFs can be incorporated into living cells for NIR imaging. Specifically, we introduce bulk and nano-Yb-phenylenevinylenedicarboxylate-3 (nano-Yb-PVDC-3), a unique MOF based on a PVDC sensitizer-ligand and Yb³⁺ NIR-emitting lanthanide cations. This material has been structurally characterized, its stability in various media has been assessed, and its luminescent properties have been studied. We demonstrate that it is stable in certain specific biological media, does not photobleach, and has an IC₅₀ of 100 μg/mL, which is sufficient to allow live cell imaging. Confocal microscopy and inductively coupled plasma measurements reveal that nano-Yb-PVDC-3 can be internalized by cells with a cytoplasmic localization. Despite its relatively low quantum yield, nano-Yb-PVDC-3 emits a sufficient number of photons per unit volume to serve as a NIR-emitting reporter for imaging living HeLa and NIH 3T3 cells. NIR microscopy allows for highly efficient discrimination between the nano-MOF emission signal and the cellular autofluorescence arising from biological material. This work represents a demonstration of the possibility of using NIR lanthanide emission for biological imaging applications in living cells with single-photon excitation.     

Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex

The signal and resolution during in vivo imaging of the mouse brain is limited by sample-induced optical aberrations. We find that, although the optical aberrations can vary across the sample and increase in magnitude with depth, they remain stable for hours. As a result, two-photon adaptive optics can recover diffraction-limited performance to depths of 450 μm and improve imaging quality over fields of view of hundreds of microns. Adaptive optical correction yielded fivefold signal enhancement for small neuronal structures and a threefold increase in axial resolution. The corrections allowed us to detect smaller neuronal structures at greater contrast and also improve the signal-to-noise ratio during functional Ca²⁺ imaging in single neurons.The ability to visualize biological systems in vivo has been a major attraction of optical microscopy, because studying biological systems as they evolve in their natural, physiological state provides relevant information that in vitro preparations often do not allow (1). However, for conventional optical microscopes to achieve their optimal, diffraction-limited resolution, the specimen needs to have identical optical properties to those of the immersion media for which the microscope objective is designed. For example, one of the most widely applied microscopy techniques for in vivo imaging, two-photon fluorescence microscopy, often uses water-dipping objectives. Because biological samples are comprised of structures (i.e., proteins, nuclear acids, and lipids) with refractive indices different from that of water, they induce optical aberrations to the incoming excitation wave and result in an enlarged focal spot within the sample and a concomitant deterioration of signal and resolution (2, 3). As a result, the resolution and contrast of optical microscopes is compromised in vivo, especially deep in tissue.Many questions related to how the brain processes information on both the neuronal circuit level and the cell biological level can be addressed by observing the morphology and activity of neurons inside a living and, preferably, awake and behaving mouse (1). In a typical experiment, an area of the skull is surgically removed and replaced with a cover glass to provide optical access to the underlying structure of interest (4). For imaging during behavior, the cover glass is often attached to an optically transparent plug embedded in the skull to improve mechanical stability and to prevent the skull from growing back and blocking the optical access (5, 6) (Fig. 1A). Before the excitation light of a two-photon microscope reaches the desired focal plane inside the brain, it has to traverse first the cranial window and then the brain tissue, both with optical properties different from water. Thus, they both impart optical aberrations on the excitation light, which leads to a distorted focus, even at the surface of the brain.Open in a separate windowFig. 1.AO improves imaging quality in vivo: (A) schematic of the geometry for in vivo imaging in the mouse brain, showing the cranial window (green) embedded in the skull (pink) to provide stability to the brain as well as optical access. (B) Lateral and axial images of a 2-μm-diameter bead 170 μm below the brain surface before and after AO correction. (C) Axial signal profiles along the white line in B before and after AO correction. (D) Lateral and axial images of GFP-expressing dendritic processes over a field centered on the bead in A. (E) Axial signal profiles along the white line in D. (F) Measured aberrated wavefront in units of excitation wavelength. (G) Lateral and axial images of GFP-expressing neurons 110 μm below the surface of the brain with and without AO correction. (H) Axial signal profiles along the white line in G. (I) Axial signal profiles along the blue line in G. (J) Aberrated wavefront measured in units of excitation wavelength. (Scale bars: 2 μm in B and 10 μm elsewhere.)These sample-induced aberrations can be corrected with adaptive optics (AO) to recover diffraction-limited resolution. In AO, a wavefront-shaping device modifies the phase of the excitation light before it enters the sample in such a way as to cancel out the phase errors induced by the sample (7). Originally developed for applications in astronomy, the most common AO setup uses a sensor to measure the wavefront after it passes through the aberrating medium (e.g., atmosphere in astronomical AO). This information is then used to control the wavefront-shaping device, which is usually a deformable mirror or a spatial light modulator (SLM) (8). However, this direct wavefront-sensing approach is not suitable for imaging in vivo. For one, it is not possible to place the wavefront sensor past the aberrating medium, which in this case would still be within the brain. Other approaches where the wavefront of the light reflected from the sample is directly measured are limited, because the strong scattering of light in brain tissue scrambles the information in the reflected wavefront (9, 10).Recently, we developed an image-based AO approach that does not require direct wavefront measurement and that is insensitive to sample scattering (11). By comparing images of the sample taken with different segments of the pupil illuminated, the local slope of the wavefront is measured from image shift. The phase offset for each segment is then either measured directly via interference or calculated by using phase reconstruction algorithms similar to those developed for astronomical AO. This pupil-segmentation-based approach as applied to two-photon fluorescence microscopy can recover diffraction-limited performance in both biological and nonbiological samples, including fixed brain slices. The question remains, however, whether the same enhancements can be achieved during two-photon imaging in the intact mouse. Issues that must be addressed include how fast optical aberrations evolve in vivo, what the magnitude and complexity of their spatial variation are, and to what degree adaptive optical correction can improve both the signal and the resolution during morphological and/or functional imaging. Here we answer these questions and demonstrate that we can recover diffraction-limited resolution at a depth of 450 μm in the cortex of the living mouse.     

