Interference Contrast Microscopy Research Articles

Exploring silica accumulation in bamboo leaves: A study on phytolith morphology and epidermal patterning in the tropical giant bamboo Dendrocalamus copelandii.

Bamboo is a grass in the Poaceae family with various applications. Bamboo leaves can accumulate high silica. However, silica deposition in bamboo has received limited study. Therefore, this research investigated silica accumulation in Dendrocalamus copelandii leaves. The study includes the localisation of silica through phytolith morphology, examination of the distribution patterns of phytoliths in epidermal tissues, analysis of silica accumulation within specialised silica cells (short cells), and analysis of silicon concentration across various leaf developmental stages. We employed imaging techniques, including Differential interference contrast and Scanning electron microscope incorporated with an energy-dispersive X-ray spectrometer, to investigate silica accumulation in bamboo leaves. We also analysed silicon concentration using Inductively coupled plasma-optical emission spectroscopy. Leaves of D. copelandii exhibited 11 phytolith morphotypes, such as BILOBATE, POLYLOBATE, SADDLE, ACUTE, ACUTE BULBOSUS, MICROHAIR, STOMATA, BULLIFORM FLABELLATE, ELONGATE SINUATE, ELONGATE ENTIRE and TRACHEARY. Most of these phytoliths were found in short cells (BILOBATE, POLYLOBATE and SADDLE) of epidermal tissues. The short cells were arranged transversely along the leaf length. BILOBATE was found in both the abaxial and adaxial epidermis, while SADDLE was found only in the abaxial epidermis. Silica accumulation in the short cells of unexpanded leaves begins at the leaf apex, spreads to the middle and base positions, and accumulates first in the abaxial before the adaxial epidermis. Moreover, bamboo leaves accumulate more silicon concentration as they age. Phytolith morphotypes and silica accumulation in epidermal short cells are key factors in understanding silica deposition. Leaf age and climate significantly impact silicon concentration in bamboo leaves. Our findings are informative for archaeological studies and for plant taxonomical classification. The results are also applicable for biotechnological applications.

Species of the Genus Paroniella Fuhrmann, 1920 (Cestoda: Davaineidae) from Bulbuls (Passeriformes: Pycnonotidae): Redescription of P. perreti (Mahon, 1954) and Description of P. neolestes n. sp. from Africa.

The purpose of the present study is to contribute to the knowledge on the diversity of cestodes of the genus Paroniella Fuhrmann, 1920 parasitising passerine birds of the family Pycnonotidae (bulbuls) in the Afrotropical Region. The only known species of this groups, Paroniella perreti (Mahon, 1954) from Pycnonotus tricolor (Hartlaub) in the Democratic Republic of the Congo, is poorly described. Therefore, it needed a detailed redescription in order to make a reliable comparison and provide differentiation among this species and newly-collected davaineid specimens. Based on new materials collected from Neolestes torquatus Cabanis in Gabon, Paroniella neolestes n. sp. is described. The type series of P. perreti from the collection of the Natural History Museum of Geneva is redescribed and figured using conventional light microscopy and interference-contrast microscopy. The specimens of the new species were stained by iron acetocarmine and mounted in Canada balsam. The present study provided more detailed and accurate data on P. perreti (compared to its original description) in relation mostly to the armament of suckers (not mentioned in the original description), the structure of the copulative apparatus, the number of rostellar hooks and the number of testes. The morphological comparison of the specimens from Neolestes torquatus from Gabon with the known 53 species of the genus Paroniella (presented in a table format) characterised them as belonging to a new species. The new combination Paroniella oitaensis (Kugi, 1990) n. comb. was proposed. Based on the present state of knowledge, P. perreti and P. neolestes seem to be specific each to a single host species, i.e. Pycnonotus tricolor and Neolestes torquatus, respectively. Further studies of davaineid cestodes from pycnonotids from Africa as well as from South and Southeast Asia may result in discovering much greater diversity of this group than currently known.

