Scanning Switch-off Microscopy for Super-Resolution Fluorescence Imaging.

Super-resolution (SR) microscopy provides a revolutionary optical imaging approach by breaking the diffraction limit of light, while the commonly required special instrumentation with complex optical setup hampers its popularity. Here, we present a scanning switch-off microscopy (SSM) concept that exploits the omnipresent switch-off response of fluorophores to enable super-resolution imaging using a commercial confocal microscope. We validated the SSM model with theoretical calculations and experiments. An imaging resolution of ∼100 nm was obtained for DNA origami nanostructures and cellular cytoskeletons using fluorescent labels of Alexa 405, Alexa 488, Cy3, and Atto 488. Notably, super-resolution imaging of live cells was realized with SSM, by employing a dronpa fluorescent protein as the fluorescent label. In principle, this SSM method can be applied to any excitation laser scanning-based microscope.

Spinning Disk Confocal Microscopy for Optimized and Quantified Live Imaging of 3D Mitochondrial Network.

Mitochondria are the energy factories of a cell, and depending on the metabolic requirements, the mitochondrial morphology, quantity, and membrane potential in a cell change. These changes are frequently assessed using commercially available probes. In this study, we tested the suitability of three commercially available probes-namely 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolo-carbocyanine iodide (JC-1), MitoTracker Red CMX Rox (CMXRos), and tetramethylrhodamine methyl ester (TMRM)-for assessing the mitochondrial quantity, morphology, and membrane potential in living human mesoangioblasts in 3D with confocal laser scanning microscope (CLSM) and scanning disk confocal microscope (SDCM). Using CLSM, JC-1, and CMXRos-but not TMRM-uncovered considerable background and variation. Using SDCM, the background signal only remained apparent for the JC-1 monomer. Repetitive imaging of CMXRos and JC-1-but not TMRM-demonstrated a 1.5-2-fold variation in signal intensity between cells using CLSM. The use of SDCM drastically reduced this variation. The slope of the relative signal intensity upon repetitive imaging using CLSM was lowest for TMRM (-0.03) and highest for CMXRos (0.16). Upon repetitive imaging using SDCM, the slope varied from 0 (CMXRos) to a maximum of -0.27 (JC-1 C1). Conclusively, our data show that TMRM staining outperformed JC-1 and CMXRos dyes in a (repetitive) 3D analysis of the entire mitochondrial quantity, morphology, and membrane potential in living cells.

In situ observation of cartilage matrix based on two-photon fluorescence microscopy.

Articular cartilage lesions remain a major challenge for clinicians and researchers. Several techniques, such as histological scoring, magnetic resonance imaging, and tissue section staining, are available for detecting cartilage degeneration and lesions and evaluating cartilage repairs. Nevertheless, these methods are complex and have numerous influencing factors, which may present obstacles to efficient communication between studies. In this study, we developed a fluorescence observation system that integrated a two-photon laser scanning confocal microscope (TPLSCM) with the second-harmonic generation (SHG) of a cartilage matrix. The observation system enabled the detection of autofluorescence emitted by the cartilage matrix without species specificity, facilitating both qualitative and quantitative analyses of the cartilage matrix. Notably, this observation could be applied three-dimensionally to a fresh specimen in situ up to a depth of 300 µm, obviating the need for traditional histological fixation, slicing, or staining. Furthermore, using this observation system, we reconstructed a three-dimensional (3D) image and a 3D model of the cartilage matrix. The utilization of the 3D fluorescence model may serve as a dependable option for the fabrication of cartilage matrix biomimetic scaffolds in future studies.

In-situ Monitoring of Photocontrollable Wrinkle Erasure in Azobenzene-based Supramolecular Systems.

