Differential Interference Contrast Research Articles

Impact of α-crystallin protein loss on zebrafish lens development

The α-crystallin small heat shock proteins contribute to the transparency and refractive properties of the vertebrate eye lens and prevent the protein aggregation that would otherwise produce lens cataracts, the leading cause of human blindness. There are conflicting data in the literature as to what role the α-crystallins may play in early lens development. In this study, we used CRISPR gene editing to produce zebrafish lines with mutations in each of the three α-crystallin genes (cryaa, cryaba and cryabb) to prevent protein production. The absence of each α-crystallin protein was analyzed by mass spectrometry, and lens phenotypes were assessed with differential interference contrast microscopy and histology. Loss of αA-crystallin produced a variety of lens defects with varying severity in larvae at 3 and 4 dpf but little substantial change in normal fiber cell denucleation. Loss of αBa-crystallin produced no substantial lens defects. Our cryabb mutant produced a truncated αBb-crystallin protein and showed no substantial change in lens development. Mutation of each α-crystallin gene did not alter the mRNA levels of the remaining two, suggesting a lack of genetic compensation. These data suggest that αA-crystallin plays some role in lens development, but the range of phenotype severity in null mutants indicates its loss simply increases the chance for defects and that the protein is not essential. Our finding that cryaba and cryabb mutants lack noticeable lens defects is congruent with insubstantial transcript levels for these genes in lens epithelial and fiber cells through five days of development. Future experiments can explore the molecular mechanisms leading to lens defects in cryaa null mutants and the impact of αA-crystallin loss during zebrafish lens aging.

Fused in sarcoma undergoes cold denaturation: Implications for phase separation.

The mediation of liquid-liquid phase separation (LLPS) for fused in sarcoma (FUS) protein is generally attributed to the low-complexity, disordered domains and is enhanced at low temperature. The role of FUS folded domains on the LLPS process remains relatively unknown since most studies are mainly based on fragmented FUS domains. Here, we investigate the effect of metabolites on full-length (FL) FUS LLPS using turbidity assays and differential interference contrast (DIC) microscopy, and explore the behavior of the folded domains by nuclear magnetic resonance (NMR) spectroscopy. FL FUS LLPS is maximal at low concentrations of glucose and glutamate, moderate concentrations of NaCl, Zn2+ , and Ca2+ and at the isoelectric pH. The FUS RNA recognition motif (RRM) and zinc-finger (ZnF) domains are found to undergo cold denaturation above 0°C at a temperature that is determined by the conformational stability of the ZnF domain. Cold unfolding exposes buried nonpolar residues that can participate in LLPS-promoting hydrophobic interactions. Therefore, these findings constitute the first evidence that FUS globular domains may have an active role in LLPS under cold stress conditions and in the assembly of stress granules, providing further insight into the environmental regulation of LLPS.

Minimizing the Optical Illusion of Nanoparticles in Single Cells Using Four-Dimensional Cuboid Multiangle Illumination-Based Light-Sheet Super-Resolution Imaging.

Although light-sheet-based super-resolution microscopy is an excellent detection technique for biological samples because of minimal photodamage, uneven light paths due to solid-angle illumination limits it, resulting in an optical illusion. Furthermore, the optical illusion limits the observations of individual molecules in diffraction. In this study, a four-dimensional cuboid multiangle illumination-based light-sheet super-resolution (4D CMLS) imaging system was developed to minimize optical illusions in cells. The lab-built 4D CMLS imaging system was integrated with total internal reflection fluorescence and a differential interference contrast microscope. A specially designed rotatable cuboid prism simply overcame the optical illusion by rotating a specimen on the prism to change the direction of light coming from an illumination lens. 4D CMLS reconstructed images of nanoparticles of different sizes were acquired in multi-illumination angles of 0°, 90°, 180°, and 270°. Additionally, a 4D multiangle illumination-based algorithm was created to select the optimal illumination angle by combining three-dimensional super-resolution imaging with multiangle observation, even in the presence of obstacles. The 4D CMLS imaging method demonstrates the in-depth 4D observation of samples at an optimum angle that can be used in various applications, such as single-molecule and subcellular organelle observations in single cells at subdiffraction limit resolutions that describe the scenario of nature.