Quantitative phase imaging by gradient retardance optical microscopy

Quantitative phase imaging (QPI) has become a vital tool in bioimaging, offering precise measurements of wavefront distortion and, thus, of key cellular metabolism metrics, such as dry mass and density. However, only a few QPI applications have been demonstrated in optically thick specimens, where scattering increases background and reduces contrast. Building upon the concept of structured illumination interferometry, we introduce Gradient Retardance Optical Microscopy (GROM) for QPI of both thin and thick samples. GROM transforms any standard Differential Interference Contrast (DIC) microscope into a QPI platform by incorporating a liquid crystal retarder into the illumination path, enabling independent phase-shifting of the DIC microscope's sheared beams. GROM greatly simplifies related configurations, reduces costs, and eradicates energy losses in parallel imaging modalities, such as fluorescence. We successfully tested GROM on a diverse range of specimens, from microbes and red blood cells to optically thick (~ 300 μm) plant roots without fixation or clearing.

A new species of Agauopsis (Acari, Halacaridae) from South Korea belonging to A. brevipalpus group

A new marine halacarid mite of the genus Agauopsis Viets, 1927 is recorded from mussels in the intertidal bedrock of the southern coast of South Korea. Agauopsis bisetosa n. sp. belonging to the A. brevipalpus group, is characterized by an ‘H’-shaped areola on the anterior dorsal plate, two pairs of costae on the posterior dorsal plate, a tectum with a truncate anterior margin, posterior epimeral plate with two ventral setae, telofemur I with one ventral and two ventromedial dentate spines, and tibia II with one ventral and one ventromedial dentate spines. Notably, the presence of two ventral setae on the posterior epimeral plate is a unique characteristic of the new species. This study deals with a description of A. bisetosa n. sp., accompanied by detailed illustrations drawn under a differential interference contrast microscope and scanning electron microscope photomicrographs. Additionally, it includes a character comparison table of the A. brevipalpus group and a key for known Agauopsis species in the northwest Pacific, both including the new species.

Isolation of phase edges using off-axis q-plate filters.

Edge-enhanced microscopes with a q-plate have attracted more attention to enhance the edges of phase-amplitude objects in biological samples due to their capacity for all-directional edge enhancement, while differential interference-contrast microscopy enhances edges in only one-direction. However, the edge-enhanced microscopes cannot distinguish the edges of phase and amplitude objects, as both edges are equally enhanced. This study introduces a novel method for isolating the edge of a phase object from an amplitude object using an off-axis q-plate filter in a 4f system. Herein, we combined off-axis q-plates with four different displacements to isolate the phase object edge from the amplitude object. To demonstrate the proposed method, we conducted experiments using two distinct samples. The first sample comprised a phase test target surrounded by an aperture, and the second sample involved an overlap between the phase test target and a white hair with non-zero transmittance. In the samples, the isolated phase object edge is in good agreement with the theoretical expectations, and the amplitude object edge was reduced by approximately 93%. The proposed method is a novel and effective approach for isolating the edge of a phase object from an amplitude object and can be useful in various biological imaging applications.

Species composition of Ostracoda in the Okinawa islands, and geographical distribution of the two genera \(\textit{Loxoconcha}\) and \(\textit{Xestoleberis}\) (Arthropoda: Crustacea) around Japan