In this contribution, dynamic photoinduced wrinkle erasure enabled by photomechanical changes in supramolecular polymer-azo complexes was characterized via confocal microscopy. Different photoactive molecules, disperse yellow 7 (DY7) and 4,4'-dihydroxyazobenzene (DHAB), were compared to 4-hydroxy-4'-dimethylaminoazobenzene (OH-azo-DMA). The characteristic erasure times of wrinkles were quickly assessed by using an image processing algorithm. The results confirm that the photoinduced movement on the topmost layer can be successfully transferred to the substrate. Furthermore, the chosen supramolecular strategy allows decoupling the effect of molecular weight of the polymer and photochemistry of the chromophore, allowing quantitative comparison of wrinkling erasure efficiency of different materials and providing a facile way to optimize the system for specific applications.

The Plasma Membrane Polarity Is Higher in the Neuronal Growth Cone than in the Cell Body of Hippocampal and Cerebellar Granule Neurons.

The polarity of the biological membrane, or lipid order, regulates many cellular events. It is generally believed that the plasma membrane polarity is regulated according to cell type and function, sometimes even within a cell. Neurons have a variety of functionally specialized subregions, each of which bears distinct proteins and lipids, and the membrane polarity of the subregions may differ accordingly. However, no direct experimental evidence of it has been presented to date. In the present study, we used a cell-impermeable solvatochromic membrane probe NR12A to investigate the local polarity of the plasma membrane of neurons. Both in hippocampal and cerebellar granule neurons, growth cones have higher membrane polarity than the cell body. In addition, the overall variation in the polarity value of each pixel was greater in the growth cone than in cell bodies, suggesting that the lateral diffusion and/or dynamics of the growth cone membrane are greater than other parts of the neuron. These tendencies were much less notably observed in the lamellipodia of a non-neuronal cell. Our results suggest that the membrane polarity of neuronal growth cones is unique and this characteristic may be important for its structure and function.

Role of VivaScope 2500 ex vivo confocal microscopy in skin pathology: Advantages, limitations, and future prospects.

BACKGROUND: Vivascope 2500 ex vivo confocal microscopy (EVCM) is an emerging optical imaging device that allows nuclear level resolution of freshly excised tissues. EVCM provides, rapid real-time pathological examination in many subspecialties of pathology including skin, prostate, breast, liver, etc. In contrast to traditional time-consuming frozen sectioning and histological analysis. AIMS: To evaluate the current state of EVCM utilization. MATERIALS AND METHODS: This study highlights the advantages, limitations, and prospects of EVCM in skin pathology. RESULTS: Our findings demonstrate that EVCM is a promising adjunctive tool to assess margins in Mohs surgery and to provide rapid, accurate diagnosis of cutaneous tumors, infectious and inflammatory diseases. CONCLUSION: EVCM is a revolutionary device that can be used as an adjunct to paraffin-fixed, hematoxylin and eosin-stained slides and frozen sectioning. Additional refinements are required before EVCM can be used as an alternative to frozen sectioning or traditional tissue processing.

Dermoscopy and reflection confocal microscope features of pigmented prurigo.

