Structured self-assembly of like-charged bridging nanoparticles driven by varying separation between two interfaces.

Wide-field fluorescence microscopy (WFFM) is widely adopted in biomedical studies. However, axial resolution in most WFFM is poor due to the absence of optical-sectioning capability. To achieve wide-field optical-sectioning, several methods have been proposed, most of which need at least two images to reconstruct one optical sectioning image. So, the frame rate of current wide-field optical sectioning microscopy is no more than half of that of conventional WFFM, which may not meet the speed requirement of fast biodynamic studies. We introduce a novel high-speed, wide-field optical sectioning method based on local contrast weighting function and two-dimensional Hilbert-Huang transform, in which only one structured image is required to reconstruct an optical sectioning image. In this way, the loss of temporal resolution in conventional wide-field optical sectioning microscopy is compensated. We validated this method with the imaging of mouse brain slices.

A combination of genetically-encoded calcium indicators and micro-optics has enabled monitoring of large-scale dynamics of neuronal activity from behaving animals. In these studies, wide-field microscopy is often used to visualize neural activity. However, this method lacks optical sectioning capability, and therefore its axial resolution is generally poor. At present, it is unclear whether wide-field microscopy can visualize activity of densely packed small neurons at cellular resolution. To examine the applicability of wide-field microscopy for small-sized neurons, we recorded calcium activity of dentate granule cells having a small soma diameter of approximately 10 micrometers. Using a combination of high numerical aperture (0.8) objective lens and independent component analysis-based image segmentation technique, activity of putative single granule cell activity was separated from wide-field calcium imaging data. The result encourages wider application of wide-field microscopy in in vivo neurophysiology.

Toxoplasma gondii is an intracellular parasite that proliferates within most nucleated cells, an important human pathogen, and a model for the study of human and veterinary parasitic infections. We used a stable yellow fluorescent protein-alpha-tubulin transgenic line to determine the structure of the microtubule cytoskeleton in T. gondii. Imaging of living yellow fluorescent protein-alpha-tubulin parasites by laser-scanning confocal microscopy (LSCM) failed to resolve the 22 subpellicular microtubules characteristic of the parasite cytoskeleton. To understand this result, we analyzed sources of noise in the LSCM and identified illumination fluctuations on time scales from microseconds to hours that introduce significant amounts of noise. We confirmed that weakly fluorescent structures could not be imaged in LSCM by using fluorescent bead standards. By contrast, wide-field microscopy (WFM) did visualize weak fluorescent standards and the individual microtubules of the parasite cytoskeleton. We therefore measured the fluorescence per unit length of microtubule by using WFM and used this information to estimate the tubulin content of the conoid (a structure important for T. gondii infection) and in the mitotic spindle pole. The conoid contains sufficient tubulin for approximately 10 microtubule segments of 0.5-microm length, indicating that tubulin forms the structural core of the organelle. We also show that the T. gondii mitotic spindle contains approximately 1 microtubule per chromosome. This analysis expands the understanding of structures used for invasion and intracellular proliferation by an important human pathogen and shows the advantage of WFM combined with image deconvolution over LSCM for quantitative studies of weakly fluorescent structures in moderately thin living cells.

In order to obtain the internal fine structure of biological tissues and living cells, the microscopic imaging technology is required to be capable of microscopy. In the wide-field fluorescence microscopy with dynamic speckle illumination, a series of dynamically changing speckle patterns are used to illuminate a biological sample in the whole field. The fluorescence sectioning images of sample’s three-dimensional structural are obtained by extracting intensely changing fluorescence signals in the focal plane. In this paper, the process of obtaining fluorescence sectioning images by the fluorescence microscopy is studied by theoretical analysis and simulation. Two main factors affecting the imaging quality of fluorescence sectioning image are analyzed, which are the number of original fluorescence images recorded by CCD and granularity of diffuser. The simulation results indicates that the imaging quality of fluorescence sectioning images first increases and then tends to saturation with the number of original fluorescence images increasing. It first increases and then decreases with the graininess of diffusers increasing. Considering the imaging quality and imaging time, when the number of original fluorescence images is 60 that is used to extract fluorescence sectioning images, and the granularity of diffuser is about 1000, the high spatial resolution fluorescence sectioning images with contrast higher than 85% can be obtained. Theoretical analysis and simulation research provide a theoretical basis and guidance for designing the system structure, implementing and optimizing the wide-field fluorescence microscopy with dynamic speckle illumination.