Uptake of bacterial lipopolysaccharide and expression of tumor necrosis factor-α-mRNA in isolated rat intrahepatic bile duct epithelial cells

AIM: To study the uptake of bacterial lipopolysaccharides (LPS) and expression of tumor necrosis factor α-mRNA (TNF-α-mRNA) with cultured rat intrahepatic bile duct epithelial cells.METHODS: By using fluorescent, immunohistochemical and in situ hybridization techniques, the uptake of Escherichia coli LPS and expression of TNF-α-mRNA with isolated rat intrahepatic bile duct epithelial cells were observed with confocal laser scanning microscopy.RESULTS: Positive reactions to LPS were found in the cytoplasm of isolated intrahepatic bile duct epithelial cells after incubation with LPS for 15 min and the FITC fluorescent intensity against LPS was significantly higher than that of the controls (121.45 μFI/μm² ± 15.62 μFI/μm² vs 32.12 μFI/μm² ± 9.64 μFI/μm², P < 0.01). After incubation with LPS for 3 h, fluorescein isocyanate (FITC) fluorescent intensities of the expression of TNF-α-mRNA with fluorescent in situ hybridization in the cytoplasm and nuclei of the cultured bile duct epithelial cells were significantly higher than those of the controls (189.15 μFI/μm² ± 21.33 μFI/μm² vs 10.00 μFI/μm² ± 8.99 μFI/μm², 64.85 μFI/μm² ± 14.99 μFI/μm² vs 21.20 μFI/μm² ± 2.04 μFI/μm², respectively (P < 0.01)). The increase of FITC fluorescent intensity of TNF-α-mRNA expression in the cytoplasm peaked at 6 h after incubation (221.38 μFI/μm² ± 22.99 μFI/μm²). At various time points after incubation with LPS, the increase of fluorescent intensities of TNF-α-mRNA in the cytoplasm were much higher than those in the nuclei (P < 0.01).CONCLUSION: LPS can act on and enter into isolated intrahepatic bile duct epithelial cells and stimulate the expression of TNF-α-mRNA.     

