Background: We previously have proved that sodium tanshinone II-A sulfonate (DS-201), a derivative of traditional Chinese medicinal herb Danshen , is an opener and vasodilator of BKCa channel in the vascular smooth muscle cells (VSMCs). Vascular tension is closely associated with Ca2+ dynamics and activation of BKCa channel may not be the sole mechanism for the relaxation of the vascular tension by DS-201. Therefore, we hypothesized that the vasorelaxing effect of DS-20 may be also related to Ca2+ channel and cytoplasmic Ca2+ level in the VSMCs.Methods: Arterial tension was measured by Danish Myo Technology (DMT) myograph system in the mesentery vessels of rats, intracellular Ca2+ level by fluorescence imaging system in the VSMCs of rats, and L-type Ca2+ current by patch clamp technique in Ca2+ channels transfected human embryonic kidney 293 (HEK-293) cells.Results: DS-201 relaxed the endothelium-denuded artery rings pre-constricted with PE or high K+ and the vasorelaxation was reversible. Blockade of K+ channel did not totally block the effect of DS-201 on vasorelaxation. DS-201 suppressed [Ca2+]i transient induced by high K+ in a concentration-dependent manner in the VSMCs, including the amplitude of Ca2+ transient, the time for Ca2+ transient reaching to the [Ca2+]i peak and the time to remove Ca2+ from the cytoplasm. DS-201 inhibited L-type Ca2+ channel with an EC50 of 59.5 μM and at about 40% efficacy of inhibition. However, DS-201did not significantly affect the kinetics of Ca2+ channel. The effect of DS-201 on L-type Ca2+ channel was rate-independent.Conclusion: The effect of DS-201 on vasorelaxation was not only via activating BKCa channel, but also blocking Ca2+ channel and inhibiting Ca2+ influx in the VSMCs of rats. The results favor the use of DS-201 and Danshen in the treatment of cardiovascular diseases clinically. Salvia miltiorrhiza), a traditional Chinese medicinal herb, is effective in the prevention and treatment of various cardiovascular diseases including angina pectoris, hyperlipidemia and acute ischemic stroke  channel is the main K+ channel in blood vessel for carrying 60-70% of the outward currents, thus it plays a pivotal role in vascular relaxation  which can come from either Ca2+ influx through L-type Ca2+ channel in the plasma membrane or receptor-mediated Ca2+ release from the intracellular Ca2+ stores including the sarcoplasmic reticulum (SR). Danshen and its derivatives were reported to have beneficial effects on stroke and ischemic diseases because of their properties of vasodilation and hypotension. For example, Lam et al  was obtained from the National Institutes for Food and Drug Control . PE, ACh, TEA, IbTX, Bay K 8644 and Nifedipine were purchased from Sigma–Aldrich Inc. . Fura-2 AM methoxy)-2-oxoethyl)amino) -5-(2-(2-(bis(2-((acetyloxy)methoxy)-2-oxoet-hyl)amino)-5-methylphenoxy)ethoxy)-2-benzofuranyl)-, (acet-yloxy)methyl ester) was purchased from Invitrogen Inc. .∗/32/Δ33) plus the subunits of β2a and α2δ and cultured in modified RPMI-1640 medium containing 10 % fetal bovine serum (FBS) and 1% Penicillin–Streptomycin solution at 37°C and 5% CO2.Human embryonic kidney 293 (HEK293) cells were transiently transfected with the smooth muscle predominant CaV1.2 channel isoform Cav1.2SM (1/8/9Six-month old specific-pathogen-free (SPF) male Sprague–Dawley (SD) rats (250–300 g) were obtained from the Animal Care Center of Southwest Medical University . The rats were housed up to four rats per cage with free access to water and food at a constant room temperature (∼25°C) in a 12-h light/12-h dark cycle. All animal experiments were performed strictly in accordance with university guidelines and an approved animal study protocol by the Committee on Use and Care of Animals of Southwest Medical University .2 1.2, CaCl2 2.4, Na-HEPES 10.0, glucose 12.0, pH 7.4). The VSMCs from each vessel used in the experiments were first detected the presence of endothelial cells and removed them in order to exclude the effect of endothelial cells. The endothelial cells were removed by the method of 0.1% Triton-100 