Fluorescence Microscopy Methods Research Articles

Protection of the Telomeric Junction by the Shelterin Complex.

Shelterin serves critical roles in suppressing superfluous DNA damage repair pathways on telomeres. The junction between double-stranded telomeric tracts (dsTEL) and single-stranded telomeric overhang (ssTEL) is the most accessible region of the telomeric DNA. The shelterin complex contains dsTEL and ssTEL binding proteins and can protect this junction by bridging the ssTEL and dsTEL tracts. To test this possibility, we monitored shelterin binding to telomeric DNA substrates with varying ssTEL and dsTEL lengths and quantified its impact on telomere accessibility using single-molecule fluorescence microscopy methods in vitro. We identified the first dsTEL repeat nearest the junction as the preferred binding site for creating the shelterin bridge. Shelterin requires at least two ssTEL repeats, while the POT1 subunit of shelterin that binds to ssTEL requires longer ssTEL tracts for stable binding to telomeres and effective protection of the junction region. The ability of POT1 to protect the junction is significantly enhanced by the 5'-phosphate at the junction. Collectively, our results show that shelterin enhances the binding stability of POT1 to ssTEL and provides more effective protection compared with POT1 alone by bridging single- and double-stranded telomeric tracts.

Supramolecular approach to obtain folate-functionalized nanoassociates

Nanoassociates containing folic acid (FA) were obtained through solubilization by colloid solutions of three betaine calixresorcinarenes (sulfobetaine tetrapentylcalixresorcinarene С5-SB, hydroxysulfobetaine tetrapentyl- С5-hSB and tetraundecylcalixresorcinarene C11-hSB). FA was solubilized in its anionic form and fraction of bound molecules of FA was of from 89 % to 99 % (FT-PGSE NMR method data). The driving forces of FA encapsulation in the nanoassociates has been considered with the help of 1Н NMR, 2D NOESY NMR, and FT-PGSE NMR data. The macrocycle-FA nanoassociates have average hydrodynamic diameters of 3–5 nm (PDI of 0.1840.238) and a zeta potential close to zero; however, they were stable for 4–8 weeks. The enhancement of the internalization of the macrocycle-FA nanoassociates into M-HeLa cells compared to unloaded macrocycles self-associates was shown by the flow cytometry and fluorescence microscopy methods. Additionally, the growth of cytotoxicity of Rhodamine B by 19 % in the presence of С5-SB+FA nanoassociates compared to С5-SB self-associates in the in vitro PDT experiment on M-HeLa cells was found. Overall, the potential of a supramolecular approach to obtaining folate-functionalized nanocontainers has been demonstrated.

Functional organization of 3D plant thylakoid membranes as seen by high resolution microscopy

In the field of photosynthesis, only a limited number of approaches of super-resolution fluorescence microscopy can be used, as the functional architecture of the thylakoid membrane in chloroplasts is probed through the natural fluorescence of chlorophyll molecules. In this work, we have used a custom-built fluorescence microscopy method called Single Pixel Reconstruction Imaging (SPiRI) that yields a 1.4 gain in lateral and axial resolution relative to confocal fluorescence microscopy, to obtain 2D images and 3D-reconstucted volumes of isolated chloroplasts, obtained from pea (Pisum sativum), spinach (Spinacia oleracea) and Arabidopsis thaliana. In agreement with previous studies, SPiRI images exhibit larger thylakoid grana diameters when extracted from plants under low-light regimes. The three-dimensional thylakoid architecture, revealing the complete network of the thylakoid membrane in intact, non-chemically-fixed chloroplasts can be visualized from the volume reconstructions obtained at high resolution. From such reconstructions, the stromal connections between each granum can be determined and the fluorescence intensity in the stromal lamellae compared to those of neighboring grana.

Unveiling nuclear chromatin distribution using IsoConcentraChromJ: A flourescence imaging plugin for IsoRegional and IsoVolumetric based ratios analysis.

