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

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

This study was performed to evaluate clinical use of laser fluorescence (LF) to identify early childhood caries lesions suitable for applying resin infiltration.20 exfoliated primary molars with proximal caries were selected and cut buccolingually cross the central pit for regarding the mesial and distal surfaces respectively. 27 specimens corresponding to ICDAS code 1 and 2 were selected and the LF values were measured. When infiltrant resin was applied, double staining for microscopy detection has done simultaneously. Tooth samples were sliced with 0.7 mm thick. The maximum lesion depth, maximum penetration depth, and average penetration rate were measured from the confocal scanning laser microscope image. Pearson correlation analysis was performed.The intraclass correlation coefficient of LF values shows excellent agreement. LF values had positive correlation with penetration rate, but not lesion depth and penetration depth. Significant correlation between LF readings and penetration rate was verified in deep enamel caries and dentin caries except shallow enamel caries.Infiltrant resin could penetrate with a higher rate and LF values could be increased in more active caries lesions. In assessing radiologically similar caries lesion, laser fluorescence might be useful for identifying caries activity.
Dental Enamel ; Dentin ; Fluorescence ; Microscopy ; Molar ; Reading ; Tooth ; Tooth, Deciduous

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

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

Two photon fluorescence microscopy and the numerous technical advances to it have served as valuable tools in biomedical research. The fluorophores (exogenous or endogenous) absorb light and emit lower energy photons than the absorption energy and the emission (fluorescence) signal is measured using a fluorescence decay graph. Additionally, high spatial resolution images can be acquired in two photon fluorescence lifetime imaging (2P-FLIM) with improved penetration depth which helps in detection of fluorescence signal in vivo. 2P-FLIM is a non-invasive imaging technique in order to visualize cellular metabolic, by tracking intrinsic fluorophores present in it, such as nicotinamide adenine dinucleotide, flavin adenine dinucleotide and tryptophan etc. 2P-FLIM of these molecules enable the visualization of metabolic alterations, non-invasively. This comprehensive review discusses the numerous applications of 2P-FLIM towards cancer, neuro-degenerative, infectious diseases, and wound healing.
Absorption ; Communicable Diseases ; Flavin-Adenine Dinucleotide ; Fluorescence ; Microscopy, Fluorescence ; NAD ; Photons ; Tryptophan ; Wound Healing

C3 glomerulopathy is a renal disorder involving dysregulation of alternative pathway complement activation. In most instances, a membranoproliferative pattern of glomerular injury with a prevalence of C3 deposition is observed by immunofluorescence microscopy. Dense deposit disease (DDD) and C3 glomerulonephritis (C3GN) are subclasses of C3 glomerulopathy that are distinguishable by electron microscopy. Highly electron-dense transformation of glomerular basement membrane is characteristic of DDD. C3GN should be differentiated from post-infectious glomerulonephritis and other immune complex-mediated glomerulonephritides showing C3 deposits.
Complement Activation ; Complement Pathway, Alternative ; Dichlorodiphenyldichloroethane ; Glomerular Basement Membrane ; Glomerulonephritis ; Glomerulonephritis, Membranoproliferative ; Microscopy, Electron ; Microscopy, Fluorescence ; Pathology ; Prevalence