Light Fluorescence Microscopy Research Articles

Double-layer coated particles formed by one-step method based on microfluidic technology

This study used a microfluidic device with a focus-wrapping structure to create double-layer calcium alginate hydrogel drug particles in a one-step process. We validated the double-layer structure of the particles using both a fluorescence and regular light microscope. Curcumin and catalase were distributed independently in each layer, and we expected that such structure could play a role in the slow release of drugs. This scheme greatly reduces the need of hydrophilic and hydrophobic modification treatment, therefore greatly simplifies the experimental process. In the meantime, the requirement for injecting drugs into osteoarthritis and other diseases is expected to be realized, expanding the use of hydrogel in the medical field, because the microsphere is easy to generate, inexpensive, and strong in internal drug substitutability.

Differential Expression of Stress Adaptation Genes in a Diatom Ulnaria acus under Different Culture Conditions.

Diatoms are a group of unicellular eukaryotes that are essential primary producers in aquatic ecosystems. The dynamic nature of their habitat necessitates a quick and specific response to various stresses. However, the molecular mechanisms of their physiological adaptations are still underexplored. In this work, we study the response of the cosmopolitan freshwater diatom Ulnaria acus (Bacillariophyceae, Fragilariophycidae, Licmophorales, Ulnariaceae, Ulnaria) in relation to a range of stress factors, namely silica deficiency, prolonged cultivation, and interaction with an algicidal bacterium. Fluorescent staining and light microscopy were used to determine the physiological state of cells under these stresses. To explore molecular reactions, we studied the genes involved in the stress response-type III metacaspase (MC), metacaspase-like proteases (MCP), death-specific protein (DSP), delta-1-pyrroline-5-carboxylate dehydrogenase (ALDH12), and glutathione synthetase (GSHS). We have described the structure of these genes, analyzed the predicted amino acid sequences, and measured their expression dynamics in vitro using qRT-PCR. We demonstrated that the expression of UaMC1, UaMC3, and UaDSP increased during the first five days of silicon starvation. On the seventh day, it was replaced with the expression of UaMC2, UaGSHS, and UaALDH. After 45 days of culture, cells stopped growing, and the expression of UaMC1, UaMC2, UaGSHS, and UaDSP increased. Exposure to an algicidal bacterial filtrate induced a higher expression of UaMC1 and UaGSHS. Thus, we can conclude that these proteins are involved in diatoms' adaptions to environmental changes. Further, these data show that the molecular adaptation mechanisms in diatoms depend on the nature and exposure duration of a stress factor.

Vesicle condensation induced by synapsin: condensate size, geometry, and vesicle shape deformations

We study the formation of vesicle condensates induced by the protein synapsin, as a cell-free model system mimicking vesicle pool formation in the synapse. The system can be considered as an example of liquid–liquid phase separation (LLPS) in biomolecular fluids, where one phase is a complex fluid itself consisting of vesicles and a protein network. We address the pertinent question why the LLPS is self-limiting and stops at a certain size, i.e., why macroscopic phase separation is prevented. Using fluorescence light microscopy, we observe different morphologies of the condensates (aggregates) depending on the protein-to-lipid ratio. Cryogenic electron microscopy then allows us to resolve individual vesicle positions and shapes in a condensate and notably the size and geometry of adhesion zones between vesicles. We hypothesize that the membrane tension induced by already formed adhesion zones then in turn limits the capability of vesicles to bind additional vesicles, resulting in a finite condensate size. In a simple numerical toy model we show that this effect can be accounted for by redistribution of effective binding particles on the vesicle surface, accounting for the synapsin-induced adhesion zone.

A correlated light and electron microscopy approach to study the subcellular localization of phosphorylated vimentin in human lung tissue.

