Application Of Confocal Microscopy Research Articles

Investigation of irregular apertures and applications in confocal microscopy and speckle imaging

Investigation of irregular apertures and applications in confocal microscopy and speckle imaging

The AMSlide for noninvasive time-lapse imaging of arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhizal (AM) symbiosis, the nutritional partnership between AM fungi and most plant species, is globally ubiquitous and of great ecological and agricultural importance. Studying the processes of AM symbiosis is confounded by its highly spatiotemporally dynamic nature. While microscopy methods exist to probe the spatial side of this plant-fungal interaction, the temporal side remains more challenging, as reliable deep-tissue time-lapse imaging requires both symbiotic partners to remain undisturbed over prolonged time periods. Here, we introduce the AMSlide: a noninvasive, high-resolution, live-imaging system optimised for AM symbiosis research. We demonstrate the AMSlide's applications in confocal microscopy of mycorrhizal roots, from whole colonisation zones to subcellular structures, over timeframes from minutes to weeks. The AMSlide's versatility for different microscope set-ups, imaging techniques, and plant and fungal species is also outlined. It is hoped that the AMSlide will be applied in future research to fill in the temporal blanks in our understanding of AM symbiosis, as well as broader root and rhizosphere processes.

Application of confocal microscopy and flow cytometry to identify physiological responses of Prorocentrum micans to the herbicide glyphosate

The physiological response of the dinoflagellate P. micans to the effect of the herbicide glyphosate at a concentration of 25–200 μg L−1 was evaluated. It has been shown that P. micans is able to grow due to the consumption of dissolved organic phosphorus formed as a result of the mineralization of glyphosate by bacteria. The addition of glyphosate to the medium inhibits the photosynthetic activity of cells; there is a pronounced inhibition of the relative electron transfer rate along the electron transport chain and the maximum quantum efficiency of the use of light energy. Morphological and ultrastructural changes in P. micans cells were evaluated at sublethal (150 μg L−1) and lethal (200 μg L−1) glyphosate concentrations. It has been shown that at a herbicide concentration of 150 μg L−1, the first signs of apoptosis appear in most P. micans cells: a decrease in lateral light scattering, cytoplasmic retraction, partial destruction of cytoplasmic organelles, a change in the morphology of nuclei, mitochondria, a change in the potential of mitochondrial membranes, and a decrease in the autofluorescence of chlorophyll in cells. At a glyphosate concentration of 200 μg L−1, P. micans showed signs of a late stage of apoptosis: violation of the integrity of intracellular organelles and chromatin organization, fragmentation of nuclei, condensation of cytoplasm, disorganization of chloroplasts in the cells, and the release of cell contents beyond the cell membrane. The effectiveness of using flow cytometry and laser scanning confocal microscopy methods for identifying signs and stages of cell apoptosis when exposed to glyphosate is discussed.

Anti-SARS-CoV-2 activity of cyanopeptolins produced by Nostoc edaphicum CCNP1411

Despite the advances in contemporary medicine and availability of numerous innovative therapies, effective treatment and prevention of SARS-CoV-2 infections pose a challenge. In the search for new anti-SARS-CoV-2 drug candidates, natural products are frequently explored. Here, fifteen cyanopeptolins (CPs) were isolated from the Baltic cyanobacterium Nostoc edaphicum and tested against SARS-CoV-2. Of these depsipeptides, the Arg-containing structural variants showed the strongest inhibition of the Delta SARS-CoV-2 infection in A549ACE2/TMPRSS2 cells. The functional assays indicated a direct interaction of the Arg-containing CP978 with the virions. CP978 also induced a significant decline in virus replication in the primary human airway epithelial cells (HAE). Of the four tested SARS-CoV-2 variants, Wuhan, Alpha, Omicron and Delta, only Wuhan was not affected by CP978. Finally, the analyses with application of confocal microscopy and with the SARS-CoV-2 pseudoviruses showed that CP978-mediated inhibition of viral infection results from the direct binding of the cyanopeptolin with the coronaviral S protein. Considering the potency of viral inhibition and the mode of action of CP978, the significance of the peptide as antiviral drug candidate should be further explored.

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae.