3D differential interference contrast microscopy using polarisation-sensitive tomographic diffraction microscopy.

Tomographic diffraction microscopy (TDM) is a generalisation of digital holographic microscopy (DHM), for which the illumination angle onto the sample is fully controlled, which has become a tool of choice for 3D, high-resolution imaging of unlabelled samples. TDM makes it possible to obtain the optical field in both amplitude and phase for each illumination angle. Proper information reallocation eventually allows for 3D reconstruction of the complex refractive index map. On the other hand, polarisation array sensors (PAS) paves new way for TDM, as vectorial information assessment about the investigated sample. In this contribution, we show an alternative use of this polarisation information based on the phase sensitive nature of TDM. Here, we demonstrated that TDM coupled with PAS can lead to a 3D differential interference contrast (DIC) microscope with almost no experimental configurationmodification.

Morphological and Molecular Characterization of Myxobolus planilizae n. sp. (Cnidaria; Myxosporea; Myxobolidae) Infecting the Largescale Mullet Planiliza macrolepis (Smith, 1846) Collected From Cochin Backwaters, India.

Myxobolus planilizae n. sp. is described from the intestinal muscles of the largescale mullet Planiliza macrolepis from Cochin backwaters, Kerala, India. Host fishes inhabiting Cochin backwaters were collected using Chinese nets/gill nets. The morphometry and morphological studies were carried out using Nomarski differential interference contrast (DIC) optics, followed by molecular and phylogenetic analyses of the small subunit ribosomal DNA gene (SSU rDNA). Plasmodia small, pale white, and infect the muscles of the intestine; measured 0.13-0.22 (0.17) × 0.09-0.14 (0.13) mm. Mature myxospores pyriform in valvular view, and biconvex in sutural and apical views with a short anterior extension, and measured 7.45-8.75 (8.40) × 6.04-6.86 (6.25) µm. Shell valves with sutural ornamentations. Polar capsules two, equal, pyriform, measured 3.96-4.54 (4.45) × 2.22-2.94 (2.52) µm. Polar filament arranged in five coils, measured 24.41-34.44 (28.52) µm when extruded. In morphological and morphometric analysis, the present species exhibit remarkable variations from other species of the genus Myxobolus. In molecular analysis, the present species revealed the highest identity of 91.85% and divergence of 9.95% with related species, underlining its molecular uniqueness. In phylogenetic analysis, species of Myxobolus infecting mullets appeared as a separate clade and the present species was positioned distinctly with a high bootstrap value. Based on morphology, morphometry, and molecular and phylogenetic analyses, along with tissue/host specificities and geographic location, the present parasite is treated as new and is reported here as M. planilizae n. sp.

Evaluating the environmental microbiota across four National Health Service hospitals within England

Hospital surfaces contaminated with microbial soiling such as dry surface biofilms (DSBs) can act as a reservoir for pathogenic microorganisms and inhibit their detection and removal during routine cleaning. Studies have recognised such increases in bioburden can hinder the impact of disinfectants and mask the detection of potential pathogens. Cleanliness within healthcare settings is often determined through routine culture-based analysis, whereby surfaces that exhibit > 2.5 colony forming units (CFU) per cm2 pose a risk to patient health and therefore, any underestimation could have detrimental effects. In this study, we quantified the microbial growth on high-touch surfaces in four hospitals within England over 19 months. This was achieved using environmental swabs to sample a variety of surfaces within close proximity to the patient and plating these onto non-specific low nutrient detection agar. The presence of DSBs were confirmed, on surfaces physically removed from the environment, using real-time imaging through episcopic differential interference contrast microscopy combined with epifluorescence. Approximately two-thirds of surfaces tested exceeded the limit for cleanliness (median: 2230 CFU/cm2) whilst 83% of surfaces imaged with BacLight™ LIVE/DEAD™ staining confirmed traces of biofilm. Despite the differences in infection control methods such as choice of surface disinfectants and cleaning personnel, this was not reflected in the microbial variation observed and resulting risk to patients. This highlights a potential limitation in the effectiveness of the current standards for all hospital cleaning and further development using representative clinical data is required to overcome this limitation.