Chromatin exhibits non-random distribution within the nucleus being arranged into discrete domains that are spatially organized throughout the nuclear space. Both the spatial distribution and structural rearrangement of chromatin domains in the nucleus depend on epigenetic modifications of DNA and/or histones and structural elements such as the nuclear envelope. These components collectively contribute to the organization and rearrangement of chromatin domains, thereby influencing genome architecture and functional regulation. This study develops an innovative, user-friendly, ImageJ-based plugin, called IsoConcentraChromJ, aimed quantitatively delineating the spatial distribution of chromatin regions in concentric patterns. The IsoConcentraChromJ can be applied to quantitative chromatin analysis in both two- and three-dimensional spaces. After DNA and histone staining with fluorescent probes, high-resolution images of nuclei have been obtained using advanced fluorescence microscopy approaches, including confocal and stimulated emission depletion (STED) microscopy. IsoConcentraChromJ workflow comprises the following sequential steps: nucleus segmentation, thresholding, masking, normalization, and trisection with specified ratios for either 2D or 3D acquisitions. The effectiveness of the IsoConcentraChromJ has been validated and demonstrated using experimental datasets consisting in nuclei images of pre-adipocytes and mature adipocytes, encompassing both 2D and 3D imaging. The outcomes allow to characterize the nuclear architecture by calculating the ratios between specific concentric nuclear areas/volumes of acetylated chromatin with respect to total acetylated chromatin and/or total DNA. The novel IsoConcentrapChromJ plugin could represent a valuable resource for researchers investigating the rearrangement of chromatin architecture driven by epigenetic mechanisms using nuclear images obtained by different fluorescence microscopy methods.

Rapid detection of salmon louse larvae in seawater based on machine learning

The proliferation of salmon lice is one of the most challenging problems in salmon farming, seriously affecting the welfare of both farmed and wild salmonids. The detection of salmon lice in seawater, especially in the (pre-) infectious stage, supports the accurate management of salmon lice proliferation. Methods for detecting salmon lice in seawater to date involve traditional light and fluorescence microscopy and Polymerase Chain Reaction (PCR) methods. However, these methods require sample pre-processing and are laborious and time-consuming, rendering them difficult to apply for the task of continuous, rapid salmon lice detection in large water volumes. This study aims to develop a rapid and easy-to-use method for the detection of salmon lice in seawater. A novel approach was introduced for the detection of salmon lice at different larval stages in seawater by leveraging machine learning. A setup for the rapid acquisition of images of salmon lice in seawater was developed, and a dataset comprising 8316 images encompassing salmon lice “Nauplius Live,” “Nauplius Shell” and “Copepodite” was created. Then 16 object detection models including one- and two-stage Convolutional Neural Network (CNN-) based models and Transformer-based models were trained, tested, and compared on this comprehensive dataset. RT-DETR showed the best performance among all models tested with a precision of 0.972 and a recall of 0.978. The YOLO series models, except for YOLOv3, showcased remarkable inference speeds (ranging from 152 to 416 frames per second) while maintaining high precision (ranging from 0.968 to 0.977) and recall (ranging from 0.964 to 0.970). To our knowledge, this is the first time machine learning has been applied to the task of rapidly detecting salmon louse larvae at different stages in seawater samples. The results demonstrate that the machine learning approaches can achieve rapid easy-to-use and cost-effective detection of salmon lice in seawater. In the future, this study has the potential for application in measuring salmon louse larvae numbers in aquatic environments, supporting the enhancement of the welfare of both farmed and wild salmon.

Can the laws of physics be circumvented? On methods of super-resolution fluorescence microscopy

Biological sciences are increasingly uncovering the foundations of life in greater detail, made possible by the development of research methods enabling exploration at the nanometer scale. Optical microscopy, a field with a significant contribution to current knowledge, is inherently limited by the Abbe limit, stemming from the fundamental wave properties of light. Through the efforts of scientists, this limit can be circumvented, as evidenced by STED and MINFLUX techniques. STED allows imaging with a resolution down to 40 nm, while MINFLUX enables resolution as fine as 2 nm. Both techniques require labelling of biological molecular targets with fluorescent markers and enable imaging in living cells, facilitating the study of dynamic biological processes. This article provides an introduction to super-resolution techniques STED and MINFLUX, demonstrating their utility through the example of studying kinesin movement along microtubules using the MINFLUX technique.

Rapid and High-Density Antibody Immobilization Using Electropolymerization of Pyrrole for Highly Sensitive Immunoassay.

