As a new kind of advanced nonlinear imaging approach, two-photon fluorescence microscopy technology is wildly used in the field of live cell and tissue imaging, especially focusing on long-term dynamic three-dimensional cell imaging. This paper firstly presents the principle and characteristic of two-photon fluorescence microscopy. Then, the paper focuses on the three key aspects of the viability of the specimen, sensitivity of detection, as well as the speed of acquisition. In the end, the future prospect of development and application of two-phonon imaging technology are predicted.
Diagnostic Imaging ; Microscopy, Fluorescence, Multiphoton ; methods

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

Live imaging has become an essential tool to investigate the coordinated activity and output of cellular networks. Within the last decade, 2 Nobel prizes have been awarded to recognize innovations in the field of imaging: one for the discovery, use, and optimization of the green fluorescent protein (2008) and the second for the development of super-resolved fluorescence microscopy (2014). New advances in both optogenetics and microscopy now enable researchers to record and manipulate activity from specific populations of cells with better contrast and resolution, at higher speeds, and deeper into live tissues. In this review, we will discuss some of the recent developments in microscope technology and in the synthesis of fluorescent probes, both synthetic and genetically encoded. We focus on how live imaging of cellular physiology has progressed our understanding of the control of gastrointestinal motility, and we discuss the hurdles to overcome in order to apply the novel tools in the field of neurogastroenterology and motility.
Awards and Prizes ; Enteric Nervous System ; Fluorescence ; Fluorescent Dyes ; Gastrointestinal Motility ; Microscopy ; Microscopy, Fluorescence ; Optogenetics ; Physiology

The purpose of this study was to evaluate the penetration pattern of dentin adhesives according to the orientation of dentinal tubules with confocal laser scanning microscopy. Specimens having perpendicular, parallel and oblique surface to dentinal tubules were fabricated. The primer of dentin adhesives (ALL BOND(R) 2, CLEARFIL(TM) SE BOND and PQ1) was mixed with fluorescent material, rhodamine B isothiocyanate (Aldrich Chem. CO., Milw., USA). It was applied to the specimens according to the instructions of manufactures. The specimens were covered with composite resin (Estelite, shade A2) and then cut to a thickness of 500 microm with low speed saw (Isomet(TM), Buehler, USA). The adhesive pattern of dentin adhesives were observed by fluorescence image using confocal laser scanning microscopy. The results were as follows. 1. For the groups with tubules perpendicular to bonded surface, funnel shape of resin tag was observed in all specimen. However, resin tags were more prominent in phosphoric acid etching system (ALL BOND(R) 2 and PQ1) than self etching system (CLEARFIL(TM) SE BOND). 2. For the groups with tubules parallel to bonded surface, rhodamine-labeled primer penetrated into peritubular dentin parallel to the orientation of dentinal tubules. But rhodamine-labeled primer of PQ1 diffused more radially into surrounding intertubular dentin than other dentin adhesive systems. 3. For the groups with tubules oblique to bonded surface, resin tags appeared irregular and discontinuous. But they penetrated deeper into dentinal tubules than other groups.
Adhesives* ; Dentin* ; Dentin-Bonding Agents ; Fluorescence ; Microscopy, Confocal* ; Rhodamines

Cyclodialysis and ciliochoroidal detachment was performed in one eye of three rabbits and in one eye of another three rabbits respectively. 0.1 ml of 10% sodium fluorescein was injected intracamerally after aspiration of aqueous humor and the eyeball was enucleated between 30 minutes and one hour after sodium fluorescein injection and prepared for fluorescence microscopy. Sodium fluorescein concentration in supraciliary space was much greater in group with cyclodialysis or ciliochoroidal detachment than in normal control group. These results suggest that in the eye with cyclodialysis, aqueous humor may gain access freely to supraciliary space through the cleft between anterior chamber and supraciliary space and then is removed rapidly and in the eye with ciliochoroidal detachment, aqueous humor may pass through uveoscleral outflow pathway.
Anterior Chamber ; Aqueous Humor* ; Fluorescein ; Microscopy, Fluorescence ; Rabbits

BACKGROUND: Ultraviolet(UV) light is one of the injurious environmental agents which is known to lead to apoptosis of cells. However, studies on UVB-induced apoptosis of melanocytes are still lacking and there are some discrepancies between researchers. OBJECTIVE: Our purpose was to evaluate the characteristics of UVB-induced apoptosis of melanocytes and G361 cells. METHODS: Cultured normal human melanocytes and malignant melanoma cell lines (G361 cells) were analyzed by several detection methods including morphological examination of propidium iodide(PI) stained cells under fluorescence microscopy, quantitation of fragmented DNA, and flow cytometric analysis. RESULTS: Both melanocytes and G361 cells showed similar rate of apoptosis with gradual increment of UVB doses by the quantitation of fragmented DNA. However, flow cytometric analysis using scatter properties and PI stainability revealed that the melanocytes were more resistant to UVB than G361 cells. CONCLUSION: We suggest that melanocytes seem to be more resistant to UVB-induced injury than G361 cells. In addition, various methods for the detection of apoptosis might be necessary for its study. (Ann Dermatol 10:(3) 147152, 1998).
Apoptosis* ; Cell Line ; DNA ; Humans* ; Melanocytes* ; Melanoma ; Microscopy, Fluorescence ; Propidium