Correlative microscopy is an important approach for bridging the resolution gap between fluorescence light and electron microscopy. Here, we describe a fast and simple method for correlative immunofluorescence and immunogold labeling on the same section to elucidate the localization of phosphorylated vimentin (P-Vim), a robust feature of pulmonary vascular remodeling in cells of human lung small arteries. The lung is a complex, soft and difficult tissue to prepare for transmission electron microscopy (TEM). Detailing the molecular composition of small pulmonary arteries (<500μm) would be of great significance for research and diagnostics. Using the classical methods of immunochemistry (either hydrophilic resin or thin cryosections), is difficult to locate small arteries for analysis by TEM. To address this problem and to observe the same structures by both light and electron microscopy, correlative microscopy is a reliable approach. Immunofluorescence enables us to know the distribution of P-Vim in cells but does not provide ultrastructural detail on its localization. Labeled structures selected by fluorescence microscope can be identified and further analyzed by TEM at high resolution. With our method, the morphology of the arteries is well preserved, enabling the localization of P-Vim inside pulmonary endothelial cells. By applying this approach, fluorescent signals can be directly correlated to the corresponding subcellular structures in areas of interest.

Morphological and lipid metabolism alterations in macrophages exposed to model environmental nanoplastics traced by high-resolution synchrotron techniques.

The release of nanoplastics (NPs) in the environment is a significant health concern for long-term exposed humans. Although their usage has certainly revolutionized several application fields, at nanometer size, NPs can easily interact at the cellular level, resulting in potential harmful effects. Micro/Nanoplastics (M/NPs) have a demonstrated impact on mammalian endocrine components, such as the thyroid, adrenal gland, testes, and ovaries, while more investigations on prenatal and postnatal exposure are urgently required. The number of literature studies on the NPs' presence in biological samples is increasing. However, only a few offer a close study on the model environmental NP-immune system interaction exploited by advanced microscopy techniques. The present study highlights substantial morphological and lipid metabolism alterations in human M1 macrophages exposed to labeled polypropylene and polyvinyl chloride nanoparticles (PP and PVC NPs) (20 μg/ml). The results are interpreted by advanced microscopy techniques combined with standard laboratory tests and fluorescence microscopy. We report the accurate detection of polymeric nanoparticles doped with cadmium selenide quantum dots (CdSe-QDs NPs) by following the Se (L line) X-ray fluorescence emission peak at higher sub-cellular resolution, compared to the supportive light fluorescence microscopy. In addition, scanning transmission X-ray microscopy (STXM) imaging successfully revealed morphological changes in NP-exposed macrophages, providing input for Fourier transform infrared (FTIR) spectroscopy analyses, which underlined the chemical modifications in macromolecular components, specifically in lipid response. The present evidence was confirmed by quantifying the lipid droplet (LD) contents in PP and PVC NPs-exposed macrophages (0-100 μg/ml) by Oil Red O staining. Hence, even at experimental NPs' concentrations and incubation time, they do not significantly affect cell viability; they cause an evident lipid metabolism impairment, a hallmark of phagocytosis and oxidative stress.

Fluorescence-guided Cryo-lift-out using an Integrated Fluorescence Light Microscope and an Optimized Sample-needle Attachment Procedure.

Fluorescence-guided Cryo-lift-out using an Integrated Fluorescence Light Microscope and an Optimized Sample-needle Attachment Procedure.

High-Throughput Correlative Light and Cryo-Electron Microscopy Pipeline Using PRIMO Micropatterning, CERES Ice Shield and the METEOR In-Chamber Fluorescence Light Microscope.

High-Throughput Correlative Light and Cryo-Electron Microscopy Pipeline Using PRIMO Micropatterning, CERES Ice Shield and the METEOR In-Chamber Fluorescence Light Microscope.

What do we know about blood-testis barrier? current understanding of its structure and physiology.