Polypyrrole (Ppy) is a biologically compatible polymer that is used as a matrix, in which drugs and enzymes can be incorporated by doping. Here, we suggest an inventive application of Ppy as a biorecognition film encapsulated with an antibody (Ab) as an alternative strategy for the on-site multistep functionalization of thiol-based self-assembled monolayers. The fabrication steps of the recognition films were followed by dropping pyrrole and Ab mixed solutions onto the electrode and obtaining a thin film by direct current electropolymerization. The efficiency of Ab immobilization was studied by using fluorescence microscopy and electrochemical (EC) methods. Finally, the Ab density was increased and immobilized in 1 min, and the sensing performance as an EC immunosensor was demonstrated using α-fetoprotein with a limit of detection of 3.13 pg/mL and sensing range from 1 pg/mL to 100 ng/mL. This study demonstrates the potential for electrochemical functionalization of biomolecules with high affinity and rapidity.

Time course of fluorescent-labelled oligonucleotide accumulation in cells with the use of folate receptor-targeted cationic liposomes

INTRODUCTION. The use of cationic liposomes is a promising approach to the delivery of therapeutic nucleic acids to target cells because liposomes can protect nucleic acids from degradation by extracellular nucleases. However, to ensure selective delivery to the site of action, this approach needs modification, including liposome surface functionalisation with targeting ligands.AIM. This study aimed to compare the time courses of the accumulation of a fluorescent-labelled oligonucleotide (FITC-ODN), which simulated a nucleic acid-based medicinal product, in cells with the use of folate receptor-targeted (F) and conventional (L) cationic liposomes.MATERIALS AND METHODS. F- and L-liposomes were prepared using the polycationic amphiphile 2X3, the zwitterionic helper lipid DOPE, and the folate lipoconjugate F12. Physicochemical characterisation of the liposomes was performed using dynamic light scattering and transmission electron microscopy. Liposome–FITC-ODN complexes were formed at various nitrogen to phosphate (N/P) charge ratios. Flow cytometry, fluorescence microscopy, and confocal microscopy methods were used to study the accumulation of liposome–FITC-ODN complexes in human cervical carcinoma (KB-3-1) and human embryonic kidney (HEK 293) cells.RESULTS. The prepared F- and L-liposomes were spherical particles with a diameter of 75–100 nm. The authors selected the optimal N/P ratio of 2/1 to obtain complexes of F- and L-liposomes with the FITC-ODN. This N/P ratio yielded homogeneous liposome–FITC-ODN complexes having a polydispersity index below 0.200 and a size of 112.4–125.1 nm. F-liposomes were 25% more efficient than L-liposomes in FITC-ODN delivery to KB-3-1 cells at 90, 120, and 240 minutes after transfection. In the first few minutes of cell transfection, fluorescence and confocal microscopy data on the distribution of liposome–FITC-ODN complexes showed that cationic liposome fluorescence signals colocalised with FITC-ODN signals. Later, FITC-ODN accumulation in the cytoplasm was observed.CONCLUSIONS. Cationic liposomes demonstrated efficient FITC-ODN delivery into the cytoplasm of cancer cells. F-liposomes enhanced the percentage of transfected cells and improved FITC-ODN delivery compared with L-liposomes. The results obtained can be used in the further development of targeted medicinal products based on therapeutic nucleic acids and liposomes.

Effect of composite collagen containing hydrogel on the functional activity of fibroblasts after acid-induced <i>in vitro</i> injury