To investigate the difference of texture exhibited on interproximal enamel surface with each different stripping method and the susceptibility of proximal enamel to demineralization after stripping and the application of a topical fluoride gel and sealant, one hundred human premolars, which were previously extracted for orthodontic reasons were evaluated by means of Scanning electron microscopy and laser fluorescence. The results were as follows : 1. No matter what the initial stripping instrument was the furrows that resulted from all the stripping methods were not completely removed by careful polishing. 2. Among the enamel surfaces that were treated with three different initial abrasive instruments, followed by the same polishing method (Sof-Lex(R)disks), the enamel surfaces that were treated with 700 crosscut carbide bur showed the smoothest surfaces. 3. The stripped teeth, no matter what the initial stripping instrument was, were less resistant to initial demineralization than untreated teeth. But no difference in caries susceptibility according to differently stripped methods was found (p<0.001). 4. Teeth treated with APF-gel or sealant were more resistant to demineralization than those treated without other treatment after stripping (p<0.001). 5. Comparing groups treated with APF-gel to groups treated with sealant, the former was more resistant to demineralization than the latter (p<0.05). In conclusion, enamel surfaces that were stripped interproximally were less resistant to demineralization even though various attempts were made to produce smooth, self-cleaning enamel surfaces. Therefore, additional treatment-sealant or calcifying/ fluoridating solution to the stripped enamel surfaces is recommended.
Bicuspid ; Dental Enamel* ; Fluorescence ; Fluorides ; Humans ; Microscopy, Electron, Scanning ; Tooth*

PURPOSE: The objective of this study was to investigate the effects of radiofrequency energy on human chondrocyte viability, and to correlate confocal laser microscopy fluorescence to sulfate uptake and to the histological integrity of articular cartilage. MATERIALS AND METHODS: The chondroplasty procedure for chondromalacic articular cartilage was performed using a 3.0-mm ArthroWand (Arthroscopic Electrosurgery System, ArthroCare Corporation) on fresh human articular cartilage. Radiofrequency energy was applied to the cartilage surface through the probe at a velocity of 10-mm per second in contact and non-contact mode. Three power settings were used. The treated cartilage was analyzed for chondrocyte viability by confocal laser microscopy and (35)S uptake. RESULTS: Confocal laser microscopy demonstrated partial-thickness chondrocyte death irrespective of treatment method. No mode of treatment or radiofrequency energy power setting resulted in full-thickness chondrocyte death. The depth of cartilage ablation was increased in the treated areas in contact mode in proportion to the power level and the time of treatment. No statistically significant difference in radiolabeled sulfate uptake of the specimens was observed with respect to the treatment modes and power settings. CONCLUSION: The extent of chondrocyte death by radiofrequency energy was not as significant as reported previously when the probe was moved at the speed of 10 mm/sec. Radiofrequency energy may be useful to treat chondromalacic cartilage in a contact mode using a proper energy level and delivery time.
Cartilage ; Cartilage, Articular* ; Chondrocytes* ; Electrosurgery ; Fluorescence ; Humans* ; Microscopy, Confocal

The cytotoxicological responses to insect growth regulator (IGR), using tebufenozide as ecdysteroid mimic, were investigated in Drosophila Kc cells. Treatment of Kc cells with tebufenozide showed significant growth inhibition and striking morphological changes including aggregation and elongation of the cells. In order to understand the cellular mechanism underlying the response of Drosophila cells to tebufenozide, immunofluorescence microscopy was performed. We found that treatment of Kc cells with tebufenozide enhanced the reorganization of f-actin and stimulated the expression of hsp27. These data suggest a possible association of filamentous actin (f-actin) and hsp27 in the cytotoxicological mechanisms of growth regulators in Drosophila cells.
Actins* ; Drosophila* ; Ecdysteroids ; Insects* ; Microscopy, Fluorescence ; Strikes, Employee

This study aimed to evaluate surface morphology and resin tag penetration of resin infiltration into primary anterior teeth after enamel deproteinization with sodium hypochlorite (NaOCl) prior to phosphoric acid (H₃PO₄) etching.Ninety primary anterior teeth with non-cavitated caries lesion were devided five groups according to enamel pretreatment as follows, group I-15% hydrochloric acid (HCl) 2min. ; group II-5.25% NaOCl 1min., 35% H₃PO₄ 1min. ; group III-5.25% NaOCl 2min., 35% H₃PO₄ 1min. ; group IV-5.25% NaOCl 1min., 35% H₃PO₄ 2min. ; group V-5.25% NaOCl 2min., 35% H3PO4 2min. Fifteen teeth were examined etched surface structure using field emission-scanning electron microscope. Seventy five teeth were infiltrated with resin, maximum penetration depth and percentage penetration were analysed using dual fluorescence confocal microscopy.As the application time of NaOCl increased, ratio of enamel type I, II were increased. Percentage penetration (PP) was higher in group V than group II, III (p < 0.05). PP of group IV, V did not show any differences.Non-cavitated caries of primary anterior teeth can be treated with resin infiltration. Enamel deproteinization with NaOCl prior to 35% H3PO4 etching could be an alternative of 15% HCl etching in resin infiltration.
Dental Enamel ; Fluorescence ; Hydrochloric Acid ; Microscopy, Confocal ; Sodium Hypochlorite ; Tooth