This study was conducted in the period of 2012 to 2015 in some locations of Japan. At each location, samplings were carried out on reef slopes using SCUBA diving, on reef flats, tidal beaches and river mouths during low tide. The specimens then were treated using a differential interference contrast microscope with a camera lucida (BX-50, OLYMPUS), a quick auto-coater (JFC-1500, Ion Sputtering Device), a scanning electron microscope (SEM) in the laboratory of Shizuoka University,… The result shows that total 139 species of recent ostracoda belonging to 28 families, 70 genera recorded in the Okinawa Islands, southern Japan are found. Most diversified families include Loxoconchidae (26 species - 18.7%), Xestoleberididae (17 species - 12.2%), Bairdiidae (13 species - 9.4%), Hemicytheridae (13 species - 9.4%), Leptocytheridae (8 species - 5.8%). Based on the distributional pattern of pore systems located below the eye tubercle of Loxoconcha and the types of pores on carapaces of Xestoleberis, the species groups of the two genera were divided into three groups, i.e., Groups A, B and C. Geographical distribution of three species groups was shown for each location in the present report. The geographical distribution of three species phylogenetic groups (i.e. Groups A, B and C) of the two genera Loxoconcha and Xestoleberis suggests that the fauna of two genera in the Okinawa islands are more similar to southern faunas (e.g., Vietnam, Philippines, Australia,…) than Japanese Island Arc fauna. Hence, the origin of two genera Loxoconcha and Xestoleberis in the Okinawa islands, southern Japan may be from the South.

Study of the Interference Color with Nomarski Prism Wedge Angle in a Differential Interference Contrast Microscopy System

Differential interference contrast microscopy systems demonstrate the phase (optical path) rather than the amplitude of a sample. Previous studies have usually approximated the optical path difference produced by the Normarski prism. We derive the mathematical expression for the optical path difference produced by the incident light at any position of the Nomarski prism. As a result, the optical path difference introduced by the differential interference contrast microscope can be calculated. Moreover, the optical path difference is linearly related to the position of the incident light. In addition, the differential interference contrast microscopy system uses a composite light source, while previous studies were basically performed at a single wavelength. The standard wavelengths d (656 nm), F (587 nm), and C (486 nm) are taken as examples to analyze the relationship between the interference color change of the observation surface under the superposition of different wavelengths and the prism wedge angle when the prism moves. When the prism moves the same distance, the larger the prism wedge Angle is, the faster the interference color change is. The analysis based on practical considerations in this paper is believed to provide a method for studying Nomarski prisms.

Reflectional quantitative differential phase microscopy using polarized wavefront phase modulation.

Quantitative phase microscopy (QPM), as a label-free and nondestructive technique, has been playing an indispensable tool in biomedical imaging and industrial inspection. Herein, we introduce a reflectional quantitative differential phase microscopy (termed RQDPM) based on polarized wavefront phase modulation and partially coherent full-aperture illumination, which has high spatial resolution and spatio-temporal phase sensitivity and is applicable to opaque surfaces and turbid biological specimens. RQDPM does not require additional polarized devices and can be easily switched from reflectional mode to transmission mode. In addition, RQDPM inherits the characteristic of high axial resolution of differential interference contrast microscope, thereby providing topography for opaque surfaces. We experimentally demonstrate the reflectional phase imaging ability of RQDPM with several samples: semiconductor wafer, thick biological tissues, red blood cells, and Hela cells. Furthermore, we dynamically monitor the flow state of microspheres in a self-built microfluidic channel by using RQDPM converted into the transmission mode.

Coupled mechanical mapping and interference contrast microscopy reveal viscoelastic and adhesion hallmarks of monocyte differentiation into macrophages.

Monocytes activated by pro-inflammatory signals adhere to the vascular endothelium and migrate from the bloodstream to the tissue ultimately differentiating into macrophages. Cell mechanics and adhesion play a crucial role in macrophage functions during this inflammatory process. However, how monocytes change their adhesion and mechanical properties upon differentiation into macrophages is still not well understood. In this work, we used various tools to quantify the morphology, adhesion, and viscoelasticity of monocytes and differentiatted macrophages. Combination of atomic force microscopy (AFM) high resolution viscoelastic mapping with interference contrast microscopy (ICM) at the single-cell level revealed viscoelasticity and adhesion hallmarks during monocyte differentiation into macrophages. Quantitative holographic tomography imaging revealed a dramatic increase in cell volume and surface area during monocyte differentiation and the emergence of round and spread macrophage subpopulations. AFM viscoelastic mapping showed important stiffening (increase of the apparent Young's modulus, E0) and solidification (decrease of cell fluidity, β) on differentiated cells that correlated with increased adhesion area. These changes were enhanced in macrophages with a spread phenotype. Remarkably, when adhesion was perturbed, differentiated macrophages remained stiffer and more solid-like than monocytes, suggesting a permanent reorganization of the cytoskeleton. We speculate that the stiffer and more solid-like microvilli and lamellipodia might help macrophages to minimize energy dissipation during mechanosensitive activities. Thus, our results revealed viscoelastic and adhesion hallmarks of monocyte differentiation that may be important for biological function.