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at the Drosophila NMJ enables researchers to acquire synaptic information, encompassing both quantitative data on synapse abundance and detailed insights into their morphology. However, the diffuse distribution and limited visual range of the TEM present challenges for the ultrastructural analysis. This study introduces an innovative and efficient sample preparation method that surpasses the conventional approach. The procedure begins by placing a metal mesh at the base of a flat-bottomed bottle or test tube, followed by positioning fixed larvae samples onto the mesh. An additional mesh is placed over the samples, ensuring that they are positioned between the two meshes. The fixed samples are thoroughly dehydrated and infiltrated before proceeding with the embedding procedure. Then embedding of the samples in epoxy resin is performed in a flat sheet manner, which allows for the preparation of muscles for positioning and sectioning. Benefiting from these steps, all the muscles of Drosophila larvae can be visualized under light microscopy, therey facilitating subsequent positioning and sectioning. Excess resin is removed after locating the 6th and 7th muscles of body segments A2 and A3. Serial ultra-thin sectioning of the 6th or 7th muscle is performed.

Fast Screening of Protein-Protein Interactions Using Förster Resonance Energy Transfer (FRET-) Based Fluorescence Plate Reader Assay in Live Cells.

Protein-protein interactions (PPIs) have great importance for intracellular signal transduction and sustaining the homeostasis of an organism. Thus, the identification of PPIs is necessary to better understand the downstream signaling functions of the proteins in healthy and pathological conditions. Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful tool for detecting PPIs in living cells. In literature, FRET analysis methods such as donor photobleaching (FLIM), acceptor photobleaching, spectral imaging, and the three-filter cube method (sensitized emission) are abundantly applied to investigate PPIs; however, they require various expensive instrumentations, and their calculation methods are very time consuming. Since confocal microscopy applications and live cell-based techniques of FRET are very costly, scientists sometimes prefer plate readers for FRET experiments. However, plate reader applications also have many disadvantages and considerations compared to confocal fluorescence microscopy, and complex calculation procedures should be performed. To overcome these problems, we propose a FRET-based high-throughput assay method with a standard monochromator-based microplate reader, which is generally available in most biochemistry laboratories, and an alternative calculation procedure. This rapid, low cost, and effective analysis method enables the scientists to prescreen PPIs in living cells as a preliminary study and quick glance at the experiment before preparing the whole experimental setup with the expensive instrumentations. Additionally, the alternative calculation procedure provides the FRET area comparison without complex bleed-through calculations in a non-conventional manner by shortening the analysis processes with this quick and uncomplicated spectral representation.

Accessible quantitative phase imaging in confocal microscopy with sinusoidal-phase synthetic optical holography.

We present a technically simple implementation of quantitative phase imaging in confocal microscopy based on synthetic optical holography with sinusoidal-phase reference waves. Using a Mirau interference objective and low-amplitude vertical sample vibration with a piezo-controlled stage, we record synthetic holograms on commercial confocal microscopes (Nikon, model: A1R; Zeiss: model: LSM-880), from which quantitative phase images are reconstructed. We demonstrate our technique by stain-free imaging of cervical (HeLa) and ovarian (ES-2) cancer cells and stem cell (mHAT9a) samples. Our technique has the potential to extend fluorescence imaging applications in confocal microscopy by providing label-free cell finding, monitoring cell morphology, as well as non-perturbing long-time observation of live cells based on quantitative phase contrast.

Brightly Fluorescent 2'-Deoxyribonucleoside Triphosphates Bearing Methylated Bodipy Fluorophore for in Cellulo Incorporation to DNA, Imaging, and Flow Cytometry.

Synthesis of cytosine, uracil, and 7-deazaadenine 2'-deoxyribonucleosides and triphosphates (dNTPs) bearing hexamethylated phenyl-bodipy fluorophore attached at position 5 of pyrimidines or at position 7 of 7-deazapurine was developed. All the title labeled nucleosides and dNTPs displayed bright green fluorescence with very high quantum yields. The modified dNmBdpTPs were good substrates to diverse DNA polymerases and were used for in vitro enzymatic synthesis of labeled DNA by primer extension or PCR. In combination with cationic cyclodextrin-peptide-based dNTP transporter, the dNmBdpTPs were successfully used for staining of genomic DNA in living cells for applications in confocal microscopy and in flow cytometry. The best performing cytosine nucleotide dCmBdpTP was used to monitor mitosis in live cells.

Ring and Tails: Exploring the Intimacy of Cyclodextrin - Membrane Interactions

Cyclodextrins (CDs) are cyclic, ring-shaped molecules made of sugar moieties, consisting of a hydrophobic cavity and a hydrophilic outer part. Such a structure allows CDs to form inclusion complexes with hydrophobic molecules, where the guest molecule is physically entrapped in the host's (CD) cavity. This feature finds a wide range of applications in drug delivery, pharmaceutical and food industries, as inclusion complexes with CDs show enhanced bioavailability and longer circulation times.Recently, αCDs have been reported to directly interact with aliphatic tails of phospholipids from biological and model bilayer membranes. As the tails are not directly accessible for molecules from the aqueous solution, this observation suggests complex interplays and possible membrane remodeling occurring at the CD/membrane/water interface. Interestingly, despite the wide applications of CDs, the essentials of this process and its possible consequences in vivo remain poorly understood.Herein, we use a combination of complementary biophysical techniques to uncover the inner workings of interactions between CDs and model lipid membranes, focusing on CDs’ membrane-perturbing properties. Specifically, we employ Quartz Crystal Microbalance (QCMB-D) to reveal variations in viscoelastic properties of a lipid membrane induced by the presence of αCD. Furthermore, the application of confocal microscopy and CryoTEM allows us to explore the role of membrane curvature-mediated interactions between αCDs and the lipid membrane.