BACKGROUND: Pigmented prurigo (PP) is a chronic and recurrent inflammatory skin disease. PP is not common clinically, but it is easily misdiagnosed because of its diversified clinical manifestations in different stages. MATERIALS AND METHODS: We retrospectively analyzed the clinical, histopathological, dermoscopy, and reflectance confocal microscopy (RCM) features of 20 patients diagnosed as PP. RESULTS: The female predominance ratio was revealed with male to female of 1:4. Seven female patients were on a diet (without staple food) and one patient had a history of diabetes. Eight cases were suffered in spring, six cases in winter, three cases in summer, and three cases in autumn. Multiple sites were involved in 13 cases. Four patients had urticarial papules and plaques. Nineteen patients had erythematous papules with reticular distribution, of which 14 cases accompanied reticulate hyperpigmentation, four cases with papulovesicle, and two cases accompanied with pustules. One patient only showed reticulate hyperpigmentation. In the early lesions, dermatoscopy showed pink oval lesions, punctate or linear vessels, and pale yellow rings around the skin lesions. RCM is characterized by spongiosis, spongy vesicle, neutrophils scattered in the epidermis, which was consistent with epidermis spongiosis, neutrophils infiltrating into the upper epidermis and necrotic keratinocytes in histopathology. In the fully developed lesions, dermatoscopy showed pink lesions with brown pigment granules in the center and linear vessels in the edge. RCM showed that demarcation of epidermis and dermis is not clear, and inflammatory cells can be seen in the upper dermis and histopathologically lesions assumed a patchy lichenoid pattern, and the inflammatory cells infiltrating the dermis were dominated by lymphocytes. In the late lesions, dermatoscopy showed grainy grayish-brown or yellowish-brown pigmentation surrounding the hair follicle merging with each other. RCM showed that pigment granules were increased on the ring of basal cells, inflammatory cells were sparsely infiltrated in the dermal papilla and superficial layer, and epidermis slightly hyperplastic, with melanophages and a few lymphocytes infiltrating the superficial dermis in histopathology. CONCLUSION: PP is easily misdiagnosed and not always occurs in those on a restrictive diet. A combination of dermatoscopy and RCM is helpful for its diagnosis of PP.

Constructing an In Vitro and In Vivo Flow Cytometry by Fast Line Scanning of Confocal Microscopy.

Composed of a fluidic and an optical system, flow cytometry has been widely used for biosensing. The fluidic flow enables its automatic high-throughput sample loading and sorting while the optical system works for molecular detection by fluorescence for micron-level cells and particles. This technology is quite powerful and highly developed; however, it requires a sample in the form of a suspension and thus only works in vitro. In this study, we report a simple scheme to construct a flow cytometry based on a confocal microscope without any modifications. We demonstrate that line scanning of microscopy can effectively excite fluorescence of flowing microbeads or cells in a capillary tube in vitro and in blood vessels of live mice in vivo. This method can resolve microbeads at several microns and the results are comparable to a classic flow cytometer. The absolute diameter of flowing samples can be indicated directly. The sampling limitations and variations of this method is carefully analyzed. This scheme can be easily accomplished by any commercial confocal microscope systems, expands the function of them, and is of promising potential for simultaneous confocal microscopy and in vivo detection of cells in blood vessels of live animals by a single system.

Cell-Based Measurement of Mitochondrial Function in Human Airway Smooth Muscle Cells.

Cellular mitochondrial function can be assessed using high-resolution respirometry that measures the O2 consumption rate (OCR) across a number of cells. However, a direct measurement of cellular mitochondrial function provides valuable information and physiological insight. In the present study, we used a quantitative histochemical technique to measure the activity of succinate dehydrogenase (SDH), a key enzyme located in the inner mitochondrial membrane, which participates in both the tricarboxylic acid (TCA) cycle and electron transport chain (ETC) as Complex II. In this study, we determine the maximum velocity of the SDH reaction (SDHmax) in individual human airway smooth muscle (hASM) cells. To measure SDHmax, hASM cells were exposed to a solution containing 80 mM succinate and 1.5 mM nitroblue tetrazolium (NBT, reaction indicator). As the reaction proceeded, the change in optical density (OD) due to the reduction of NBT to its diformazan (peak absorbance wavelength of 570 nm) was measured using a confocal microscope with the pathlength for light absorbance tightly controlled. SDHmax was determined during the linear period of the SDH reaction and expressed as mmol fumarate/liter of cell/min. We determine that this technique is rigorous and reproducible, and reliable for the measurement of mitochondrial function in individual cells.

An approach for live imaging of first cleavage in mouse embryos using fluorescent chemical probes for DNA, microtubules, and microfilaments.