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.

Antibacterial and Anti-inflammatory Activity of Extracts and Major Constituents Derived from Stachytarpheta indica Linn. Leaves and Their Potential Implications for Wound Healing.

Wounds of various types continue to have a severe socioeconomic impact on the cost of health care. Globally, there has been increased interest surrounding the identification of bioactive compounds that promote or modulate the wound healing process. Stachytarpheta indica Linn. is traditionally used to heal wounds and relieve inflammation; however, the theorised pharmacological properties have not yet been scientifically validated. In this study, dried and ground plant leaves were extracted with water and methanol, which were then subjected to various analyses. The antimicrobial activity of the plant extracts and isolated compounds was determined using well diffusion assays, while the minimum inhibitory concentrations were determined with a colorimetric assay. Morphological changes of human keratinocytes in response to plant extracts were observed with differential interference contrast microscope imaging. Cell viability, proliferation, and migratory effects post-treatment with the plant extracts were also evaluated via colorimetric cytotoxicity assays and a real-time cell analyser protocol. Anti-inflammatory effects of plant extracts and isolated compounds were evaluated by flow cytometry and cyclooxygenase and lipoxygenase enzyme inhibition assays. Three active compounds, i.e. ipolamiide, verbascoside and iso-verbascoside, were isolated from S. indica leaves. Verbascoside demonstrated broad-range antibacterial activity and imposed strong inhibition at 9.77μg/mL against Staphylococci spp. S. indica extracts (0.1-0.2mg/mL) were shown to improve human keratinocyte proliferation up to 60% and induce morphological changes by producing cytoplasmic projections at concentrations higher than 0.4mg/mL. Plant extracts (6.25-100μg/mL) and individual compounds (3.125-50μg/mL) elicited strong anti-inflammatory effects by suppressing the expression of interleukin-8 and inhibiting cyclooxygenase-1 and 5-lipoxygenase enzymes. Collectively, these results indicate that plant extracts and isolated compounds derived from S. indica have the potential to inhibit bacterial growth, promote tissue regeneration and reduce inflammation, hence, potentially providing the basis for a novel therapeutic for the treatment of wounds.

In situ thermal-microstructure characterization of a phase-transforming alloy satisfying cofactor conditions

We establish an experimental platform to combine the differential scanning calorimetry (DSC) with an in situ observation of microstructure by differential interference contrast microscope (DInM). We carry out the experiment on a phase-transforming alloy Au31Cu24Zn45 that closely satisfies the cofactor conditions - the strongest crystallographic compatibility constraints between phases. We confirm that the phase formation events observed by DInM agree well with the heat exchange profile characterized by DSC. We also observe different morphology of twins evolves differently, depending on the twin spacing. Through the quantitative analysis of various morphological domains in the temporal space, we discover that the scaling law between the twin evolution time and the fineness is linear, by which the characteristic transformation time for single variant martensite can be determined for this compatible martensite alloy as 0.636s.

Characterization of the Growth and Morphology of a BSL-2 Coccidioides posadasii Strain That Persists in the Parasitic Life Cycle at Ambient CO2.