It is well known that the acidity of the microenvironment has a significant impact on the functional activity of cells involved in tissue repair. To date, the effect of biomaterials containing components of the human dermis intercellular matrix on fibroblasts under acidosis has not been fully studied.The aim of the study. To evaluate the effect of a composite hydrogel containing dermal components on the functional activity of intact fibroblasts and fibroblasts with acid-induced dysfunction.Materials and methods. To simulate the physicochemical conditions of acute inflammation, human fibroblasts were incubated for an hour at 39 °C in a nutrient medium overacidified to pH = 6. The morphometric characteristics of human skin fibroblasts were assessed using light and fluorescence microscopy methods. The number of apoptotic cells and proliferation index were assessed using flow cytometry.Results. It has been established that exposure of fibroblasts to an acidic medium inhibits their adhesive properties and reduces the rate of cell growth. The introduction of hydrogel into a cell suspension with damaged cells restores adhesive properties and cell growth. In the fibroblast population, after acid exposure, the number of living cells decreases, and the number of apoptotic cells increases. Cultivation of damaged fibroblasts in the presence of a composite hydrogel increases the number of living cells in the population and reduces the number of apoptotic cells. Acid-induced fibroblast damage reduces the proliferative activity index. It was revealed that the introduction of hydrogel into the culture medium stimulates the proliferative activity of both intact and damaged fibroblasts. The results obtained indicate that a composite hydrogel consisting of extracellular matrix components is capable of restoring the functional activity of fibroblasts damaged as a result of exposure to an acidic medium. The results obtained can be used to create biomaterials that increase the efficiency of skin regeneration in cases of excessively intense acute inflammation.

Collagen hydrogel protects intestinal epithelial cells from indomethacin-induced damage: results of an in vitro experiment

INTRODUCTION. Indomethacin is a derivative of indoleacetic acid and has anti-inflammatory, analgesic and antipyretic effects. However, the results of numerous studies show that indomethacin, like many other nonsteroidal anti-inflammatory drugs (NSAIDs), have an inhibitory effect on the viability and functional activity of enterocytes. In this regard, the search for new ways to reduce the severity of side effects from the use of NSAIDs remains relevant. One of these approaches may be to enrich patients’ diets with non-drug biologically active compounds, including proteins. However, the effect of dietary proteins and biologically active peptides on NSAID-induced damage to the wall of the small intestine and stomach has not been sufficiently studied. AIM. To evaluate the ability of a collagen-containing dietary supplement to protect human duodenal epithelial cells (HuTu-80 line) from indomethacin-induced damage. MATERIALS AND METHODS. The composite collagen-containing hydrogel was provided by «FIRST ALIVE COLLAGEN» LLC (Russia) and is a registered dietary supplement. The work used a commercial culture of human skin fibroblast cells and human duodenal epithelial cells (line HuTu-80). The viability of intestinal cells and fibroblasts was assessed using light and fluorescence microscopy and flow cytometry methods. RESULTS AND DISCUSSION. It has been established that indomethacin inhibits cell growth, causes apoptosis and death of enterocytes, and also leads to the accumulation of cells in the S-phase, which indicates a disruption in the regulation of the cell cycle. It was revealed that collagen hydrogel prevents cell death caused by indomethacin and reduces the number of apoptotic cells in the population. The protective effect of collagen hydrogel is characterized by normalization of the cell cycle of enterocytes and restoration of their growth and proliferative activity. CONCLUSION. Thus, collagen hydrogel, in vitro, is able to reduce the pathogenic effect of indomethacin on human intestinal epithelial cells. The protective effect of collagen hydrogel is characterized by maintaining viability, inhibiting apoptotic processes, and maintaining cell cycle stability. The results obtained indicate the prospects of using a dietary supplement based on a composite collagen hydrogel as a prophylactic agent to reduce the risk of NSAID-associated gastrointestinal diseases. However, to confirm the therapeutic effectiveness of the dietary supplement, further research is necessary, both using experimental animal modeling of NSAID-associated diseases of the human gastrointestinal tract, and clinical studies.

A statistical resolution measure of fluorescence microscopy with finite photons

First discovered by Ernest Abbe in 1873, the resolution limit of a far-field microscope is considered determined by the numerical aperture and wavelength of light, approximately λ2NA\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\frac{\\lambda }{2{NA}}$$\\end{document}. With the advent of modern fluorescence microscopy and nanoscopy methods over the last century, this definition is insufficient to fully describe a microscope’s resolving power. To determine the practical resolution limit of a fluorescence microscope, photon noise remains one essential factor yet to be incorporated in a statistics-based theoretical framework. We proposed an information density measure quantifying the theoretical resolving power of a fluorescence microscope in the condition of finite photons. The developed approach not only allows us to quantify the practical resolution limit of various fluorescence and super-resolution microscopy modalities but also offers the potential to predict the achievable resolution of a microscopy design under different photon levels.