Cetirizine more potently exerts mast cell-stabilizing property than diphenhydramine.

Cetirizine, a second-generation antihistamine, and diphenhydramine, a first-generation antihistamine, are among the most widely used anti-allergic drugs. In addition to longer duration of action and less incidence of sedative side effects, recent clinical studies also indicate a higher potency of cetirizine than diphenhydramine in the treatment or prevention of allergic disorders. In the present study, using the differential-interference contrast (DIC) microscopy, we examined the effects of cetirizine and diphenhydramine (1 μM to 1 mM) on the degranulation from rat peritoneal mast cells. Using fluorescence imaging of a water-soluble dye, lucifer yellow, we also examined their effects on the deformation of the plasma membrane. At relatively higher concentrations (100 μM, 1 mM), both cetirizine and diphenhydramine significantly reduced the numbers of degranulating mast cells. Of note, at 1 mM, cetirizine more markedly reduced the number than diphenhydramine, almost entirely suppressing the degranulation of mast cells. Additionally, 1 mM cetirizine and levocetirizine, another second-generation antihistamine, almost totally inhibited the process of exocytosis in mast cells and washed out the trapping of the lucifer yellow on the cell surface, while diphenhydramine and chlorpheniramine, another first-generation antihistamine, did not. This study provided in vitro evidence for the first time that cetirizine more potently inhibited the process of exocytosis in mast cells than diphenhydramine, indicating its higher potency as a mast cell-stabilizer. Such mast cell-stabilizing property of cetirizine could be ascribed to its counteracting effect on the plasma membrane deformation in degranulating mast cells.

A huge undescribed diversity of the subgenus Hystricochaetonotus (Gastrotricha, Chaetonotidae, Chaetonotus) in Central Europe

The subgenus Hystricochaetonotus Schwank, 1990 is one of the most species-rich subgenera of Chaetonotus Ehrenberg, 1830. It has a worldwide distribution and encompasses 37 species predominantly living in the benthos and periphyton of limnetic habitats. We have discovered further nine new species in running and stagnant waters in Slovakia (Central Europe): Ch. (H.) arcanus sp. nov., Ch. (H.) avarus sp. nov., Ch. (H.) gulosus sp. nov., Ch. (H.) iratus sp. nov., Ch. (H.) luxus sp. nov., Ch. (H.) mirabilis sp. nov., Ch. (H.) optabilis sp. nov., Ch. (H.) slavicus sp. nov., and Ch. (H.) superbus sp. nov. Their morphology was studied using differential interference contrast microscopy and subsequent morphometric analyses were carried out. In addition, the primary and secondary structures of their 18S, ITS2, and 28S rRNA molecules as well as their barcoding mitochondrial gene encoding for cytochrome c oxidase (COI) were analyzed. Species boundaries were tested also using the compensatory base change analysis. The new species could be well separated both morphologically and molecularly. The present barcoding analyses revealed that the nuclear ITS2 sequences represent a powerful DNA barcode in addition to the mitochondrial COI gene. According to the multi-gene phylogenetic analyses, the lineage leading to the last common ancestor of the ‘Hystricochaetonotus’ clade is the longest internal branch within the family Chaetonotidae Gosse, 1864. Since members of the subgenus Hystricochaetonotus are morphologically highly heterogeneous, parallel evolution of Chaetonotus-like and/or Hystricochaetonotus-like characters of scales and spines occurred during its radiation.

(Invited) Growth of Power-Grade GaN Substrates By the Near Equilibrium Ammonothermal (NEAT) Method and Expansion of the Size to 100 Mm

