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

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

BACKGROUND: Silica particles (SPs) induce cell proliferation and osteogenic differentiation. We reported that SPs in the scaffold induced early stage osteogenic differentiation. METHODS: A polycaprolactone (PCL) scaffold was fabricated with a 10 wt% SPs. The surface of PCL scaffold was coated with a 10 µg/mL collagen solution. Next, the scaffold was conjugated with 2 µM SPs, 2 µg/mL bone morphogenetic protein 2 (BMP2), or 2 µM BMP2-conjugated SPs (BCSPs). Green fluorescent protein-coupled BMP2 was applied to fabricate the scaffold. The fluorescence intensity was analyzed by confocal microscopy. The mRNA levels of the early osteogenic differentiation marker, alkaline phosphatase (ALP), were analyzed by real-time quantitative polymerase chain reaction. Levels of BMP2, RUNX2, ERK1/2, and AKT were assessed by western blotting. RESULTS: ALP mRNA levels were significantly higher in the BCSP-conjugated scaffold than in the other scaffolds. In the early stage of osteogenic differentiation, the protein levels of BMP2, RUNX2, ERK1/2, and AKT in cells were significantly higher in the BCSP-conjugated scaffold than in other scaffolds. Thus, the BCSP composite scaffold induced rapid osteogenic differentiation. CONCLUSION: These results suggest that BCSP composite can be used to promote early stage osteogenic differentiation and show promise as a material for use in scaffolds for bone regeneration.
Alkaline Phosphatase ; Blotting, Western ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; Bone Regeneration ; Cell Proliferation ; Collagen ; Fluorescence ; Microscopy, Confocal ; Polymerase Chain Reaction ; RNA, Messenger ; Silicon Dioxide ; Stem Cells

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

OBJECTIVE: The aim of this study was to investigate the effects of 2 anti-malarial drugs, chloroquine (CQ) and hydroxychloroquine (HCQ), on inhibition of vascular smooth muscle cell (VSMC) proliferation both in vivo and in vitro via Adenosine monophosphate-activated protein kinase (AMPK) activation. METHODS: Protein and mRNA levels were determined by western blot analysis and real-time reverse transcription-polymerase chain reaction in primary rat VSMCs treated with CQ and HCQ, respectively. Cell proliferation was measured by flow cytometry and cell counting. Mice carotid arteries were ligated and treated with CQ or HCQ every other day for 3 weeks. Pathological changes of carotid arteries were visualized by both microscopy and fluorescence microscopy. RESULTS: CQ and HCQ increase AMPK phosphorylation in VSMCs. Both CQ and HCQ decrease platelet-derived growth factor-induced VSMC proliferation and cell cycle progression in an AMPK-dependent manner. In addition, CQ and HCQ inhibit Smad3 phosphorylation and VSMC proliferation induced by transforming growth factor-β1. Moreover, CQ and HCQ diminished neointimal proliferation in a mouse model of carotid artery ligation-induced neointima formation. CONCLUSION: The results demonstrated that CQ and HCQ inhibit cell proliferation and cell cycle progression in VSMCs via the AMPK-dependent signaling pathway. Carotid artery ligation-induced intima thickness was reduced in mouse arteries treated with CQ or HCQ, suggesting a role for antimalarial drugs in treating atherosclerosis and restenosis.
Adenosine ; AMP-Activated Protein Kinases ; Animals ; Antimalarials ; Arteries ; Atherosclerosis ; Blotting, Western ; Carotid Arteries ; Cell Count ; Cell Cycle ; Cell Proliferation ; Chloroquine ; Flow Cytometry ; Hydroxychloroquine ; In Vitro Techniques ; Mice ; Microscopy ; Microscopy, Fluorescence ; Muscle, Smooth, Vascular ; Neointima ; Phosphorylation ; Protein Kinases ; Rats ; RNA, Messenger

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