Features of the DNA Escherichia coli RecN interaction revealed by fluorescence microscopy and single-molecule methods

The SOS response is a condition that occurs in bacterial cells after DNA damage. In this state, the bacterium is able to reсover the integrity of its genome. Due to the increased level of mutagenesis in cells during the repair of DNA double-strand breaks, the SOS response is also an important mechanism for bacterial adaptation to the antibiotics. One of the key proteins of the SOS response is the SMC-like protein RecN, which helps the RecA recombinase to find a homologous DNA template for repair. In this work, the localization of the recombinant RecN protein in living Escherichia coli cells was revealed using fluorescence microscopy. It has been shown that the RecN, outside the SOS response, is predominantly localized at the poles of the cell, and in dividing cells, also localized at the center. Using in vitro methods including fluorescence microscopy and optical tweezers, we show that RecN predominantly binds single-stranded DNA in an ATP-dependent manner. RecN has both intrinsic and single-stranded DNA-stimulated ATPase activity. The results of this work may be useful for better understanding of the SOS response mechanism and homologous recombination process.

A novel variational approach for multiphoton microscopy image restoration: from PSF estimation to 3D deconvolution

In multi-photon microscopy (MPM), a recent in-vivo fluorescence microscopy system, the task of image restoration can be decomposed into two interlinked inverse problems: firstly, the characterization of the point spread function (PSF) and subsequently, the deconvolution (i.e. deblurring) to remove the PSF effect, and reduce noise. The acquired MPM image quality is critically affected by PSF blurring and intense noise. The PSF in MPM is highly spread in 3D and is not well characterized, presenting high variability with respect to the observed objects. This makes the restoration of MPM images challenging. Common PSF estimation methods in fluorescence microscopy, including MPM, involve capturing images of sub-resolution beads, followed by quantifying the resulting ellipsoidal 3D spot. In this work, we revisit this approach, coping with its inherent limitations in terms of accuracy and practicality. We estimate the PSF from the observation of relatively large beads (approximately 1 μm in diameter). This goes through the formulation and resolution of an original non-convex minimization problem, for which we propose a proximal alternating method along with convergence guarantees. Following the PSF estimation step, we then introduce an innovative strategy to deal with the high level multiplicative noise degrading the acquisitions. We rely on a heteroscedastic noise model for which we estimate the parameters. We then solve a constrained optimization problem to restore the image, accounting for the estimated PSF and noise, while allowing a minimal hyper-parameter tuning. Theoretical guarantees are given for the restoration algorithm. These algorithmic contributions lead to an end-to-end pipeline for 3D image restoration in MPM, that we share as a publicly available Python software. We demonstrate its effectiveness through several experiments on both simulated and real data.

Salt Tolerance Assessment in Triticum Aestivum and Triticum Durum.

Salt stress is a multicomponent phenomenon; it includes many processes that directly or indirectly affect the plant. Attempts have been made to comprehensively consider the processes of salt stress in plants Triticum aestivum (variety Orenburgskaya 22) and Triticum durum (variety Zolotaya). The study used methods of light and fluorescence microscopy, methods of immunofluorodetection, expression of DNA methyltransferase genes, genes of the ion transporter and superoxide dismutase families, as well as biochemical determination of total antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent. According to morphometric indicators, the Orenburgskaya 22 variety showed greater tolerance to the action of 150 mM NaCl than the Zolotaya variety. The level of expression of genes of the HKT ion transporter family in the Orenburgskaya 22 variety is higher than in the Zolotaya variety. It was found that the expression of the DNA methyltransferase gene DRM2.1, which post-translationally methylates cytosine residues, is 22.3 times higher in Zolotaya compared to Orenburg 22 when exposed to salt. The accumulation of toxic ions is accompanied by an increase in reactive oxygen species (ROS) and increased damage to root tissue, especially in the Zolotaya variety. Using fluorescence microscopy using the Carboxy-H2DFF marker in the Orenburgskaya 22 variety at high NaCl concentrations, the highest fluorescence intensity was determined in the cap zone; in the Zolotaya variety-in the zones of the cap and root meristem. Excess ROS is more successfully removed in the Orenburgskaya 22 variety, which has a higher level of antioxidant activity (AOA), as well as the level of expression of the Cu/ZnSOD and MnSOD superoxide dismutase genes. Using programmed cell death (PCD) markers based on the release of cytochrome c from mitochondria into the cytoplasm, DNA breakage and the release of phosphatidylserine from mitochondria, the degree of damage to root cells was assessed in both wheat varieties. It has been proven that wheat cell death occurs through the mitochondrial pathway. It was noted that the salt-sensitive variety Zolotaya had a significant number of necrotic cells. Based on the data obtained, it was concluded that the Orenburgskaya 22 variety exhibits greater resistance to salinity than the Zolotaya variety. These data may be of practical importance for enhancing protective mechanisms under abiotic stress.