Widely accessible method for superresolution fluorescence imaging of living systems

Superresolution fluorescence microscopy overcomes the diffraction resolution barrier and allows the molecular intricacies of life to be revealed with greatly enhanced detail. However, many current superresolution techniques still face limitations and their implementation is typically associated with a steep learning curve. Patterned illumination-based superresolution techniques [e.g., stimulated emission depletion (STED), reversible optically-linear fluorescence transitions (RESOLFT), and saturated structured illumination microscopy (SSIM)] require specialized equipment, whereas single-molecule-based approaches [e.g., stochastic optical reconstruction microscopy (STORM), photo-activation localization microscopy (PALM), and fluorescence-PALM (F-PALM)] involve repetitive single-molecule localization, which requires its own set of expertise and is also temporally demanding. Here we present a superresolution fluorescence imaging method, photochromic stochastic optical fluctuation imaging (pcSOFI). In this method, irradiating a reversibly photoswitching fluorescent protein at an appropriate wavelength produces robust single-molecule intensity fluctuations, from which a superresolution picture can be extracted by a statistical analysis of the fluctuations in each pixel as a function of time, as previously demonstrated in SOFI. This method, which uses off-the-shelf equipment, genetically encodable labels, and simple and rapid data acquisition, is capable of providing two- to threefold-enhanced spatial resolution, significant background rejection, markedly improved contrast, and favorable temporal resolution in living cells. Furthermore, both 3D and multicolor imaging are readily achievable. Because of its ease of use and high performance, we anticipate that pcSOFI will prove an attractive approach for superresolution imaging.     

Material Removal Capability and Profile Quality Assessment on Silicon Carbide Micropillar Fabrication with a Femtosecond Laser

Silicon carbide (SiC) has a variety of applications because of its favorable chemical stability and outstanding physical characteristics, such as high hardness and high rigidity. In this study, a femtosecond laser with a spiral scanning radial offset of 5 μm and a spot radius of 6 μm is utilized to process micropillars on a SiC plate. The influence of pulsed laser beam energies and laser translation velocities on the micropillar profiles, dimensions, surface roughness Ra, and material removal capability (MRC) of micropillars was investigated. The processing results indicate that the micropillar has the best perpendicularity, with a micropillar bottom angle of 75.59° under a pulsed beam energy of 50 μJ in the range of 10–70 μJ, with a pulsed repetition rate of 600 kHz and a translation velocity of 0.1 m/s. As the laser translation velocity increases between 0.2 m/s and 1.0 m/s under a fixed pulsed beam energy of 50 μJ and a constant pulsed repetition rate of 600 kHz, the micropillar height decreases from 119.88 μm to 81.79 μm, with the MRC value increasing from 1.998 μm³/μJ to 6.816 μm³/μJ, while the micropillar bottom angle increases from 68.87° to 75.59°, and the Ra value diminishes from 0.836 μm to 0.341 μm. It is suggested that a combination of a higher pulsed laser beam energy with a faster laser translation speed is recommended to achieve micropillars with the same height, as well as an improved processing efficiency and surface finish.     

Ceramic and Composite Polishing Systems for Milled Lithium Disilicate Restorative Materials: A 2D and 3D Comparative In Vitro Study