Blood-testis barrier (BTB) creates a particular compartment in the seminiferous epithelium. Contacting Sertoli cell-Sertoli cell plasma membranes possess specialized junction proteins which present a complex dynamic of formation and dismantling. Thus, these specialized structures facilitate germ cell movement across the BTB. Junctions are constantly rearranged during spermatogenesis while the BTB preserves its barrier function. Imaging methods are essential to studying the dynamic of this sophisticated structure in order to understand its functional morphology. Isolated Sertoli cell cultures cannot represent the multiple interactions of the seminiferous epithelium and in situ studies became a fundamental approach to analyze BTB dynamics. In this review, we discuss the contributions of high-resolution microscopy studies to enlarge the body of morphofunctional data to understand the biology of the BTB as a dynamic structure. The first morphological evidence of the BTB was based on a fine structure of the junctions, which was resolved with Transmission Electron Microscopy. The use of conventional Fluorescent Light Microscopy to examine labelled molecules emerged as a fundamental technique for elucidating the precise protein localization at the BTB. Then laser-scanning confocal microscopy allowed the study of three-dimensional structures and complexes at the seminiferous epithelium. Several junction proteins, like the transmembrane, scaffold and signaling proteins, were identified in the testis using traditional animal models. BTB morphology was analyzed in different physiological conditions as the spermatocyte movement during meiosis, testis development, and seasonal spermatogenesis, but also structural elements, proteins, and BTB permeability were studied. Under pathological, pharmacological, or pollutant/toxic conditions, there are significant studies that provide high-resolution images which help to understand the dynamic of the BTB. Notwithstanding the advances, further research using new technologies is required to gain information on the BTB. Super-resolution light microscopy is needed to provide new research with high-quality images of targeted molecules at a nanometer-scale resolution. Finally, we highlight research areas that warrant future studies, pinpointing new microscopy approaches and helping to improve our ability to understand this barrier complexity.

Teucrium fruticans L., a Multi-Scale Study: From Trichomes to Essential Oil.

This work represents the first multi-scale study on Teucrium fruticans L. cultivated at the Ghirardi Botanic Garden (Lombardy, Northern Italy), combining a micromorphological and a phytochemical survey on the plant's aerial parts. Micromorphological investigations, performed by Light Microscopy, Fluorescence Microscopy and Scanning Electron Microscopy, highlighted the presence of five trichomes morphotypes, distinguished by a different distribution pattern: peltates, short-stalked and ball-like medium-stalked capitates, ubiquitous on the whole plant, medium-stalked and long-stalked capitates, exclusive to the floral whorls. Both peltates and medium-stalked capitates were recognized as the main terpene production sites. Phytochemical characterization focused on the essential oils (EOs), obtained by Clevenger-type hydrodistillation in February and April 2022 and characterized by Gas Chromatography-Mass Spectrometry (GC/MS), which resulted mainly formed by sesquiterpene hydrocarbons. The February EO profile was characterized by β-caryophyllene (28.30 %) and germacrene D (19.16 %) as main compounds, while in April β-myrcene was detected at high percentage (13.77 %), in addition to the previous two components (15.72 % and 11.55 %, respectively). Literature data, dealing with the biological activities of the main oil constituents, highlighted an anti-microbial, anti-inflammatory, and anti-tumor potential, due to the high content in sesquiterpenes and, particularly, of β-caryophyllene and germacrene D.

The Bacterial Nucleoid: From Electron Microscopy to Polymer Physics-A Personal Recollection.

In the 1960s, electron microscopy did not provide a clear answer regarding the compact or dispersed organization of the bacterial nucleoid. This was due to the necessary preparation steps of fixation and dehydration (for embedding) and freezing (for freeze-fracturing). Nevertheless, it was possible to measure the lengths of nucleoids in thin sections of slow-growing Escherichia coli cells, showing their gradual increase along with cell elongation. Later, through application of the so-called agar filtration method for electron microscopy, we were able to perform accurate measurements of cell size and shape. The introduction of confocal and fluorescence light microscopy enabled measurements of size and position of the bacterial nucleoid in living cells, inducing the concepts of "nucleoid occlusion" for localizing cell division and of "transertion" for the final step of nucleoid segregation. The question of why the DNA does not spread throughout the cytoplasm was approached by applying polymer-physical concepts of interactions between DNA and proteins. This gave a mechanistic insight in the depletion of proteins from the nucleoid, in accordance with its low refractive index observed by phase-contrast microscopy. Although in most bacterial species, the widely conserved proteins of the ParABS-system play a role in directing the segregation of newly replicated DNA strands, the basis for the separation and opposing movement of the chromosome arms was proposed to lie in preventing intermingling of nascent daughter strands already in the early replication bubble. E. coli, lacking the ParABS system, may be suitable for investigating this basic mechanism of DNA strand separation and segregation.