Tetrodotoxins in Tissues and Cells of Different Body Regions of Ribbon Worms Kulikovia alborostrata and K. manchenkoi from Spokoynaya Bay, Sea of Japan.

Nemerteans, or ribbon worms, possess tetrodotoxin and its analogues (TTXs), neurotoxins of bacterial origin, which they presumably use for capturing prey and self-defense. Most TTXs-containing nemertean species have low levels of these toxins and, therefore, have usually been neglected in studies of TTXs functions and accumulation. In the present study, Kulikovia alborostrata and K. manchenkoi, two closely related species, were analyzed for TTXs distribution in the body using the HPLC-MS/MS and fluorescence microscopy methods. The abundance of TTXs-positive cells was determined in the proboscis, integument, and digestive system epithelium. As a result, six TTXs-positive cell types were identified in each species; however, only four were common. Moreover, the proportions of the toxins in different body parts were estimated. According to the HPLC-MS/MS analysis, the TTXs concentrations in K. alborostrata varied from 0.91 ng/g in the proboscis to 5.52 ng/g in the precerebral region; in K. manchenkoi, the concentrations ranged from 7.47 ng/g in the proboscis to 72.32 ng/g in the posterior body region. The differences observed between the two nemerteans in the distribution of the TTXs were consistent with the differences in the localization of TTXs-positive cells. In addition, TTXs-positive glandular cell types were found in the intestine and characterized for the first time. TTXs in the new cell types were assumed to play a unique physiological role for nemerteans.

Large-field and fast super-resolution microscopic imaging method based on laser interferometry

In recent years, structured illumination microscopy (SIM) has been drawing great attention for both technique development and application. However, conventional SIM, which uses a spatial light modulator (SLM) for fringe projection, often has a limited field of view. To meet the demand for high-throughput microscopic imaging in biomedicine research, a large-field super-resolution (SR) fluorescence microscopic imaging method based on laser interferometry was proposed. The method that combines a two-dimensional (2D) grating for fringe pattern projection and an SLM for selecting fringe orientation can break the limitation of fringe number limited by the digital projection devices. A spatial-domain reconstruction algorithm was developed to improve the computational speed of super-resolution imaging. Finally, an experimental platform for SIM microscopy was established. A large-field view of 1380 μm × 1035 μm under a 20×/NA0.75 objective is experimentally demonstrated, and an enhancement of 1.8-fold resolution is realized. The spatial-domain reconstruction algorithm can significantly improve the computational speed by approximately 10 times faster compared to the traditional frequency-domain algorithm.

The intracellular visualization of exogenous DNA in fluorescence microscopy

In the development of non-viral gene delivery vectors, it is essential to reliably localize and quantify transfected DNA inside the cell. To track DNA, fluorescence microscopy methods are commonly applied. These mostly rely on fluorescently labeled DNA, DNA binding proteins fused to a fluorescent protein, or fluorescence in situ hybridization (FISH). In addition, co-stainings are often used to determine the colocalization of the DNA in specific cellular compartments, such as the endolysosomes or the nucleus. We provide an overview of these DNA tracking methods, advice on how they should be combined, and indicate which co-stainings or additional methods are required to draw precise conclusions from a DNA tracking experiment. Some emphasis is given to the localization of exogenous DNA inside the nucleus, which is the last step of DNA delivery. We argue that suitable tools which allow for the nuclear detection of faint signals are still missing, hampering the rational development of more efficient non-viral transfection systems.Graphical abstractThe intracellular visualization of exogenous DNA in fluorescence microscopy. Created with BioRender.com.