Coccidioides is a dimorphic fungus responsible for Valley Fever and is the cause of severe morbidity and mortality in the infected population. Although there is some insight into the genes, pathways, and growth media involved in the parasitic to saprophytic growth transition, the exact determinants that govern this switch are largely unknown. In this work, we examined the growth and morphology of a Coccidioides posadasii strain (C. posadasii S/E) that efficiently produces spherules and endospores and persists in the parasitic life cycle at ambient CO2. We demonstrated that C. posadasii S/E remains virulent in an insect infection model. Surprisingly, under spherule-inducing conditions, the C. posadasii S/E culture was found to be completely hyphal. Differential interference contrast (DIC) and transmission electron microscopy (TEM) revealed unexpected cellular changes in this strain including cell wall remodeling and formation of septal pores with Woronin bodies. Our study suggests that the C. posadasii S/E strain is a useful BSL-2 model for studying mechanisms underlying the parasitic to saprophytic growth transition—a morphological switch that can impact the pathogenicity of the organism in the host.

A Versatile, Incubator-Compatible, Monolithic GaN Photonic Chipscope for Label-Free Monitoring of Live Cell Activities.

The ability to quantitatively monitor various cellular activities is critical for understanding their biological functions and the therapeutic response of cells to drugs. Unfortunately, existing approaches such as fluorescent staining and impedance‐based methods are often hindered by their multiple time‐consuming preparation steps, sophisticated labeling procedures, and complicated apparatus. The cost‐effective, monolithic gallium nitride (GaN) photonic chip has been demonstrated as an ultrasensitive and ultracompact optical refractometer in a previous work, but it has never been applied to cell studies. Here, for the first time, the so‐called GaN chipscope is proposed to quantitatively monitor the progression of different intracellular processes in a label‐free manner. Specifically, the GaN‐based monolithic chip enables not only a photoelectric readout of cellular/subcellular refractive index changes but also the direct imaging of cellular/subcellular ultrastructural features using a customized differential interference contrast (DIC) microscope. The miniaturized chipscope adopts an ultracompact design, which can be readily mounted with conventional cell culture dishes and placed inside standard cell incubators for real‐time observation of cell activities. As a proof‐of‐concept demonstration, its applications are explored in 1) cell adhesion dynamics monitoring, 2) drug screening, and 3) cell differentiation studies, highlighting its potential in broad fundamental cell biology studies as well as in clinical applications.

A class I histone deacetylase HDA-2 is essential for embryonic development and size regulation of fertilized eggs in Caenorhabditis elegans.

Caenorhabditis elegans encodes three class I histone deacetylases (HDACs), HDA-1, HDA-2, and HDA-3. Although HDA-1 is known to be involved in embryogenesis, the regulatory roles of HDA-2 and HDA-3 in embryonic development remain unexplored. To elucidate the functional roles of the three class I HDACs in C. elegans embryonic development. The roles of Class I HDACs, HDA-1, HDA-2, and HDA-3 in Caenorhabditis elegans during embryogenesis were investigated through the analysis of embryonic lethality via gene knockdown or deletion mutants. Additionally, the size of these knockdown and mutant eggs was observed using a differential interference contrast microscope. Finally, expression pattern and tissue-specific role of hda-2 and transcriptome of the hda-2 mutant were analyzed. Here, we report that HDA-1 and HDA-2, but not HDA-3, play essential roles in Caenorhabditis elegans embryonic development. Our observations of the fertilized egg size variance demonstrated that HDA-2 is involved in regulating the size of fertilized eggs. Combined analysis of expression patterns and sheath cell-specific rescue experiments indicated that the transgenerational role of HDA-2 is involved in the viability of embryonic development and fertilized egg size regulation. Furthermore, transcriptome analysis of hda-2 mutant embryos implies that HDA-2 is involved in epigenetic regulation of embryonic biological processes by downregulating and upregulating the gene expression. Our finding suggests that HDA-2 regulates the embryonic development in Caenorhabditis elegans by controling a specific subset of genes, and this function might be mediated by transgenerational epigenetic effect.