Purpose: This study aims to evaluate the effectiveness of two ceramic and two composite polishing systems for a novel chairside computer-aided design/computer-aided manufacturing (CAD/CAM) lithium disilicate ceramic with three-dimensional and two-dimensional microscopy images. This ceramic material can be used for implant-supported or tooth-borne single-unit prostheses. Materials and Methods: Sixty flat samples of novel chairside CAD/CAM reinforced lithium disilicate ceramic (Amber Mill, Hass Bio) were divided into five groups (n = 15/group) and treated as follows: Group 1 (NoP), no polished treatment; group 2 (CeDi), polished with ceramic Dialite LD (Brasseler USA); group 3, (CeOp) polished with ceramic OptraFine (Ivoclar Vivadent); group 4, (CoDi) polished with composite DiaComp (Brasseler USA), and group 5 (CoAs), polished with composite Astropol (Ivoclar Vivadent). The polished ceramic surface topography was observed and measured with three-dimensional and two-dimensional images. Results: All polishing systems significantly reduced the surface roughness compared with the non-polished control group (Sa 1.15 μm). Group 2 (CeDi) provided the smoothest surface arithmetical mean eight with 0.32 μm, followed by group 3 (CeOp) with 0.34 μm. Group 5 (CoAs) with 0.52 μm provided the smoothest surface among the composite polishing kits. Group 4 (CoDi) with 0.66 μm provided the least smooth surface among all polishing systems tested. Conclusions: Despite the effectiveness of ceramic polishing systems being superior to composite polishing systems of the CAD/CAM lithium disilicate restorative material, both polishing systems significantly improved the smoothness.     

A Study of Defects in InAs/GaSb Type-II Superlattices Using High-Resolution Reciprocal Space Mapping

In this paper, the study of defects in InAs/GaSb type-II superlattices using high-resolution an x-ray diffraction method as well as scanning (SEM) and transmission (TEM) electron microscopy is presented. The investigated superlattices had 200 (#SL200), 300 (#SL300), and 400 (#SL400) periods and were grown using molecular beam epitaxy. The growth conditions differed only in growth temperature, which was 370 °C for #SL400 and #SL200, and 390 °C for #SL300. A wings-like diffuse scattering was observed in reciprocal space maps of symmetrical (004) GaSb reflection. The micrometer-sized defect conglomerates comprised of stacking faults, and linear dislocations were revealed by the analysis of diffuse scattering intensity in combination with SEM and TEM imaging. The following defect-related parameters were obtained: (1) integrated diffuse scattering intensity of 0.1480 for #SL400, 0.1208 for #SL300, and 0.0882 for #SL200; (2) defect size: (2.5–3) μm × (2.5–3) μm –#SL400 and #SL200, (3.2–3.4) μm × (3.7–3.9) μm –#SL300; (3) defect diameter: ~1.84 μm –#SL400, ~2.45 μm –#SL300 and ~2.01 μm –#SL200; (4) defect density: 1.42 × 10⁶ cm⁻² –#SL400, 1.01 × 10⁶ cm⁻² –#SL300, 0.51 × 10⁶ cm⁻² –#SL200; (5) diameter of stacking faults: 0.14 μm and 0.13 μm for #SL400 and #SL200, 0.30 μm for #SL300.     

Large-Scale Growth of Tubular Aragonite Whiskers through a MgCl2-Assisted Hydrothermal Process

In this paper, we have developed a facile MgCl₂-assissted hydrothermal synthesis route to grow tubular aragonite whiskers on a large scale. The products have been characterized by powder X-ray diffraction (XRD), optical microscopy, and scanning electronic microscopy (SEM). The results show the as-grown product is pure tubular aragonite crystalline whiskers with a diameter of 5–10 μm and a length of 100–200 μm, respectively. The concentration of Mg²⁺ plays an important role in determining the quality and purity of the products. Furthermore, the method can be extended to fabricate CaSO₄ fibers. The high quality of the product and the mild conditions used mean that the present route has good prospects for the growth of inorganic crystalline whiskers.     

Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules

Superresolution imaging techniques based on the precise localization of single molecules, such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), achieve high resolution by fitting images of single fluorescent molecules with a theoretical Gaussian to localize them with a precision on the order of tens of nanometers. PALM/STORM rely on photoactivated proteins or photoswitching dyes, respectively, which makes them technically challenging. We present a simple and practical way of producing point localization-based superresolution images that does not require photoactivatable or photoswitching probes. Called bleaching/blinking assisted localization microscopy (BaLM), the technique relies on the intrinsic bleaching and blinking behaviors characteristic of all commonly used fluorescent probes. To detect single fluorophores, we simply acquire a stream of fluorescence images. Fluorophore bleach or blink-off events are detected by subtracting from each image of the series the subsequent image. Similarly, blink-on events are detected by subtracting from each frame the previous one. After image subtractions, fluorescence emission signals from single fluorophores are identified and the localizations are determined by fitting the fluorescence intensity distribution with a theoretical Gaussian. We also show that BaLM works with a spectrum of fluorescent molecules in the same sample. Thus, BaLM extends single molecule-based superresolution localization to samples labeled with multiple conventional fluorescent probes.     

Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes

Accurate determination of the relative positions of proteins within localized regions of the cell is essential for understanding their biological function. Although fluorescent fusion proteins are targeted with molecular precision, the position of these genetically expressed reporters is usually known only to the resolution of conventional optics ( approximately 200 nm). Here, we report the use of two-color photoactivated localization microscopy (PALM) to determine the ultrastructural relationship between different proteins fused to spectrally distinct photoactivatable fluorescent proteins (PA-FPs). The nonperturbative incorporation of these endogenous tags facilitates an imaging resolution in whole, fixed cells of approximately 20-30 nm at acquisition times of 5-30 min. We apply the technique to image different pairs of proteins assembled in adhesion complexes, the central attachment points between the cytoskeleton and the substrate in migrating cells. For several pairs, we find that proteins that seem colocalized when viewed by conventional optics are resolved as distinct interlocking nano-aggregates when imaged via PALM. The simplicity, minimal invasiveness, resolution, and speed of the technique all suggest its potential to directly visualize molecular interactions within cellular structures at the nanometer scale.     

DNA typing in thirty seconds with a microfabricated device

We report the development of a practical ultrafast allelic profiling assay for the analysis of short tandem repeats (STRs) by using a highly optimized microfluidic electrophoresis device. We have achieved baseline-resolved electrophoretic separations of single-locus STR samples in 30 sec. Analyses of PCR samples containing the four loci CSF1PO, TPOX, THO1, and vWA (abbreviated as CTTv) were performed in less than 2 min. This constitutes a 10- to 100-fold improvement in speed relative to capillary or slab gel systems. The separation device consists of a microfabricated channel 45 μm × 100 μm in cross section and 26 mm in length, filled with a replaceable polyacrylamide matrix operated under denaturing conditions at 50°C. A fluorescently labeled STR ladder was used as an internal standard for allele identification. Samples were prepared by standard procedures and only 4 μl was required for each analysis. The device is capable of repetitive operation and is suitable for automated high-speed and high-throughput applications.     

Active Tactile Sensibility of Brånemark Protocol Prostheses: A Case–Control Clinical Study

Few studies have assessed active tactile sensibility in patients rehabilitated with implants. Improved knowledge about functional tactile sensibility will contribute to several clinical applications, such as protocols for immediate loading, prosthesis design, occlusal improvement in implantology, and physiological integration of implant-supported prostheses. The present study evaluated active tactile sensibility in patients rehabilitated with Brånemark-type mandibular prostheses that impede the total mucosa-supported maxillary prosthesis. Thirty-five subjects participated in this study. The experimental group (n = 18) inclusion criteria were as follows: Brånemark-type prosthesis and a total mucosa-supported maxillary prosthesis. The control group (n = 17) was composed of participants with complete healthy dentition. Carbon foils with different thicknesses (12 μm, 24 μm, 40 μm, 80 μm, and 200 μm) were placed in the premolar region to evaluate the brink of active oral tactile sensibility. The researchers assessed the participants 120 times. After evaluation, we observed a statistical difference (p < 0.05) between the groups. Additionally, the degree of sensibility was found for all thicknesses, except for 12 μm, on both sides. There was a more significant increase in perception in the control group as the carbon thickness increased. The tactile sensibility threshold was 2.5 times greater for participants with prostheses. Thus, the tactile sensibility for mandibular implant-supported and maxillary mucosa-supported prostheses is significantly lower than that of dentate patients, which was detected above the thickness of 80 μm; in patients with natural dentition, different thicknesses were seen starting from 24 μm.