Purpose: Dynamic morphological changes in the chromosome and cytoskeleton occur in mammals and humans during early embryonic development, and abnormalities such as embryonic chromosomal aneuploidy occur when development does not proceed normally. Visualization of the intracellular organelles and cytoskeleton allows elucidation of the development of early mammalian embryos. The behavior of the DNA and cytoskeleton in early mammalian embryos has conventionally been observed by injecting target molecule mRNAs, incorporating a fluorescent substance-expressing gene, into embryos. In this study, we visualized the chronological behavior of male and female chromosome condensation in mouse embryos, beginning in the two-pronuclear zygote, through the first division to the two-cell stage, using fluorescent chemical probes to visualize the behavior of DNA, microtubules, and microfilaments. Method: Mouse two-pronuclear stage embryo were immersed in medium containing fluorescent chemical probes to visualize DNA, microtubules, and microfilaments. Observation was performed with a confocal microscope. Results: This method allowed us to observe how chromosome segregation errors in first somatic cell divisions in mouse embryos and enabled dynamic analysis of a phenomenon called lagging chromosomes. Conclusions: By applying this method, we can observe any stage of embryonic development, which may provide new insights into embryonic development in other mammals.

In vivo confocal microscopy of sub-basal corneal nerves and corneal densitometry after three kinds of refractive procedures for high myopia.

PURPOSE: To observe corneal nerve fibers and densitometry after small incision lenticule extraction (SMILE), femtosecond laser-assisted laser in situ keratomileusis (FS-LASIK) and laser-assisted subepithelial keratomileusis (LASEK) for high myopia. METHODS: This is a prospective, cross-sectional research study. Patients with high myopia (equivalent spherical lens: -6.00 and -11.00D) who underwent laser corneal refractive surgery were divided into three groups: SMILE, FS-LASIK and LASEK. Scheimpflug imaging of corneal nerves in five areas was observed by confocal microscopy before and 6, 12 months after surgery. Corneal densitometry was measured by Pentacam anterior segment analysis system. RESULTS: Overall, 59 patients were enrolled. The nerve density in the central area did not recover to the preoperative level in three groups until 12 months. The density and length of corneal nerves in central and lower area were better in the SMILE group 6 months postoperatively (p = 0.01), while nerve density did not differ significantly among three groups 12 months postoperatively (p = 0.18). Nerve fibers in central and temporal region were wider in LASEK than that in other two groups at 6- and 12-month follow-up. Corneal densitometry in the central 6 mm diameter was significantly higher in the LASEK group compared with other two groups 6 months postoperatively (p = 0.04). Twelve months postoperatively, corneal densitometry in range of all zone was lower in SMILE than in FS-LASIK and LASEK (p = 0.01, 0.03, 0.04). CONCLUSIONS: Compared with FS-LASIK and LASEK, SMILE-treated eyes with high myopia had certain advantages in nerve density, length and nerve connection way and had better corneal transparency after operation.

[Treatment of rheumatoid arthritis by injection of sinomenine solid lipid nanoparticles under a fluorescence endoscopic laser confocal microscope].