Single-shot quantitative phase imaging with polarization differential interference contrast

We present single-shot quantitative phase imaging with polarization differential interference contrast (PDIC) for a differential interference contrast microscope which records the unfiltered Stokes vector of the differential interference pattern with a polarization camera. PDIC enables high spatial resolution phase imaging in real-time, applicable to either absorptive or transparent samples and integrates simply with epi-fluorescence imaging. An algorithm with total variation regularization is further introduced to solve the quantitative phase map from the partial derivative along one single axis, improving the accuracy and the image quality from the Fourier transform solution. After quantifying the accuracy of PDIC phase imaging with numerical simulations and phantom measurements, we demonstrate the biomedical applications by imaging the quantitative phase of both stained and unstained histological tissue sections and visualizing the fission yeast Schizosaccharomyces pombe's cytokinesis.

Automated Confluence Measurement Method for Mesenchymal Stem Cell from Brightfield Microscopic Images

Cell confluence is an important metric in cell culture, as proper timing is essential to maintain cell phenotype and culture quality. To estimate cell confluence, transparent cells are observed under a phase-contrast or differential interference contrast microscope by a biologist, whose estimations are error-prone and subjective. To overcome the necessity of using the phase-contrast microscope and reducing intra- and inter-observer errors, we have proposed an algorithm that automatically measures cell confluence by using a commonly used brightfield microscope. The proposed method consists of a transport-of-intensity equation-based brightfield microscopic image processing, an image reconstruction method, and an adaptive image segmentation method based on edge detection, entropy filtering, and range filtering. Experimental results have shown that our method has outperformed several popular algorithms, with an F-score of 0.84 ± 0.07, in images with various cell confluence values. The proposed algorithm is robust and accurate enough to perform confluence measurement with various lighting conditions under a low-cost brightfield microscope, making it simple and cost-effective to use for a fully automated cell culture process.

Sources of infestation of the rice sheath mite, Steneotarsonemus spinki Smiley (Acari: Tarsonemidae), in West Bengal, India

The Rice Sheath Mite, Steneotarsonemus spinki Smiley (Acari: Tarsonemidae) is a pest of Kharif Rice (wet rice) of West Bengal, causing losses every year. It initially infests leaf sheaths, moving later onto the grains. It is not known how the mite survives in the off season, which is important for devising management strategies. The present investigation was conducted to find out the sources of infestation of S. spinki. It was concentrated on four types of substrates: rice associated weeds, soil, rice grains of different ages (before and after harvesting) and rice stubbles. Mites detected in any of those possible sources were slide mounted in modified Berlese medium and examined under phase and differential interference contrast microscope for identification. Tarsonemid mites were found on some grassy weeds, but not Steneotarsonemus spinki Smiley. No tarsonemid mite was found in soil samples from infested fields. Steneotarsonemus spinki was found on rice grains of different ages and in stubbles. Hence, both rice grains and rice stubbles could be important sources of infestation of S. spinki to new plants at each season.

Minimizing scattering-induced phase errors in differential interference contrast microscopy.

.Significance: Differential interference contrast (DIC) microscopes allow noninvasive in vivo observation of transparent microstructures in tissue without the use of fluorescent dyes or genetic modification. We show how to modify a DIC microscope to measure the sample phase distribution accurately and in real-time even deep inside sample tissue.Aim: Our aim is to improve the DIC microscope’s phase measurement to remove the phase bias that occurs in the presence of strong scattering.Approach: A quarter-wave plate was added in front of the polarization camera, allowing a modified phase calculation to incorporate all four polarization orientation angles (0 deg, 45 deg, 90 deg, and 135 deg) captured simultaneously by the polarization camera, followed by deconvolution.Results: We confirm that the proposed method reduces phase measurement error in the presence of scattering and demonstrate the method using in vivo imaging of a beating heart inside a medaka egg and the whole-body blood circulation in a young medaka fish.Conclusions: Modifying a polarization-camera DIC microscope with a quarter-wave plate allows users to image deep inside samples without phase bias due to scattering effects.