Cytoplasmic contractile injection systems mediate cell death in Streptomyces

Contractile injection systems (CIS) are bacteriophage tail-like structures that mediate bacterial cell–cell interactions. While CIS are highly abundant across diverse bacterial phyla, representative gene clusters in Gram-positive organisms remain poorly studied. Here we characterize a CIS in the Gram-positive multicellular model organism Streptomyces coelicolor and show that, in contrast to most other CIS, S. coelicolor CIS (CISSc) mediate cell death in response to stress and impact cellular development. CISSc are expressed in the cytoplasm of vegetative hyphae and are not released into the medium. Our cryo-electron microscopy structure enabled the engineering of non-contractile and fluorescently tagged CISSc assemblies. Cryo-electron tomography showed that CISSc contraction is linked to reduced cellular integrity. Fluorescence light microscopy furthermore revealed that functional CISSc mediate cell death upon encountering different types of stress. The absence of functional CISSc had an impact on hyphal differentiation and secondary metabolite production. Finally, we identified three putative effector proteins, which when absent, phenocopied other CISSc mutants. Our results provide new functional insights into CIS in Gram-positive organisms and a framework for studying novel intracellular roles, including regulated cell death and life-cycle progression in multicellular bacteria.

High-resolution and high-speed 3D tracking of microrobots using a fluorescent light field microscope.

Imaging and tracking are crucial for microrobots which navigate through complex 3D environments. Fluorescent imaging (FI) by microscope offers a high-resolution and high-sensitive imaging method to study the property of microrobots. However, conventional microscope suffers from shallow depth of field (DOF) and lacks 3D imaging capability. We proposed a high-resolution and high-speed 3D tracking method for microrobots based on a fluorescent light field microscope (FLFM). We designed the FLFM system according to the size of a representative helical microrobot (150 μm body length, 50 μm screw diameter), and studied the system's performance. We also proposed a 3D tracking algorithm for microrobots using digital refocusing. We validated the method by simulations and built an FLFM system to perform the tracking experiments of microrobots with representative size. Our 3D tracking method achieves a 30 fps data acquisition rate, 10 μm lateral resolution and approximately 40 μm axial resolution over a volume of 1,200×1,200×326 μm3. Results indicate that the accuracy of the method can reach about 9 μm. Compared with the FI by a conventional microscope, the FLFM-based method gains wider DOF and 3D imaging capability with a single-shot image. The tracking method succeeds in providing the trajectory of the microrobot with a good lateral resolution.

Feeding rates of malaria vectors from a prototype attractive sugar bait station in Western Province, Zambia: results of an entomological validation study

BackgroundAttractive targeted sugar bait (ATSB) stations are a promising new approach to malaria vector control that could compliment current tools by exploiting the natural sugar feeding behaviors of mosquitoes. Recent proof of concept work with a prototype ATSB® Sarabi Bait Station (Westham Co., Hod-Hasharon, Israel) has demonstrated high feeding rates and significant reductions in vector density, human biting rate, and overall entomological inoculation rate for Anopheles gambiae sensu lato (s.l.) in the tropical savannah of western Mali. The study reported here was conducted in the more temperate, rainier region of Western Province, Zambia and was designed to confirm the primary vector species in region and to estimate corresponding rates of feeding from prototype attractive sugar bait (ASB) Sarabi Bait Stations.MethodsThe product evaluated was the Sarabi v1.1.1 ASB station, which did not include insecticide but did include 0.8% uranine as a dye allowing for the detection, using UV fluorescence light microscopy, of mosquitoes that have acquired a sugar meal from the ASB. A two-phase, crossover study design was conducted in 10 village-based clusters in Western Province, Zambia. One study arm initially received 2 ASB stations per eligible structure while the other initially received 3. Primary mosquito sampling occurred via indoor and outdoor CDC Miniature UV Light Trap collection from March 01 through April 09, 2021 (Phase 1) and from April 19 to May 28, 2021 (Phase 2).ResultsThe dominant vector in the study area is Anopheles funestus s.l., which was the most abundant species group collected (31% of all Anophelines; 45,038/144,5550), had the highest sporozoite rate (3.16%; 66 positives out of 2,090 tested), and accounted for 94.3% (66/70) of all sporozoite positive specimens. Of those An. funestus specimens further identified to species, 97.2% (2,090/2,150) were An. funestus sensu stricto (s.s.). Anopheles gambiae s.l. (96.8% of which were Anopheles arabiensis) is a likely secondary vector and Anopheles squamosus may play a minor role in transmission. Overall, 21.6% (9,218/42,587) of An. funestus specimens and 10.4% (201/1,940) of An. gambiae specimens collected were positive for uranine, translating into an estimated daily feeding rate of 8.9% [7.7–9.9%] for An. funestus (inter-cluster range of 5.5% to 12.7%) and 3.9% [3.3–4.7%] for An. gambiae (inter-cluster range of 1.0–5.2%). Feeding rates were no different among mosquitoes collected indoors or outdoors, or among mosquitoes from clusters with 2 or 3 ASBs per eligible structure. Similarly, there were no correlations observed between feeding rates and the average number of ASB stations per hectare or with weekly rainfall amounts.ConclusionsAnopheles funestus and An. gambiae vector populations in Western Province, Zambia readily fed from the prototype Sarabi v1.1.1 ASB sugar bait station. Observed feeding rates are in line with those thought to be required for ATSB stations to achieve reductions in malaria transmission when used in combination with conventional control methods (IRS or LLIN). These results supported the decision to implement a large-scale, epidemiological cluster randomized controlled trial of ATSB in Zambia, deploying 2 ATSB stations per eligible structure.