[Invited Paper]This paper reviews the growth of power-grade GaN crystals by the near-equilibrium ammonothermal (NEAT) method and reports the evaluation of a 4" bulk GaN crystal. Ammonothermal growth is a proven method of producing low-dislocation, GaN crystals with practical productivity. [1, 2] By choosing a near-equilibrium growth condition, consistent growth with maintained crystal quality has been achieved. [3] As the development of GaN-based power devices shifts from GaN-on-Si horizontal architecture to GaN-on-GaN vertical architecture, the development of low-cost, large-area, low-dislocation, pit-free GaN substrates has become an urgent agenda. SixPoint started the development of 4" power-grade GaN substrates with reduced dislocation density to meet the increasing demand of GaN substrates for the development of power devices.Bulk GaN crystals are grown on HVPE-grown GaN substrates (HVPE seeds) by the NEAT method. The Ga-face of HVPE seed is covered so that growth only occurs on the N-face. In one batch of growth, many seed crystals, including 2" and 4" sizes, are loaded in a high-pressure autoclave. The details of the growth conditions are found elsewhere [4]. After growth, the crystals are sliced into wafers and polished to be "epi-ready" substrates.A 4" bulk GaN was grown on a 4" HVPE seed. The grown crystal was evaluated with Nomarski microscopy, mapping of full-width half maximums (FWHMs) of 002 and 201 X-ray diffraction rocking curves (X-ray mapping), and X-ray topography. To evaluate the dislocation density of the grown bulk crystal, X-ray topography was measured on the top as-grown N-face and the exposed Ga-face of the HVPE-seed (i.e., the backside of the bulk crystal).Both X-ray mapping and X-ray topography showed improved microstructure quality. The dislocation density measured on the as-grown N-face was 2-5 x 105 cm-2, whereas that measured on the Ga-face of the HVPE seed was too high to be separated in the topography image. The estimated dislocation density of the HVPE seed was at the order of 106 cm-2. In addition, the grown 4" bulk crystal does not have any macro defects (i.e., pits and holes). The observed reduction of dislocation density in the growth of the 4" crystal was similar to what was seen in the growth of 2" crystals. A typical dislocation density of 2" NEAT GaN substrates is low-105 cm-2. The result was quite encouraging to apply the NEAT method to the production of large-are power-grade GaN substrates.

Photonic Spin-Hall Differential Microscopy

The visualization of objects such as transparent living cells and tissues plays an important role in biological research. However, weak scattering and absorption of ``phase objects'' make it challenging to obtain high-contrast images, and phase-contrast techniques are needed. This study proposes differential microscopy based on the photonic spin Hall effect at a simple glass interface. The combination of the photonic spin Hall effect and a bright-field microscope can perform spatial differentiation on the phase distribution, which leads to a low-cost differential interference contrast (DIC) system.

O-Net: A Fast and Precise Deep-Learning Architecture for Computational Super-Resolved Phase-Modulated Optical Microscopy.

We present a fast and precise deep-learning architecture, which we term O-Net, for obtaining super-resolved images from conventional phase-modulated optical microscopical techniques, such as phase-contrast microscopy and differential interference contrast microscopy. O-Net represents a novel deep convolutional neural network that can be trained on both simulated and experimental data, the latter of which is being demonstrated in the present context. The present study demonstrates the ability of the proposed method to achieve super-resolved images even under poor signal-to-noise ratios and does not require prior information on the point spread function or optical character of the system. Moreover, unlike previous state-of-the-art deep neural networks (such as U-Nets), the O-Net architecture seemingly demonstrates an immunity to network hallucination, a commonly cited issue caused by network overfitting when U-Nets are employed. Models derived from the proposed O-Net architecture are validated through empirical comparison with a similar sample imaged via scanning electron microscopy (SEM) and are found to generate ultra-resolved images which came close to that of the actual SEM micrograph.

Liquid to solid transition of elastin condensates

Liquid-liquid phase separation of tropoelastin has long been considered to be an important early step in the complex process of elastin fiber assembly in the body and has inspired the development of elastin-like peptides with a wide range of industrial and biomedical applications. Despite decades of study, the material state of the condensed liquid phase of elastin and its subsequent maturation remain poorly understood. Here, using a model minielastin that mimics the alternating domain structure of full-length tropoelastin, we examine the elastin liquid phase. We combine differential interference contrast (DIC), fluorescence, and scanning electron microscopy with particle-tracking microrheology to resolve the material transition occurring within elastin liquids over time in the absence of exogenous cross-linking. We find that this transition is accompanied by an intermediate stage marked by the coexistence of insoluble solid and dynamic liquid phases giving rise to significant spatial heterogeneities in material properties. We further demonstrate that varying the length of the terminal hydrophobic domains of minielastins can tune the maturation process. This work not only resolves an important step in the hierarchical assembly process of elastogenesis but further contributes mechanistic insight into the diverse repertoire of protein condensate maturation pathways with emerging importance across biology.

