This paper reviews the research progress on live-cell super-resolution fluorescence microscopy, discusses the current research status and hotspots in this field, and summarizes the technological application of super-resolution fluorescence microscopy for live-cell imaging. To date, this field has gained progress in numerous aspects. Specifically, the structured illumination microscopy, stimulated emission depletion microscopy, and the recently introduced minimal photon fluxes microscopy are the current research hotspots. According to the current progress in this field, future development trend is likely to be largely driven by artificial intelligence as well as advances in fluorescent probes and relevant labelling methods.
Artificial Intelligence ; Microscopy, Fluorescence ; Fluorescent Dyes ; Technology

BACKGROUND/OBJECTIVES: Alzheimer's disease is a neurodegenerative disease that induces symptoms such as a decrease in motor function and cognitive impairment. Increases in the aggregation and deposition of amyloid beta protein (Aβ) in the brain may be closely correlated with the development of Alzheimer's disease. In this study, the effects of an adzuki bean extract on the aggregation of Aβ were examined; moreover, the anti-Alzheimer's activity of the adzuki extract was examined. MATERIALS/METHODS: First, we undertook thioflavin T (ThT) fluorescence analysis and transmission electron microscopy (TEM) to evaluate the effect of an adzuki bean extract on Aβ42 aggregation. To evaluate the effects of the adzuki extract on the symptoms of Alzheimer's disease in vivo, Aβ42-overexpressing Drosophila were used. In these flies, overexpression of Aβ42 induced the formation of Aβ42 aggregates in the brain, decreased motor function, and resulted in cognitive impairment. RESULTS: Based on the results obtained by ThT fluorescence assays and TEM, the adzuki bean extract inhibited the formation of Aβ42 aggregates in a concentration-dependent manner. When Aβ42-overexpressing flies were fed regular medium containing adzuki extract, the Aβ42 level in the brain was significantly lower than that in the group fed regular medium only. Furthermore, suppression of the decrease in motor function, suppression of cognitive impairment, and improvement in lifespan were observed in Aβ42-overexpressing flies fed regular medium with adzuki extract. CONCLUSIONS: The results reveal the delaying effects of an adzuki bean extract on the progression of Alzheimer's disease and provide useful information for identifying novel prevention treatments for Alzheimer's disease.
Alzheimer Disease ; Amyloid beta-Peptides ; Brain ; Cognition Disorders ; Diptera ; Drosophila ; Fluorescence ; Microscopy, Electron, Transmission ; Neurodegenerative Diseases

BACKGROUND: The objectives of this study were to design polycaprolactone nanofibers with a radial pattern using a modified electrospinning method and to evaluate the effect of radial nanofiber deposition on mechanical and biological properties compared to non-patterned samples. METHODS: Radially patterned polycaprolactone nanofibers were prepared with a modified electrospinning method and compared with randomly deposited nanofibers. The surface morphology of samples was observed under scanning electron microscopy (SEM). The tensile properties of nanofibrous mats were measured using a tabletop uniaxial testing machine. Fluorescence-stained human bone marrow stem cells were placed along the perimeter of the radially patterned and randomly deposited. Their migration toward the center was observed on days 1, 4, and 7, and quantitatively measured using ImageJ software. RESULTS: Overall, there were no statistically significant differences in mechanical properties between the two types of polycaprolactone nanofibrous mats. SEM images of the obtained samples suggested that the directionality of the nanofibers was toward the central area, regardless of where the nanofibers were located throughout the entire sample. Florescence images showed stronger fluorescence inside the circle in radially aligned nanofibers, with significant differences on days 4 and 7, indicating that migration was quicker along radially aligned nanofibers than along randomly deposited nanofibers. CONCLUSIONS: In this study, we successfully used modified electrospinning to fabricate radially aligned nanofibers with similar mechanical properties to those of conventional randomly aligned nanofibers. In addition, we observed faster migration along radially aligned nanofibers than along randomly deposited nanofibers. Collectively, the radially aligned nanofibers may have the potential for tissue regeneration in combination with stem cells.
Bandages ; Bone Marrow ; Fluorescence ; Humans ; Methods ; Microscopy, Electron, Scanning ; Nanofibers ; Polymers ; Regeneration ; Stem Cells ; Wound Healing ; Wounds and Injuries