Single-Molecule Spectroscopy and Super-Resolution Mapping of Physicochemical Parameters in Living Cells.

By superlocalizing the positions of millions of single molecules over many camera frames, a class of super-resolution fluorescence microscopy methods known as single-molecule localization microscopy (SMLM) has revolutionized how we understand subcellular structures over the past decade. In this review, we highlight emerging studies that transcend the outstanding structural (shape) information offered by SMLM to extract and map physicochemical parameters in living mammalian cells at single-molecule and super-resolution levels. By encoding/decoding high-dimensional information-such as emission and excitation spectra, motion, polarization, fluorescence lifetime, and beyond-for every molecule, and mass accumulating these measurements for millions of molecules, such multidimensional and multifunctional super-resolution approaches open new windows into intracellular architectures and dynamics, as well as their underlying biophysical rules, far beyond the diffraction limit.

Effect of Syzygium cumini on Oxidative Stress Induced Cardiac Cellular Anomalies.

Doxorubicin (Dox), an antineoplastic agent is used as a primary anticancerous drug against various types of cancers. However, its associated toxicity to the cardiovascular system is major. Literature has recorded the cases of mortality due to poor validation and lack of prediagnosis of Dox-induced cardiotoxicity. Therapeutic interventions using natural products having cardioprotective properties with low toxic outcomes hold therapeutic potential for future cardio-oncological therapies. Syzygium cumini (Black berry), a traditional Indian herbal plant, has been researched and found to exert cardioprotective, anti-inflammatory, and antioxidant activities, which have been credited due to the presence of polyphenols, flavonoids, and tannins. In the current research, we investigated the cardioprotective potential of Syzygium cumini against Doxorubicin-induced cardiotoxicity (DIC) in H9C2 cardiomyocytes. Methanolic seed extract preparation of Syzygium cumini was performed using the Soxhlet apparatus. Cell viability and cell death assays were performed to determine the cardiotoxic doses of Doxorubicin. Furthermore, the cardioprotective potential of Syzygium cumini extract against DIC was studied. Morphological and nuclear alterations in H9C2 cells were studied by microscopic assays using Giemsa, Haematoxylin-Eosin stain, and PI. The intracellular stress level and ROS production were studied using DCFH-DA followed by mitochondrial integrity analysis using fluorescent microscopic methods. In the results, we investigated that Dox exerted a dose and time-dependent cardiotoxicity on H9C2 cardiomyocytes. Moreover, we observed that morphological and nuclear alterations caused by doxorubicin in dose-dependent manner were prevented by supplementing with Syzygium cumini polyphenols and it attenuated the oxidative stress in H9C2 cardiomyocytes effectively. Conclusively, Syzygium cumini possesses cardioprotective potential in H9C2 cardiomyocytes in dox-induced cardiotoxicity.

Optically Imaging In Situ Effects of Electrochemical Cycling on Single Nanoparticle Electrocatalysis.

Single-nanoparticle studies often need one or a series of nanoparticle populations that are designed with differences in a nominally particular structural parameter to clarify the structure-activity relationship (SAR). However, the heterogeneity of various properties within any population would make it rather difficult to approach an ideal one-parameter control. In situ modification ensures the same nanoparticle to be investigated and also avoids complicating effects from the otherwise often needed ex situ operations. Herein, we apply electrochemical cycling to single platinum nanoparticles and optically examine their SAR. An electrocatalytic fluorescent microscopic method is established to evaluate the apparent catalytic activity of a number of single nanoparticles toward the oxygen reduction reaction. Meanwhile, dark-field microscopy with the substrate electrode under a cyclic potential control is found to be able to assess the electrochemically active surface area (ECSA) of single nanoparticles via induced chloride redox electrochemistry. Consequently, nanoparticles with drastically increased catalytic activity are discovered to have larger ECSAs upon potential regulation, and interestingly, there are also a few particles with decreased activity, as opposed to the overall trend, that all develop a smaller ECSA in the process. The deactivated nanoparticles against the overall enhancement effects of potential cycling are revealed for the first time. As such, the SAR of single nanoparticles when subjected to an in situ structural control is optically demonstrated.