Optical imaging of voltage indicators based on green fluorescent proteins (FPs) or archaerhodopsin has emerged as a powerful approach for detecting the activity of many individual neurons with high spatial and temporal resolution. Relative to green FP-based voltage indicators, a bright red-shifted FP-based voltage indicator has the intrinsic advantages of lower phototoxicity, lower autofluorescent background, and compatibility with blue-light-excitable channelrhodopsins. Here, we report a bright red fluorescent voltage indicator (fluorescent indicator for voltage imaging red; FlicR1) with properties that are comparable to the best available green indicators. To develop FlicR1, we used directed protein evolution and rational engineering to screen libraries of thousands of variants. FlicR1 faithfully reports single action potentials (∼3% ΔF/F) and tracks electrically driven voltage oscillations at 100 Hz in dissociated Sprague Dawley rat hippocampal neurons in single trial recordings. Furthermore, FlicR1 can be easily imaged with wide-field fluorescence microscopy. We demonstrate that FlicR1 can be used in conjunction with a blue-shifted channelrhodopsin for all-optical electrophysiology, although blue light photoactivation of the FlicR1 chromophore presents a challenge for applications that require spatially overlapping yellow and blue excitation.

The MS2 system has been widely used, in organisms ranging from bacteria to higher eukaryotes, to image single mRNAs in intact cells with high precision. We have recently re-engineered the MS2 system for accurate detection of mRNAs in living Saccharomyces cerevisiae. Previous MS2 systems affected the degradation of the tagged mRNA, which led to accumulation of MS2 fragments and to erroneous conclusions about mRNA localization and expression. Here we describe a step-by-step protocol for the use of our latest MS2 system (MBSV6) for detecting endogenously tagged mRNAs using wide-field fluorescent microscopy in living yeast. The procedure is divided into three stages: tagging of endogenous gene with MBSV6 (~2 weeks), a two-color single-molecule RNA fluorescent in situ hybridization (smFISH) procedure to quantitatively assess whether mRNAs tagged with MS2 and MS2-coat protein (MCP) behave like untagged mRNAs (2 d, plus additional time for quantification), and a procedure to quantify single mRNAs by live imaging using wide-field microscopy (1 d, plus additional time for quantification). With this method it is now possible to interrogate all phases of mRNA expression, from transcription through decay. The described protocol is designed for S. cerevisiae; however, we think that our approach and the considerations discussed here can be extended to Escherichia coli, Drosophila, Caenorhabditis elegans, and mammalian cells.

Background: Multi-color fluorescence microscopy is dependent on the spectral specificity of the dyes and probes used for localization. One of the most commonly used fluorescent DNA dyes is DAPI, which is usually excited by UV light to emit in the blue visible light range. Herein, we describe a pattern of decreasing DAPI fluorescence upon extended UV exposure, closely followed by an increase in emission maxima in the green range.
 Methods: UV-induced photo-conversion of DAPI to green-fluorescing photoproducts was studied on Chinese hamster ovary cells using wide-field fluorescence microscopy, at different UV exposure times and intensities. Imaging was done in repetitive cycles, by alternating between a DAPI and FITC filter cube and following this with a 1 second UV excitation time. The effect of differing UV light intensities on the photo- conversion process was not previously described in the literature.
 Results: Upon image analysis from a large sample of cells, the rate of photo-conversion was shown to be dependent on both the duration of UV excitation and the intensity of the UV light source. Both the process of DAPI depletion and photo-product growth showed biphasic exponential patterns of change. Furthermore, the level of DAPI fluorescence intensity was found to be negatively correlated with the green fluorescence of the photo-product.
 Limitations: This study did not examine the effect of differing mounting media or a variation in DAPI concentration on the rate of DAPI photo-conversion. Also, the exact light dosage to the system was not measured from the 100W Hg bulb. Photo-bleaching of green fluorescence in cells not stained with DAPI was not measured to control for bleaching of endogenous cell molecules.
 Conclusion: Based on our findings, a set of recommendations was formulated in order to help reduce the effects of UV-induced DAPI photo-conversion.

Tracking particles in fluorescent microscopy image is essential and challenging for many biological researches. The existing methods encounter difficulties and challenges for 3D dynamic process observation, especially for high density subcellular particles. In this paper we propose an automatic 3D detection method with wide-field fluorescence microscopy instead of confocal microscopy for high density particles. We employ deconvolution to improve the resolution, while the PSF of wide-field microscopy is measured by taking microscopic fluorescent beads as pointolites. For the complex situations of high density subcellular particles, we apply the matching scaled isotropic undecimated wavelet filtering and a global thresholding to find block candidates that contain at least one particle. Among the blocks we pick out the larger ones and perform an adaptive local thresholding based on 3D watershed. Experiments show that our method is of high accuracy and performs better than others.

Simultaneous coordination-association and electrostatic-repulsion interactions play critical roles in the construction and stabilization of enzymatic function metal centers in water media. These interactions are promising for construction and self-assembly of artificial aqueous polymer single-chain nanoparticles (SCNPs). Herein, the construction and self-assembly of dative-bonded aqueous SCNPs are reported via simultaneous coordination-association and electrostatic-repulsion interactions within single chains of histamine-based hydrophilic block copolymer. The electrostatic-repulsion interactions are tunable through adjusting the imidazolium/imidazole ratio in response to pH, and in situ Cu(II)-coordination leads to the intramolecular association and single-chain collapse in acidic water. SCNPs are stabilized by the electrostatic repulsion of dative-bonded block and steric shielding of nonionic water-soluble block, and have a huge specific surface area of function metal centers accessible to substrates in acidic water. Moreover, SCNPs can assemble into micelles, networks, and large particles programmably in response to the solution pH. These unique media-sensitive phase-transformation behaviors provide a general, facile, and versatile platform for the fabrication of enzyme-inspired smart aqueous catalysts.