A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals

Crystallizing G protein-coupled receptors (GPCRs) in lipidic cubic phase (LCP) often yields crystals suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, sample preparation is challenging. Embedded crystals cannot be targeted topologically. Here, we use an integrated fluorescence light microscope (iFLM) inside of a focused ion beam and scanning electron microscope (FIB-SEM) to identify fluorescently labeled GPCR crystals. Crystals are targeted using the iFLM and LCP is milled using a plasma focused ion beam (pFIB). The optimal ion source for preparing biological lamellae is identified using standard crystals of proteinase K. Lamellae prepared using either argon or xenon produced the highest quality data and structures. MicroED data are collected from the milled lamellae and the structures are determined. This study outlines a robust approach to identify and mill membrane protein crystals for MicroED and demonstrates plasma ion-beam milling is a powerful tool for preparing biological lamellae.

Improving cryo-electron tomography data quality and throughput by streamlining the workflow

Cryo-electron tomography (cryo-ET) is an extremely powerful technique that allows studies of the cellular landscape at high resolution in a near-native state. In recent years the technique has gained popularity and an increasing number of research groups are implementing it in their lab. The power of cryo-ET is undisputed but the fact that the workflow is very error-prone has limited the data output [1]. In the workflow, there are two main challenges: keeping the sample ice contamination-free and targeting the region of interest (ROI). At Delmic we developed two workflow solutions called CERES and METEOR to streamline the entire cryo-ET preparation workflow. The CERES Ice Defence System is based on the tools developed by Tacke et al. [2] and consists of multiple innovative tools that are tailored to minimize ice contamination during sample handling (Clean Station), transfer (Vitri-Lock) and FIB milling (Ice Shield). METEOR is an integrated fluorescence light microscope (FLM) that greatly enhances the ROI targeting inside the cryo-FIB/SEM while reducing the number of handling steps required [3]. We show that combining these innovative tools can lead to improved cryo-ET data quality and throughput.

A Flexible and Efficient Microfluidics Platform for the Characterization and Isolation of Novel Bacteriophages.

Bacteriophages are viruses that infect bacteria. This property makes them highly suitable for varied uses in industry or in the development of the treatment of bacterial infections. However, the conventional methods that are used to isolate and analyze these bacteriophages from the environment are generally cumbersome and time consuming. Here, we adapted a high-throughput microfluidic setup for long-term analysis of bacteriophage-bacteria interaction and demonstrate isolation of phages from environmental samples. IMPORTANCE Bacteriophages are gaining increased attention for their potential application as agents to combat antibiotic-resistant infections. However, isolation and characterization of new phages are time consuming and limited by currently used methods. The microfluidics platform presented here allows the isolation and long-term analysis of phages and their effect on host cells with fluorescent light microscopy imaging. Furthermore, this new workflow allows high-throughput characterization of environmental samples for the identification of phages alongside gaining detailed insight into the host response. Taken together, this microfluidics platform will be a valuable tool for phage research, enabling faster and more efficient screening and characterization of host-phage interactions.