Generation and manipulation of super-resolution spherical magnetization chains.

Based on the inverse Faraday effect, the light-induced magnetization field distributions are investigated for a 4π tight focusing configuration with azimuthally polarized beams. It is found that a superlong (16λ) magnetization chain, composed of 19 subwavelength (0.44λ) spherical spots with longitudinal magnetization field, can be achieved in the focal volume of the objective. Moreover, the magnetic force on a magnetic particle or particle trains produced by tightly focused azimuthally polarized beams are calculated and exploited for the stable trapping of magnetic particles. These unique focal field distributions may find potential applications in confocal microscopy, atom control, and magneto-optical data storage.

Abstract 2457: Mechanisms of membrane binding of K-Ras4B farnesylated hypervariable region

Abstract K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. Kras is frequently mutated in cancer. K-Ras4B modulates downstream signaling at different lipid microdomains. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. HVR farnesylation plays an important role in Ras signaling, but the structural mechanisms and consequences of farnesyl-induced membrane binding are poorly understood and the basis for Ras localization to certain parts of plasma membrane is unknown. Application of confocal microscopy, surface plasmon resonance, and molecular dynamics (MD) simulations revealed that K-Ras4B is distributed between rigid lipid rafts and disordered non-raft regions of the plasma membrane. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains containing unsaturated phospholipids, while the rigid microdomains composed of tightly packed saturated lipids restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion towards the cell interior allows oligomerization of K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids, but the farnesyl's membrane orientation. Phosphorylation of Ser181 prohibits spontaneous farnesyl membrane insertion. We speculate that if the farnesyl group is not inserted into the membrane, it may serve in cooperative binding of other K-Ras4B molecules, thus leading to dimerization. In this case, the protein binds the membrane using electrostatic interaction between its positively charged HVR and the negatively charged membrane phospholipids. In the case of disordered phospholipid domains, the farnesyl group burial in the phospholipids tends to hinder its interactions. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting to microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling. Elucidating the mechanism for K-Ras4B localization at different membrane microdomains is important for in-depth understanding of Ras-effector interactions mediating signaling pathways. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E. Citation Format: Hyunbum Jang, Sherwin J. Abraham, Tanmay S. Chavan, Ben Hitchinson, Lyuba Khavrutskii, Nadya I. Tarasova, Ruth Nussinov, Vadim Gaponenko. Mechanisms of membrane binding of K-Ras4B farnesylated hypervariable region. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2457. doi:10.1158/1538-7445.AM2015-2457

The application of confocal microscopy in the diagnosis of small corneal foreign body

Objective To discuss the clinical effect of diagnosis of corneal foreign body with diameter≤ 1 mm by confocal microscope.Methods Small corneal foreign body or suspected foreign body were examined under confocal microscopy.Results Of 23 eyes,22 eyes were diagnosed accurately by confocal microscopy.Conclusion Small foreign bodies can be located precisely with confocal microscopy and the value of confocal microscope in the diagnosis and treatment of small corneal foreign body is significate. Key words: Microscopy, confocal; Body, foreign, corneal; Diagnosis

Sharper focal spot below λ/4 of azimuthally polarized illumination phase-encoded by the binary 0/π phase plate

The intensity distribution in the focal region for the azimuthally polarized beam phase-encoded by the binary 0/π phase plate is calculated on the basis of the vector diffraction theory. With the annular pupil aperture employed, the resolution of the focal spot will be improved remarkably. We demonstrate a sharper focal spot with full width at half maximum (FWHM) of 0.223λ (below λ/4), significantly smaller that of linear, circular and radial polarization beam under the same condition. The focusing phenomena for illumination beam with various polarization status and beam shapes are analyzed explicitly. This analysis could have potential applications in confocal microscopy and two-photo microscopy for polarization difference imaging.