Microsatellite instability (MSI) which resulted from the deficiency of DNA mismatch repair (MMR), is an important clinical significance in the related solid tumors, such as colorectal cancer and endometrial cancer. There are several methods to detect MSI status, including immunohistochemistry for MMR protein, multiplex fluorescent polymerase chain reaction (PCR) for microsatellite site and MSI algorithm based on next generation sequencing (NGS). The consensus elaborates the definition and clinical significance of MSI as well as the advantages and disadvantages of the three detection methods. Through this expert consensus, we hope to promote the screening which based on MSI status in malignant tumors and improve the acknowledge of clinicians about various testing methods. Thereby, they could interpret the results more accurately and provide better clinical services to patients.
Antineoplastic Agents ; administration & dosage ; adverse effects ; therapeutic use ; China ; Colorectal Neoplasms ; genetics ; pathology ; Consensus ; DNA Mismatch Repair ; DNA Sequence, Unstable ; Delivery of Health Care ; standards ; Endometrial Neoplasms ; Female ; Humans ; Immunohistochemistry ; Microsatellite Instability ; Microsatellite Repeats ; Microscopy, Fluorescence ; Polymerase Chain Reaction ; Practice Guidelines as Topic

Sparse labeling of neurons contributes to uncovering their morphology, and rapid expression of a fluorescent protein reduces the experiment range. To achieve the goal of rapid and sparse labeling of neurons in vivo, we established a rapid method for depicting the fine structure of neurons at 24 h post-infection based on a mutant virus-like particle of Semliki Forest virus. Approximately 0.014 fluorescent focus-forming units of the mutant virus-like particle transferred enhanced green fluorescent protein into neurons in vivo, and its affinity for neurons in vivo was stronger than for neurons in vitro and BHK21 (baby hamster kidney) cells. Collectively, the mutant virus-like particle provides a robust and convenient way to reveal the fine structure of neurons and is expected to be a helper virus for combining with other tools to determine their connectivity. Our work adds a new tool to the approaches for rapid and sparse labeling of neurons in vivo.
Animals ; Cells, Cultured ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Immunohistochemistry ; methods ; Male ; Mice, Inbred C57BL ; Microscopy, Fluorescence ; methods ; Neurons ; cytology ; metabolism ; Purkinje Cells ; cytology ; metabolism ; Semliki forest virus ; genetics

The complex spatial and temporal organization of neural activity in the brain is important for information-processing that guides behavior. Hence, revealing the real-time neural dynamics in freely-moving animals is fundamental to elucidating brain function. Miniature fluorescence microscopes have been developed to fulfil this requirement. With the help of GRadient INdex (GRIN) lenses that relay optical images from deep brain regions to the surface, investigators can visualize neural activity during behavioral tasks in freely-moving animals. However, the application of GRIN lenses to deep brain imaging is severely limited by their availability. Here, we describe a protocol for GRIN lens coating that ensures successful long-term intravital imaging with commercially-available GRIN lenses.
Animals ; Biocompatible Materials ; Brain ; physiology ; Hippocampus ; cytology ; Lenses ; Mice, Inbred C57BL ; Mice, Transgenic ; Microscopy, Fluorescence ; methods ; Neuroimaging ; instrumentation ; methods ; Neurons ; physiology

The aim of the present study was to establish a cell model of volume-regulated anion channel subunit LRRC8A and investigate the physiological characteristics of LRRC8A. The eukaryotic expression vectors of LRRC8A and YFP-H148Q/I152L were constructed and transfected into Fischer rat thyroid (FRT) cells by Lipofectamine 2000. The FRT cell lines co-expressing LRRC8A and YFP-H148Q/I152L were obtained by antibiotic screening. The expression of LRRC8A and YFP-H148Q/I152L in FRT cells was detected by the inverted fluorescence microscope. The fluorescence quenching kinetic experiment was done to verify the function and effectiveness of the cell model. Then the cell model was utilized to study the physiological characteristics of LRRC8A, such as the characteristics of anion transport, the opening of LRRC8A by osmotic pressure, the effect of anion transport velocity, and the effect of chloride channel inhibitors on LRRC8A anion channel. The results of the inverted fluorescence microscope showed that LRRC8A was expressed on the cell membrane and YFP-H148Q/I152L was expressed in the cytoplasm. The results of fluorescence quenching kinetic test showed that under the condition of low osmotic state, LRRC8A could transport some kinds of anions, such as iodine and chloride ions. Osmotic pressure played a key role in the regulation of LRRC8A volume-regulated anion channel opening. Chloride channel inhibitors inhibited ion transport of LRRC8A channel in a dose-dependent manner. It is suggested that LRRC8A has the characteristics of classic volume-regulated anion channels by using the cell model of FRT cells co-expressing LRRC8A and YFP-H148Q/I152L.
Animals ; Anions ; Cells, Cultured ; Chloride Channels ; antagonists & inhibitors ; Ion Transport ; Membrane Proteins ; physiology ; Microscopy, Fluorescence ; Rats ; Rats, Inbred F344 ; Thyroid Gland ; cytology ; Transfection