A fluorescence endoscopic laser confocal microscope(FELCM) was used to direct the injection of sinomenine solid lipid nanoparticles(Sin-SLN) into the joint, and the in vitro effectiveness of Sin-SLN in the treatment of rheumatoid arthritis(RA) was evaluated. Sin-SLN was prepared with the emulsion evaporation-low temperature curing method. The Sin-SLN prepared under the optimal conditions showed the encapsulation efficiency of 64.79%±3.12%, the drug loading of 3.84%±0.28%, the average particle size of(215.27±4.21) nm, and the Zeta potential of(-32.67±0.84) mV. Moreover, the Sin-SLN demonstrated good stability after sto-rage for 30 days. The rabbit model of RA was established by the subcutaneous injection of ovalbumin and complete Freund's adjuvant. Five groups were designed, including a control group, a model group, a Sin(1.5 mg·kg~(-1)) group, a Sin-SLN(1.5 mg·kg~(-1)) group, and a dexamethasone(positive drug, 1.0 mg·kg~(-1), ig) group. The control group and the model group only received puncture treatment without drug injection. After drug administration, the local skin temperature and knee joint diameter were monitored every day. The knee joint diameter and the local skin temperature were lower in the drug administration groups than in the model group(P<0.05, P<0.01). FELCM recorded the morphological alterations of the cartilage of knee joint. The Sin-SLN group showed compact tissue structure and smooth surface of the cartilage. Enzyme-linked immunosorbent assay(ELISA) was employed to determine the serum le-vels of interleukin-1(IL-1) and tumor necrosis factor-α(TNF-α). The findings revealed that the Sin-SLN group had lower IL-1 and TNF-α levels than the model group(P<0.05, P<0.01). Hematoxylin-eosin(HE) staining was employed to reveal the pathological changes of the synovial tissue, which were significantly mitigated in the Sin-SLN group. The prepared Sin-SLN had uniform particle size and high stability. Through joint injection administration, a drug reservoir was formed. Sin-SLN effectively alleviate joint swelling and cartilage damage of rabbit, down-regulated the expression of inflammatory cytokines, and inhibited the epithelial proliferation and inflammatory cell infiltration of the synovial tissue, demonstrating the efficacy in treating RA.

Wavefront-sensorless adaptive optics with a laser-free spinning disk confocal microscope.

Adaptive optics is being applied widely to a range of microscopies in order to improve imaging quality in the presence of specimen-induced aberrations. We present here the first implementation of wavefront-sensorless adaptive optics for a laser-free, aperture correlation, spinning disk microscope. This widefield method provides confocal-like optical sectioning through use of a patterned disk in the illumination and detection paths. Like other high-resolution microscopes, its operation is compromised by aberrations due to refractive index mismatch and variations within the specimen. Correction of such aberrations shows improved signal level, contrast and resolution.

Active Aberration Correction with Adaptive Coefficient SPGD Algorithm for Laser Scanning Confocal Microscope.

A laser scanning confocal microscope (LSCM) is an effective scientific instrument for studying sub-micron structures, and it has been widely used in the field of biological detection. However, the illumination depth of LSCMs is limited due to the optical aberrations introduced by living biological tissue, which acts as an optical medium with a non-uniform refractive index, resulting in a significant dispersion of the focus of LSCM illumination light and, hence, a loss in the resolution of the image. In this study, to minimize the effect of optical aberrations, an image-based adaptive optics technology using an optimized stochastic parallel gradient descent (SPGD) algorithm with an adaptive coefficient is applied to the optical path of an LSCM system. The effectiveness of the proposed aberration correction approach is experimentally evaluated in the LSCM system. The results illustrate that the proposed adaptive optics system with an adaptive coefficient SPGD algorithm can effectively reduce the interference caused by aberrations during depth imaging.

Evaluation of Various Polishing Systems for Lithium Disilicate Glass-Ceramics.

OBJECTIVE: The aim of this study was to investigate the surface roughness of lithium disilicates (LS2s) polished using various polishing systems. MATERIALS AND METHODS: Two types of LS2 (A, Amber Mill and E, IPS e.max CAD) were polished using LS2-specific polishing systems (L-Edenta, L-Jota), a zirconia-specific polishing system (Z-Jota), and a conventional ceramic polishing system (P-Shofu) (n = 8 per group). The compositions of different polishing systems were analyzed using EDS. Surface roughness was measured using confocal laser scanning microscopy and analyzed using EDS and SEM. ANOVA and Tukey's tests were used for the statistical analyses (p = 0.05). RESULTS: The polishing systems were mainly composed of C, O, and Si. The L-Jota group exhibited rougher surfaces than the other groups. Amber Mill exhibited higher surface roughness than IPS e.max CAD (p⟨0.001). Among the polishing systems, the L-Jota group presented the highest roughness value (pp⟨0.001). The surface roughness of the AL-Jota group was higher than that of the other groups. CONCLUSIONS: A sufficiently smooth surface can be achieved without a LS2-specific polishing system. Further, the same polishing system can have different effects depending on the type of LS2.