We investigate the influence of three common definitions of the solute/solvent dielectric boundary (DB) on the accuracy of the electrostatic solvation energy ΔGel computed within the Poisson Boltzmann and the generalized Born models of implicit solvation. The test structures include small molecules, peptides and small proteins; explicit solvent ΔGel are used as accuracy reference. For common atomic radii sets BONDI, PARSE (and ZAP9 for small molecules) the use of van der Waals (vdW) DB results, on average, in considerably larger errors in ΔGel than the molecular surface (MS) DB. The optimal probe radius ρw for which the MS DB yields the most accurate ΔGel varies considerably between structure types. The solvent accessible surface (SAS) DB becomes optimal at ρw ~ 0.2 Å (exact value is sensitive to the structure and atomic radii), at which point the average accuracy of ΔGel is comparable to that of the MS-based boundary. The geometric equivalence of SAS to vdW surface based on the same atomic radii uniformly increased by ρw gives the corresponding optimal vdW DB. For small molecules, the optimal vdW DB based on BONDI + 0.2 Å radii can yield ΔGel estimates at least as accurate as those based on the optimal MS DB. Also, in small molecules, pairwise charge-charge interactions computed with the optimal vdW DB are virtually equal to those computed with the MS DB, suggesting that in this case the two boundaries are practically equivalent by the electrostatic energy criteria. In structures other than small molecules, the optimal vdW and MS dielectric boundaries are not equivalent: the respective pairwise electrostatic interactions in the presence of solvent can differ by up to 5 kcal/mol for individual atomic pairs in small proteins, even when the total ΔGel are equal. For small proteins, the average decrease in pairwise electrostatic interactions resulting from the switch from optimal MS to optimal vdW DB definition can be mimicked within the MS DB definition by doubling of the solute dielectric constant. However, the use of the higher interior dielectric does not eliminate the large individual deviations between pairwise interactions computed within the two DB definitions. It is argued that while the MS based definition of the dielectric boundary is more physically correct in some types of practical calculations, the choice is not so clear in some other common scenarios.

In this paper we present a novel approach for the analysis of microtubules in wide-field fluorescence microscopy. Microtubules are flexible elongated structures and part of the cytoskeleton, a cytoplasmic scaffolding responsible for cell stability and motility. The method allows for precise measurements of microtubule length and orientation under different conditions despite a high variability of image data and in the presence of artefacts. Application of the proposed method to demonstrate the effect of the protein GAR22 on the rate of polymerisation of microtubules illustrates the potential of our approach.KeywordsSegmentation ResultHigh Dynamic RangeDirectional Cell MotilityMicrotubule LengthRidge DetectorThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

We present a new technique for the correction of optical aberrations in wide-field fluorescence microscopy. Segmented-Pupil Image Interferometry (SPII) uses a liquid crystal spatial light modulator placed in the microscope's pupil plane to split the wavefront originating from a fluorescent object into an array of individual beams. Distortion of the wavefront arising from either system or sample aberrations results in displacement of the images formed from the individual pupil segments. Analysis of image registration allows for the local tilt in the wavefront at each segment to be corrected with respect to a central reference. A second correction step optimizes the image intensity by adjusting the relative phase of each pupil segment through image interferometry. This ensures that constructive interference between all segments is achieved at the image plane. Improvements in image quality are observed when Segmented-Pupil Image Interferometry is applied to correct aberrations arising from the microscope's optical path.

Spatial quantification of hydrogels swelling using wide-field fluorescence microscopy

We present a versatile extended depth-of-field (EDOF) wide-field fluorescence microscopy using a new, to the best of our knowledge, active device, micro-mirror array lens system (MALS) for calibration-free and orientation-insensitive EDOF imaging. The MALS changed the focal plane during image acquisition, and the system could be operated in any orientation. Two EDOF imaging modes of high-speed accumulation and low-speed surface sectioning were implemented. The performance was demonstrated in non-contact imaging of conjunctival goblet cells in live mice and depth-resolved cellular examination of ex-vivo human cancer specimens. MALS-based EDOF microscopy has potential for versatile cellular examination.

Defocused imaging in wide-field fluorescence microscopy provides information on the 3D orientationof the transition dipole moment of single molecules with nanometer spatial resolution. In this chapter,the theoretical background of defocused imaging will be presented, followed by two experimental applications.As a first example, defocused imaging was used along with fluorescence lifetime measurements to provethe fact that the molecular orientation of dye molecules in a thin film has a significant affecton their fluorescence lifetime. This is attributed to the electromagnetic boundary condition effect. Asa second example, the power of the technique for following 3D molecular rotational reorientation (molecularrotational diffusion) in thin polymer films is demonstrated. Since many molecules can be monitored in parallel,both the temporal and the spatial heterogeneity of polymer dynamics can be addressed.