Agrobacterium tumefaciens-mediated transformation (ATMT), as a simple and versatile method, achieves successful transformation in the yeast-like cells (YLCs) of Tremella fuciformis with lower efficiency. Establishment of a more efficient transformation system of YLCs is important for functional genomics research and biotechnological application. In this study, an enzymolysis-assisted ATMT method was developed. The degradation degree of YLCs depends on the concentration and digestion time of Lywallzyme. Lower concentration (≤0.1%) of Lywallzyme was capable of formation of limited wounds on the surface of YLCs and has less influence on their growth. In addition, there is no significant difference of YLCs growth among groups treated with 0.1% Lywallzyme for different time. The binary vector pGEH under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter was utilized to transform the enzymolytic wounded YLCs with different concentrations and digestion time. The results of PCR, Southern blot, quantitative real-time PCR (qRT-PCR) and fluorescence microscopy revealed that the T-DNA was integrated into the YLCs genome, suggesting an efficient enzymolysis-assisted ATMT method of YLCs was established. The highest transformation frequency reached 1200 transformants per 106 YLCs by 0.05% (w/v) Lywallzyme digestion for 15 min, and the transformants were genetically stable. Compared with the mechanical wounding methods, enzymolytic wounding is thought to be a tender, safer and more effective method.
Agrobacterium ; Blotting, Southern ; Digestion ; Genome ; Genomics ; Methods ; Microscopy, Fluorescence ; Oxidoreductases ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Wounds and Injuries

Confirming the direct link between neural circuit activity and animal behavior has been a principal aim of neuroscience. The genetically encoded calcium indicator (GECI), which binds to calcium ions and emits fluorescence visualizing intracellular calcium concentration, enables detection of in vivo neuronal firing activity. Various GECIs have been developed and can be chosen for diverse purposes. These GECI-based signals can be acquired by several tools including two-photon microscopy and microendoscopy for precise or wide imaging at cellular to synaptic levels. In addition, the images from GECI signals can be analyzed with open source codes including constrained non-negative matrix factorization for endoscopy data (CNMF_E) and miniscope 1-photon-based calcium imaging signal extraction pipeline (MIN1PIPE), and considering parameters of the imaged brain regions (e.g., diameter or shape of soma or the resolution of recorded images), the real-time activity of each cell can be acquired and linked with animal behaviors. As a result, GECI signal analysis can be a powerful tool for revealing the functions of neuronal circuits related to specific behaviors.
Animals ; Behavior, Animal ; Brain ; Calcium Channels ; Calcium ; Carisoprodol ; Endoscopy ; Fires ; Fluorescence ; Ions ; Microscopy ; Neuronal Calcium-Sensor Proteins ; Neurons ; Neurosciences ; Statistics as Topic

BACKGROUND: Lonocyte-derived multipotential cells (MOMCs) include progenitors capable of differentiation into multiple cell lineages and thus represent an ideal autologous transplantable cell source for regenerative medicine. In this study, we cultured MOMCs, generated from mononuclear cells of peripheral blood, on the surface of nanocomposite thin films. METHODS: For this purpose, nanocomposite Poly(e-caprolactone) (PCL)-based thin films containing either 2.5 wt% silica nanotubes (SiO2ntbs) or strontium hydroxyapatite nanorods (SrHAnrds), were prepared using the spin-coating method. The induced differentiation capacity of MOMCs, towards bone and endothelium, was estimated using flow cytometry, real-time polymerase chain reaction, scanning electron microscopy and fluorescence microscopy after cells' genetic modification using the Sleeping Beauty Transposon System aiming their observation onto the scaffolds. Moreover, Wharton's Jelly Mesenchymal Stromal Cells were cultivated as a control cell line, while Human Umbilical Vein Endothelial Cells were used to strengthen and accelerate the differentiation procedure in semi-permeable culture systems. Finally, the cytotoxicity of the studied materials was checked with MTT assay. RESULTS: The highest differentiation capacity of MOMCs was observed on PCL/SiO2ntbs 2.5 wt% nanocomposite film, as they progressively lost their native markers and gained endothelial lineage, in both protein and transcriptional level. In addition, the presence of SrHAnrds in the PCL matrix triggered processes related to osteoblast bone formation. CONCLUSION: To conclude, the differentiation of MOMCs was selectively guided by incorporating SiO2ntbs or SrHAnrds into a polymeric matrix, for the first time.
Autografts ; Beauty ; Cell Line ; Cell Lineage ; Durapatite ; Endothelium ; Flow Cytometry ; Human Umbilical Vein Endothelial Cells ; Mesenchymal Stromal Cells ; Methods ; Microscopy, Electron, Scanning ; Microscopy, Fluorescence ; Nanocomposites ; Nanotubes ; Osteoblasts ; Osteogenesis ; Polymers ; Real-Time Polymerase Chain Reaction ; Regenerative Medicine ; Silicon Dioxide ; Strontium ; Wharton Jelly