Effect of different irrigation activation methods on non-infected dentinal tubule penetration of medicaments: A CLSM study.

Background: The whole root canal disinfection is crucial in terms of long-term endodontic success. In this context, applying medicaments following effective irrigation activation procedures becomes an important point. Aims: The purpose of this study was to evaluate and compare the efficacy of various irrigation activation techniques on non-infected dentinal tubule penetration of calcium hydroxide (CH), double (DAP), and triple (TAP) antibiotic pastes. Materials and Methods: A total of 180 extracted human permanent mandibular premolar single-rooted teeth were selected and randomly divided into a control group and four main groups according to the irrigation activation procedures (n = 36) as KTP laser irradiation, conventional needle, NaviTip FX, sonic (SI) and ultrasonic activation (UI) procedures. Each group was randomly subdivided into three subgroups according to the medicament type (n = 12); CH, DAP, and TAP. After the activation procedures, the root canals were filled with CH, TAP, and DAP mixed with 0.1% fluorescent rhodamine B isothiocyanate. Specimens were sectioned at 2, 5, and 8 mm from the apex, and all the sections were examined under a confocal laser scanning microscope to calculate the dentinal tubule penetration. Data were analyzed using a three-way analysis of variance and Tukey's post hoc tests (P = 0.05). Results: TAP provided a statistically significant greater penetration than the other groups (P < 0.05). UI provided a statistically significant higher dentinal tubules penetration area than other activation procedures (P < 0.05). Conclusion: Medicament penetration depends on the type of medicament, activation procedures, and root canal level. The TAP may be preferred following the ultrasonic activation in terms of long term root canal treatment success.

High-Speed Excitation-Spectral Microscopy Uncovers In Situ Rearrangement of Light-Harvesting Apparatus in Chlamydomonas during State Transitions at Submicron Precision.

Photosynthetic organisms adjust to fluctuating natural light under physiological ambient conditions through flexible light-harvesting ability of light-harvesting complex II (LHCII). A process called state transition is an efficient regulation mechanism to balance the excitations between photosystem II (PSII) and photosystem I (PSI) by shuttling mobile LHCII between them. However, in situ observation of the migration of LHCII in vivo remains limited. In this study, we investigated the in vivo reversible changes in the intracellular distribution of the chlorophyll (Chl) fluorescence during the light-induced state transitions in Chlamydomonas reinhardtii. The newly developed noninvasive excitation-spectral microscope provided powerful spectral information about excitation-energy transfer between Chl-a and Chl-b. The excitation spectra were detected through the fluorescence emission in the 700-750-nm spectral range, where PSII makes the main contribution, though PSI still makes a non-negligible contribution at room temperature. The technique is sensitive to the Chl-b spectral component specifically bound to LHCII. Using a PSI-specific 685-nm component also provided visualization of the local relative concentration of PSI within a chloroplast at room temperature. The decrease in the relative intensity of the Chl-b band in state 2 was more conspicuous in the PSII-rich region than in the PSI-rich region, reflecting the dissociation of LHCII from PSII. We observed intracellular redistributions of the Chl-b-related light-harvesting abilities within a chloroplast during the state transitions. This observation implies the association of the state transitions with the morphological changes in the thylakoid membrane.

Real-time confocal microscopy imaging of corneal cytoarchitectural changes induced by different stresses.