Root canal filling material in periapical lesions: A polarized and fluorescent light microscopy case series

Overfilling of the root canal can cause foreign body reactions and lead to pathophysiological responses. The aim of the study was to investigate the morphological properties of filling materials associated with periapical lesions through light, polarized, and fluorescent light microscopy analysis. This study reports two cases of root canal overfilling associated with unsuccessful endodontic procedures. In case #1, a 36-year-old woman complained of persistent pain in the region of tooth maxillary right first premolar, after extraction. Clinically, the patient was sensitive to any contact in the region. On radiography, a radiopaque area of 4 mm was observed in the apical region. In case #2, a 35-year-old man reported discomfort when pressing the left nostril, with a history of extraction of tooth maxillary left lateral incisor. Clinically, painless and immobile edema was observed in the overlying region. Radiographically, a radiolucent image with defined edges and a radiopaque dot within the radiolucent was visualized. In histopathology, case #1 was conclusive with foreign body granuloma, while case #2 with residual cyst. In both cases, an excisional biopsy of the lesion was performed, with complete resolution of the cases after the follow-up period. Recognizing the morphological and optical properties of endodontic lesions mixed with endodontic restorative materials is essential for an accurate and reliable histopathological report.

Correlative light and electron microscopy to explore the lytic immunological synapse between natural killer cells and cancer cells.

Cytotoxic lymphocytes, such as natural killer (NK) cells and cytotoxic T cells, can recognize and kill tumor cells by establishing a highly specialized cell-cell contact called the immunological synapse. The formation and lytic activity of the immunological synapse are accompanied by local changes in the organization, dynamics and molecular composition of the cell membrane, as well as the polarization of various cellular components, such as the cytoskeleton, vesicles and organelles. Characterization and understanding of the molecular and cellular processes underlying immunological synapse formation and activity requires the combination of complementary types of information provided by different imaging modalities, the correlation of which can be difficult. Correlative light and electron microscopy (CLEM) allows for the accurate correlation of functional information provided by fluorescent light microscopy with ultrastructural features provided by high-resolution electron microscopy. In this chapter, we present a detailed protocol describing each step to generate cell-cell conjugates between NK cells and cancer cells, and to analyze these conjugates by CLEM using separate confocal laser-scanning and transmission electron microscopes.

Measuring Cellular Ploidy In Situ by Light Microscopy.

Determining cellular DNA content is valuable in the study of numerous biological processes, including organ development and injury repair. While FACS analysis of dissociated cells is a widely used method for assaying ploidy in a tissue cell population, for many tissue samples, it is possible and convenient to measure ploidy in situ using light microscopy. Here, we present two protocols for measuring cellular ploidy in tissues. These protocols are based on our studies in Drosophila melanogaster, but these are applicable to other settings as well. We present example results from Drosophila hindgut, midgut, and wing imaginal disc as examples. The first protocol focuses on measuring DNA content from decondensed interphase nuclei, while the second protocol details the visualization of condensed chromosomes for ploidy determination, either from mitotic cells or from interphase cells with drug-induced chromosome condensation. These techniques can be completed in 1 day and require standard lab supplies as well as a fluorescence light microscope.

In Situ Analysis of Mitochondrial DNA Synthesis Using Metabolic Labeling Coupled to Fluorescence Microscopy.

Metabolic labeling with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU) enables the selective labeling of DNA synthesis in live cells. Newly synthesized EdU-containing DNA can be covalently modified after extraction or in fixed cells using copper-catalyzed azide-alkyne cycloaddition "click chemistry" reactions, enabling bioconjugation to various substrates including fluorophores for imaging studies. While often used to study nuclear DNA replication, EdU labeling can also be leveraged to detect the synthesis of organellar DNA in the cytoplasm of Eukaryotic cells. In this chapter, we outline methods for the application of EdU labeling to the study of mitochondrial genome synthesis in fixed cultured human cells, using fluorescent labeling and superresolution light microscopy.