Locally adaptive image denoising by a statistical multiresolution criterion

It is shown how to choose the smoothing parameter in image denoising by a statistical multiresolution criterion, both globally and locally. Using inhomogeneous diffusion and total variation regularization as examples for localized regularization schemes, an efficient method for locally adaptive image denoising is presented. As expected, the smoothing parameter serves as an edge detector in this framework. Numerical examples together with applications in confocal microscopy illustrate the usefulness of the approach.

Vascular Imaging Using Confocal Microscopy

Blood vessels are dynamic structures and capable of structural changes in response to various challenges, which is called vascular remodeling. Since vascular remodeling is important for cardiovascular pathophysiology, monitoring of structural changes of blood vessels has been drawn attention. Physiological methods allow gross vessel structure identification at physiological conditions, but technical constraints limit the analysis of detailed structure of blood vessels. For an analysis of blood vessels and its interaction to periendothelial cells, visualization of intact blood vessels with various thicknesses is critical. Recent development of confocal microscopy enables us to reveal three-dimensional structures of specimens by analyzing relatively thick specimens with optical sectioning. Especially the three-dimensional visualization combined with multi-fluorescent immunohistochemistry technique reveals the detailed structures of blood vessels as well as interactions between endothelial cells and periendothelial cells. Here we will discuss the application of confocal microscopy to imaging of blood vessels.

Confocal Microscopy in Ophthalmology

To describe the principles, capabilities, and applications of confocal microscopy in vivo in ophthalmology. Perspective, literature review, and commentary. Review and synthesis of selected recent literature, with interpretation and perspective. Confocal microscopy imaging has led to a better understanding of the cellular microstructure in the normal, postsurgical, and diseased cornea by enabling quantitative analysis of the cellular response in the human cornea in vivo. At present, the major role of confocal microscopy is in research of corneal surgery and disease. Clinical applications are limited to facilitating the diagnosis of Acanthamoeba and deep fungal keratitis, measuring residual bed thickness after laser in situ keratomileusis, and measuring endothelial cell density in high-light-scattering situations. In addition to providing qualitative data, confocal microscopy is valuable for quantitative analysis of the cornea and will enable the investigation of pharmacologic and surgical modifications of corneal wound healing, nerve regeneration, and cellular responses. Prospective, quantitative analyses require individual calibration of confocal microscopes for lateral and axial dimensions of images, for image depth, and for light intensity.

Biological Bulletin Virtual Symposium: Biology of Marine Invertebrate Larvae

Larval forms have been central to the study of evolution-ary and developmental biology since the inception of thesefields in the mid-19th century, yet they still offer contem-porary biologists tremendous opportunities for decipheringfundamental biological principles. Larvae are found be-tween the embryonic and juvenile stages of those metazoanswith indirect development, which includes the vast majorityof marine invertebrates and many marine fishes. As devel-opmental stages that interact behaviorally with their envi-ronments, they play important roles in development, ecol-ogy, and evolution and are therefore of interest to a widecommunity of biologists. The recent rise of “Evo Devo”(Evolutionary Developmental Biology) has propelled thestudy of larval forms to center stage in organismal biology.However, many marine invertebrate larvae are still unde-scribed and the variety of developmental types is greatlyunderestimated, especially in phyla that historically re-ceived little attention. Increased application of confocalmicroscopy in combination with fluorescent dyes is nowrevealing a previously unsuspected diversity of develop-ment and facilitating comparative studies of larval neuro-genesis, myogenesis, and anatomy.Most larvae are small, on the order of 0.5 to a fewmillimeters in size, and they generally occupy differenthabitats from those of the juveniles into which they meta-morphose. For planktonic larvae, small size and slow ratesof movement with cilia or muscle-driven appendages re-quire them to have shapes and functions different fromthose of juveniles and also leave them more or less at themercy of the water currents that disperse and mix them.Because many marine invertebrates have planktonic larvalstages and benthic adults, larvae link adult populationsgenetically and contribute to local population sizes anddynamics. A recent renewed interest in marine conservationfocuses on larvae as the dispersal stage that provides theall-important connectivity among metapopulations, includ-ing those in marine reserves. Larval stages are also key tothe understanding of recruitment, a critical issue in this ageof declining fisheries resources.As transitional stages between embryos and juveniles,larvae have structures, physiology, and behaviors that allowthem to operate in their own environments while developinganatomical and physiological attributes that will be used bythe juvenile stages in a new environment. In addition tosimilarities and differences of invertebrate larval formsamong major clades such as phyla and classes, many lowerlevel clades also show large variations in larval form, lengthof larval life, and timing of expression of traits, all of whichprovide opportunities to study how the differences arose.In the context outlined above, larval biology is a verybroad, very active, and very rich area for exploration ofbasic scientific questions, so it is fitting that the fourthvirtual symposium in