Maintenance of the corneal refractive power and tissue transparency is essential for normal vision. Real-time characterization of changes in corneal cells during suffering stresses or wound healing may provide a way to identify novel targets, whose therapeutic manipulation can improve the outcome of this response induced by injury. Here we describe a novel user friendly and effective confocal real-time confocal microscopy attachment that monitors the effects of anisoosmotic stress on cell morphology and corneal thickness in situ. Corneal epithelial nuclei gradually became highly reflective in the isotonic group and the corneal stroma was slightly thickened as compared with that seen prior to 60 min exposure to a hypotonic solution. After 30 min of exposure to hypertonic stress, the corneal stromal cells became crenate and shriveled. The hyper-reflective area of the corneal stroma in the hypo-osmotic group was significantly larger than that in the other two groups, as demonstrated by 3D reconstruction imaging. The hypotonic fresh chlorinated pool water was observed to cause atrophy of corneal epithelial nuclei, while the isosmotic bee venom solution caused high reflection of the corneal stroma layer and corneal endothelial cell damage. With the microscopic attachment, the inward movement of corneal epithelial cells toward the denuded central region was detected in the serum-treated group. The microscopy attachment is an effective system for obtaining a more detailed understanding of the time dependent losses in the corneal cell structure and tissue architecture of full thickness corneas induced by osmotic stress or cytotoxic agents.

Three-dimensional visualization of coronary microvasculature in rats with myocardial infarction.

BACKGROUND: Assessment of the coronary microcirculation remains challenging. OBJECTIVE: we explored the feasibility of evaluating the coronary microvasculature in rats with myocardial infarction (MI) using a three-dimensional visualization technique. METHODS: Animals were divided into the sham operation group (S), MI 45 min group (M45), and MI 180 min group (M180). Opened microvessels were labelled with the fluorescent dye DiI (1, 1'-dioctadecyl-3, 3, 3'3'-tetramethylindo carbocyanine perchlorate) using a heart perfusion method. The microvascular distribution and opening status were observed under laser scanning confocal microscopy, which was adjusted to facilitate evaluation of subjects around 6 to 20 µm. RESULTS: Microvascular vessels (6-20 µm) were successfully labelled by DiI. Intact and clear three-dimensional microvascular structures were observed in myocardium of sham rats and remote non-infarct myocardial tissue of MI rats, while there was almost no microvascular structure in the infarct area of the M45 group, and only a small amount of microvascular visualization was visualized in the infarct area of the M180 group. The microvascular area and microvascular density in M45 group and M180 group in the infarct border zone were significantly lower than corresponding area in S group. CONCLUSION: Three-dimensional visualization of opened coronary microvascular vessels is feasible in DiI-labelled myocardium in this rat MI model. This novel technique might be useful for defining the underlying mechanisms of coronary microvascular diseases and observe the efficacy of various therapy strategies on coronary microvessels.

The Effects of Different Fluorescent Indicators in Observing the Changes of the Mitochondrial Membrane Potential during Oxidative Stress-Induced Mitochondrial Injury of Cardiac H9c2 Cells.

We evaluated the ability of different fluorescent indicators by various analytical instruments, including a laser scanning confocal microscope (LSCM), fluorescence plate reader, and flow cytometer (FCM), to measure the mitochondrial membrane potential (ΔΨm) of cardiac H9c2 cells during oxidative stress-induced mitochondrial injury. The mitochondrial oxygen consumption rate and a transmission electron microscope were used to detect changes in mitochondrial functions and morphology, respectively. Cardiac H9c2 cells were exposed to H2O2 (500, 750, 1000, and 1250 µM) to induce mitochondrial oxidative stress injury, and fluorescent indicators including tetramethyl rhodamine ethyl ester (TMRE), 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1), and rhodamine 123 (R123) were used to detect changes in ΔΨm using an LSCM, fluorescence plate reader, and FCM. The decrease in ΔΨm caused by H2O2 was determined by endpoint and dynamic analyses after staining with JC-1 or TMRE. With the R123 probe, the LSCM could only detect the change in ΔΨm caused by 1000 µM H2O2. Moreover, R123 was less effective than JC-1 and TMRE for measurement of ΔΨm by the LSCM. Our data indicated that an LSCM is the most suitable instrument to detect dynamic changes in ΔΨm, whereas all three instruments can detect ΔΨm at the endpoint.