Fourier Ptychography Part II: Phase Retrieval and High-Resolution Image Formation

Abstract: This article is the second within a three-part series on Fourier ptychography, which is a computational microscopy technique for high-resolution, large field-of-view imaging. While the first article laid out the basics of Fourier ptychography, this second part sheds light on its algorithmic ingredients. We present a non-technical discussion of phase retrieval, which allows for the synthesis of high-resolution images from a sequence of low-resolution raw data. Fourier ptychographic phase retrieval can be carried out on standard, widefield microscopy platforms with the simple addition of a low-cost LED array, thus offering a convenient alternative to other phase-sensitive techniques that require more elaborate hardware such as differential interference contrast and digital holography.

An apparatus for qualitative assessment of the shading ratio of oblique illumination and real-time high-contrast imaging.

Oblique illumination imaging can significantly improve the contrast of transparent thin samples. However, in traditional oblique illumination methods, either the condenser is offset or a block is added to the condenser, which makes it complicated and challenged to build a stable oblique illumination imaging. Herein, we present a method to measure the optimal shading ratio of oblique illumination in an inverted microscope, and develop an apparatus for stable high-speed high-contrast imaging with uniform brightness. At optimal shading ratio, the oblique illumination imaging has better imaging quality than differential interference contrast, which characteristic is independent on sample. In oblique illumination with low magnification objective, the images have uneven brightness. According to target brightness, we have developed a brightness unevenness correction algorithm to form uniform background brightness for oblique illumination. Integrating the algorithm with imaging acquisition, corrected oblique illumination microscopy is appropriate to observe living cells with high contrast.

Extracellular matrix regulation of stress response genes during larval development in Caenorhabditis elegans.

Mutation or loss of 6 extracellular matrix collagen genes disrupts annular furrows in adult C. elegans cuticles, causes a wide "Dumpy" body morphology, and activates osmotic, detoxification, and antimicrobial defense genes. High environmental osmolarity reduces internal turgor pressure, physically distorts the epidermis, and activates the same stress responses. Collagen gene mutations that cause Dumpy without furrow disruption do not activate stress responses. These results are consistent with an extracellular damage sensor associated with furrows in the adult cuticle that regulates environmental stress responses in adjacent cells. Several cuticle characteristics change between molts, but all stages have annular furrows and express furrow collagen genes. We compared body shape, furrow organization imaged with differential interference contrast microscopy, and stress response gene expression in furrow collagen gene mutants at all postembryonic stages. We find that most body shape and furrow disorganization phenotypes start at the L3 stage and increase in severity with each molt afterwards. Stress response genes were induced the strongest in adults, correlating with the greatest Dumpy and furrow phenotypes. Although weaker than in adults, osmolyte transporter gene hmit-1.1 and antimicrobial gene nlp-29 were also induced in some early larvae that had weak or undetectable cuticle phenotypes. Our data are consistent with progressive cuticle phenotypes in which each new cuticle is at least partially directed by organization of the former cuticle. Gene expression and cuticle data support the role of furrow disruption as a signal in L4 larvae and adults, but also suggest a role for other cuticle organization or epidermal cell effects in early larvae.

Strongyloides spp. eliminate male-determining sperm post-meiotically

If normal male meiosis occurs, it would be expected that 50 % of sperm lack an X chromosome (nullo X) and hence upon fertilisation, result in male progeny. However, for sexual reproduction within the free-living stages of Strongyloides spp. male offspring are absent. We had shown earlier by quantitative whole genome sequencing that within Strongyloides spp., nullo-X sperm are either absent (S. papillosus) or underrepresented (S. ratti) among mature sperm. To investigate how and when this elimination of male-determining sperm occurs, we characterised spermatogenesis and the dynamic localisation of important molecular players such as tubulin, actin and major sperm protein by DIC microscopy, immunohistochemistry, and fluorescent in situ hybridization (FISH) in S. ratti, S. papillosus and Parastrongyloides trichosuri. We found that meiotic divisions in these parasites proceeded as expected for organisms with XO males, resulting in four equally sized spermatocytes, two with and two without an X chromosome. However, mature sperm were found to almost always contain an X chromosome. We also observed structures that contained protein constituents of sperm, such as actin and major sperm protein (MSP) but no DNA. These structures resemble C. elegans residual bodies in appearance and may assume their function. We hypothesize that spermatocytes without an X-chromosome undergo some form of programmed cell death and transform into these residual body-like structures. As in C. elegans, MSP is found in fibrous body-membranous organelles (FB-MOs). Knocking down MSP by RNAi showed that MSP is essential for fertility in S. ratti, as it is in C. elegans.