Morphological changes of cornea in iridocorneal endothelial syndrome under the confocal microscopy

To evaluate the morphological changes of cornea in iridocorneal endothelial syndrome (ICE) under the examination of in vivo confocal microscopy. The experimental design was retrospective observation case series. Twenty-three eyes of 23 consecutive patients, each diagnosed as ICE, had their both eyes examined with the in vivo confocal microscopy (NIDEK, confoscan 3.0). The images were recorded and analyzed by software NAVIS. Measurements were performed on endothelium density, average endothelial area, the percentage of hexagonal cells and the percentage of endothelium with nuclei, and the ANOVA was done to assess the differences. In vivo confocal microscopy highlighted two main patterns of endothelial changes: "kite-like" and "epithelial-like" abnormal endothelium, characterised by marked hyperreflective nuclei and loss of regularity in cellular size and shape. With the progression of disease, the endothelium density and the percentage of hexagonal endothelial cells decreased, which were (1687.1 +/- 122.6), (1210.6 +/- 168.7), (947.3 +/- 145.2), (856.8 +/- 73.4) cells/mm2 and (51.5 +/- 6.3)%, (39.8 +/- 9.2)%, (32.7 +/- 8.1)%, (24.1 +/- 5.6)% respectively in detail. In contrast, the average endothelial area the percentage of endothelium with nuclei increased, which were (678.3 +/- 56.3), (928.7 +/- 96.2), (1188.5 +/- 72.6), (1337.5 +/- 60.8) microm2 and (12.6 +/- 1.4)%, (56.8 +/- 3.7)%, (78.7 +/- 5.6)%, (84.3 +/- 2.8)%. The differences all had statistical significance (F = 7.158, 7.736, 6.876, 14.452 respectively, P = 0.000). The morphology of keratocyte and endothelium were normal, compared with the contralateral healthy eyes. However, the stromal nerves became thicker and more tortuous with the disease advancement. The application of confocal microscopy indicates that the ICE is characterised by epithelial-like endothelial cells with hyperreflective nuclei. The technique has great potential in diagnosing ICE, especially in evaluating the disease progression.

In-vivo confocal microscopy of iridocorneal endothelial syndrome

We carried out a study by in-vivo confocal microscopy to investigate the appearance of iridocorneal endothelial (ICE) syndrome, and discuss its diagnostic potential. Twelve patients, each with unilateral ICE syndrome, had both their eyes examined by in-vivo confocal microscopy. The images were recorded and analyzed by the use of proprietary software. Endothelium density, average endothelial area, coefficient of variation of cell size, percentage of hexagonal cells, and nerve fiber diameter were measured in both the anterior and posterior stroma. Corneal thickness was also measured for both eyes. A non-parametric test was used to compare differences between the affected eye and the contralateral healthy one. In-vivo confocal microscopy highlighted two main patterns of abnormal "epithelioid-like" endothelium, both characterized by marked hyperreflective nuclei and loss of regularity in cellular size and shape. The first pattern was relatively regular cell size and shape, conserving a pattern similar to that of normal endothelial cells. However, the cells lost normal hexagonality and presented prominent uniform "cobblestone-like" nuclei occupying the central area of the cells. The second type was more irregular in cellular size and shape, with hyperreflective diversely shaped nuclei adjacent to the boundaries of the cells. Cells with two nuclei could be found in both types. Compared with the contralateral eye, the stromal nerve fibers in affected eyes were unusually thicker and distorted. Nerve diameters in the anterior stroma of affected eyes and contralateral eyes were 5.7 +/- 0.5 microm and 3.2 +/- 0.2 microm, respectively; those in the posterior stroma were 10.8 +/- 0.3 microm and 6.6 +/- 0.4 microm, respectively (both P < 0.001). Application of confocal microscopy indicates that ICE syndrome is characterized by pleomorphic epithelioid-like endothelial cells with hyperreflective nuclei. The technique has great potential in diagnosing ICE syndrome, especially in cases with corneal edema.

Confocal laser scanning microscopy. Using new technology to answer old questions in forensic investigations

Confocal laser scanning microscopy (CLSM) is a relatively new technique for microscopic imaging. It has found a wide field of application in the general sphere of biological sciences. It has completely changed the study of cells and tissues by allowing greater resolution, optical sectioning of the sample and three-dimensional sanoke reconstruction. Confocal microscopy represents a valid, precious and useful tool capable of providing data (images) of unrivalled clearness and definition. This review discusses the possible applications of confocal microscopy in specific fields of forensic investigation, with specific regard to ballistics, forensic histopathology and toxicological pathology.