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

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

PURPOSE: Long intergenic non-protein coding RNA 665 (LINC00665) plays a vital role in the development of cancer. Its function in hepatocellular carcinoma (HCC), however, remains largely unknown. MATERIALS AND METHODS: The expressions of LINC00665, miR-186-5p, and MAP4K3 were determined by qRT-PCR. Cell viability and apoptosis were evaluated by MTT and flow cytometry, respectively. Autophagic puncta formation was observed by fluorescence microscopy. Bioinformatics analysis, luciferase reporter assay, RNA immunoprecipitation, and RNA pulldown were performed to identify associations among LINC00665, miR-186-5p, and MAP4K3. Western blot was utilized to examine the expressions of MAP4K3, Beclin-1, and LC3. Tumor growth was evaluated in a xenograft model. RESULTS: Elevations in LINC00665 were observed in HCC tissues and cells. The overall survival of HCC patients with high levels of LINC00665 was shorter than those with low levels. In vitro, LINC00665 depletion inhibited viability and induced apoptosis and autophagy. miR-186-5p interacted with LINC00665 and was downregulated in HCC tissues and cells. Upregulation of miR-186-5p inhibited viability and induced apoptosis and autophagy, which were attenuated by upregulation of LINC00665. MAP4K3 was found to possess binding sites with miR-186-5p and was upregulated in HCC tissues and cells. MAP4K3 depletion inhibited viability and induced apoptosis and autophagy, which were attenuated by miR-186-5p inhibitor. In vivo, miR-186-5p expression was negatively correlated with LINC00665 or MAP4K3 in HCC tissues, while LINC00665 was positively correlated with MAP4K3. LINC00665 knockdown suppressed tumor growth. CONCLUSION: LINC00665 was involved in cell viability, apoptosis, and autophagy in HCC via miR-186-5p/MAP4K3 axis, which may provide a new approach for HCC treatment.
Apoptosis ; Autophagy ; Binding Sites ; Blotting, Western ; Carcinoma, Hepatocellular ; Cell Survival ; Computational Biology ; Flow Cytometry ; Heterografts ; Humans ; Immunoprecipitation ; In Vitro Techniques ; Luciferases ; Microscopy, Fluorescence ; RNA ; RNA, Long Noncoding ; Up-Regulation

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

Canine adenovirus type 1 (CAV-1) infection results in hepatitis in dogs. In this study, we investigated the biologic and genetic characteristics of the CAV-1 vaccine strain (CAV1V) to improve quality control about CAV vaccine. The identity of CAV1V as CAV-1 was confirmed based on its cytopathic effects and the results of hemagglutination (HA) and immunofluorescence assays, and electron microscopy. The CAV1V strain reached 10(7.5) TCID(50)/mL in MDCK cells at 4 days post-inoculation and exhibited hemmagglutination activity of 256 U using guinea pig erythrocytes. Intranuclear fluorescence in the infected cells was observed and typical adenoviruses were observed in electon microscope. CAV1V strain was identified as a CAV-1 strain by nucleotide sequence analysis. In a comparison of the nucleotide sequences of the fiber genes of several CAV strains, CAV1V showed the highest similarity (99.8%) with the GLAXO strain, which was isolated in Canada. Our biological characterization of CAV1V will facilitate quality control of the canine hepatitis vaccine.
Adenoviridae ; Adenoviruses, Canine ; Animals ; Base Sequence ; Canada ; Dogs ; Erythrocytes ; Fluorescence ; Fluorescent Antibody Technique ; Guinea Pigs ; Hemagglutination ; Hepatitis ; Madin Darby Canine Kidney Cells ; Microscopy, Electron ; Quality Control

OBJECTIVES: The purpose of this study was to investigate changes in the composition of artificial cariogenic biofilms using a Streptococcus mutans biofilm model over a period of time. METHODS: We analyzed the dry weight, colony forming unit (CFU) number, extracellular polysaccharide (EPS) biovolume, and acid production rate of S. mutans biofilms formed on saliva-coated hydroxyapatite discs after 26 h, 50 h, 74 h, 98 h, 171 h, and 195 h. In addition, we performed a laser scanning confocal fluorescence microscopy to determine the bacterial volume, EPS biovolume, and biofilm thickness. We calculated the biofilm density using dry weight and EPS biovolume. RESULTS: Over a period of time, there was no change in the CFU number and acid production rate of S. mutans biofilms, but there was an increase in the dry weight and EPS biovolume of S. mutans biofilms. The bacterial volume, EPS biovolume, and biofilm thickness only increased in the 50-h-old biofilm; however, no change was observed in 50-195-h-old biofilms. In addition, an increase in the biofilm density was observed over time. CONCLUSIONS: These results suggest that the acid production ability of cariogenic biofilms does not change, but the biofilm density increases over time. However, due to scientific information, further research needs to be conducted in the field of dentistry to get further insights on the progression of cariogenic biofilms over time.
Biofilms ; Dentistry ; Durapatite ; Microscopy, Fluorescence ; Stem Cells ; Streptococcus mutans