Computing Metasurfaces Enabled Broad-Band Vectorial Differential Interference Contrast Microscopy

Differential interference contrast (DIC) microscopy provides the ability to visualize transparent phase samples such as living cells and tissues, which plays an essential role in applications in modern biology and medicine. However, existing DIC microscopy requires the introduction of complicated phase-contrast devices and is still limited to one-dimensional imaging. To mitigate these challenges, we propose a two-dimensional (2D) vectorial DIC microscopy based on computing metasurfaces to extract the 2D edge information for weakly scattering cells. Here, the Wollaston prism in a conventional DIC microscope is replaced by the computing metasurface, enabling our vectorial DIC microscope system to be miniaturized because of its compactness and multifunctionality. Imaging experiments with phase samples verify the capability to clearly observe a 2D vectorial DIC microscopy image with high contrast and its application in biological imaging. Particularly, because the designed computing metasurface is based on the Pancharatnam–Berry phase, our scheme may provide real-time, broad-band, and high-contrast phase imaging of living biomedical systems.

Inspired By Shinya Inoue: Quantitative Orientation-Independent Differential Interference Contrast Microscopy

Inspired By Shinya Inoue: Quantitative Orientation-Independent Differential Interference Contrast Microscopy

Contamination-induced inhomogeneity of noise sources distribution in Al2O3-passivated quasi-free-standing graphene on 4H-SiC(0001)

In this report, we introduce a novel method based on low-frequency noise analysis for the assessment of quality and pattern of inhomogeneity in intentionally-aged Hall effect sensors featuring hydrogen-intercalated quasi-free-standing epitaxial Chemical Vapor Deposition graphene mesa on semi-insulating high-purity on-axis 4H-SiC(0001), all passivated with a 100-nm-thick atomic-layer-deposited Al2O3 layer. Inferring from the comparison of the measured noise and one calculated for a homogeneous sensor, we hypothesize about possible unintentional contamination of the sensors’ active regions. Following in-depth structural characterization based on Nomarski interference contrast optical imaging, confocal micro-Raman spectroscopy, high-resolution Transmission Electron Microscopy and Secondary Ion Mass Spectrometry, we find out that the graphene’s quasi-free-standing character and p-type conductance make the Al2O3/graphene interface exceptionally vulnerable to uncontrolled contamination and its unrestrained lateral migration throughout the entire graphene mesa, eventually leading to the blistering of Al2O3. Thus, we prove the method’s suitability for the detection of these contaminants’ presence and location, and infer on its applicability to the investigation of any contamination-induced inhomogeneity in two-dimensional systems.

A cytoskeletal vortex drives phage nucleus rotation during jumbo phage replication in E.coli.

Nucleus-forming jumbo phages establish an intricate subcellular organization, enclosing phage genomes within a proteinaceous shell called the phage nucleus. During infection in Pseudomonas, some jumbo phages assemble a bipolar spindle of tubulin-like PhuZ filaments that positions the phage nucleus at midcell and drives its intracellular rotation. This facilitates the distribution of capsids on its surface for genome packaging. Here we show that the Escherichia coli jumbo phage Goslar assembles a phage nucleus surrounded by an array of PhuZ filaments resembling a vortex instead of a bipolar spindle. Expression of a mutant PhuZ protein strongly reduces Goslar phage nucleus rotation, demonstrating that the PhuZ cytoskeletal vortex is necessary for rotating the phage nucleus. While vortex-like cytoskeletal arrays are important in eukaryotes for cytoplasmic streaming and nucleus alignment, this work identifies a coherent assembly of filaments into a vortex-like structure driving intracellular rotation within the prokaryotic cytoplasm.