perfusion after comparison of the mechanical method (fine wire slide into the lumen of the blood vessel) to prove that it was easier to be controlled with more stable effect. The artery rings were mounted in a Danish Myo Technology (DMT) myograph under a normalized tension after removal of the endothelial cells as previously described . The maximal vasoconstriction was detected by high K+ solution after 1 h balance. One micromole ACh was added to detect endothelial cells when the vasocontraction reached the maximum and stable state. The arteries with less than 10% relaxation induced by ACh were used for subsequent experiments. The gradient concentrations for the maximal response of artery rings to PE were tested and determined 3 μM as the optimal concentration, thus it was selected for subsequent experiments. Vascular responses to DS-201 (20 to 200 μM) were observed following preconstriction with PE (3 μM) and high K+ solution. The vasoreactivity of DS-201 was also investigated by incubating the artery rings with 5 mM TEA or 200 nM IbTX (a BKCa channel blocker) for 10 min. The maximal contraction induced by PE and high K+ solution was defined as 100%. The percentage of relaxation at each DS-201 concentration was used to draw the concentration-response curve and the curve was fitted with the dose-response function to obtain the half maximal effective concentration (EC50). The X axes in the dose-response curves were log transformed in such cases and the curves were typically sigmoidal, with the steepest portion in the middle, so to visually imply a threshold concentration and EC50.Rats were anesthetized with 1% pentobarbital sodium (50 mg/kg) and the mesenteric arteries were isolated and the artery rings were used for the measurement of arterial tension. Briefly, artery rings (2–3 mm long) were quickly obtained from the secondary and tertiary branches of the mesenteries in rats under a binocular microscope and placed in ice-cold normal Tyrode’s solution  for 8–10 min, followed by a fresh Ca2+-free Tyrode’s solution containing 1.25 mg/mL collagenase XI  for 6-8 min at 37°C with gentle agitation. The isolated VSMCs were kept in 0.1 mM [Ca2+] Tyrode’s solution at 4°C, and were freshly used for the measurement of [Ca2+]i.Single VSMC was enzymatically isolated from the mesentery arteries of SD rats as described previously . Briefly2+ transients were measured with fura-2 fluorescence at room temperature (21 ± 2°C) by a dual excitation wavelength fluorescence method as described previously (2+ concentration ([Ca2+]i) was calculated using the following equation: 2+]i = Kd ∗(Sf2/Sb2)∗ (R – Rmin)/(Rmax – R) solution (10 mM [Ca2+] Tyrode’s solution), and Sf2/Sb2 as the ratio of emission fluorescence evoked by 380 nm excitation in Ca2+-free Tyrode’s solution and saturating [Ca2+] solution. A Kd value of 224 nM was used for the calculation. Ionomycin (10 μM) was added in the solution for the measurement of the values of Rmax and Rmin.Intracellular Caeviously  using th+-induced Ca2+ transients in the VSMCs of rats were obtained by applying of 60 mM high K+ solution for 10 s using a drug delivery system . The effect of DS-201 on high K+-induced Ca2+ transients was observed after 10 min pre-incubation of the cells with DS-201 and then applied high K+ (60 mM) for 10 s. The cells were continuously washed out with Tyrode’s solution during the 10-min interval. High K+-evoked Ca2+ transient was presented as the change of [Ca2+]i from the base level to the peak after the treatment of high K+ solution for 10 s. Ca2+ transient rise time was defined as the time from the base level to the peak of [Ca2+]i. Ca2+ transient decay time was defined as the time for 90% reduction from the peak of [Ca2+]i.High K2+ (LCa) channel-transfected HEK-293 cells were placed in a small chamber on an inverted microscope  and perfused with bath solution. L-type Ca2+ current  was measured with the whole-cell patch clamp technique. Voltage commands were given to elicit Ca2+ currents. The Ca2+ currents were measured 15 min after the formation of whole-cell configuration to allow equilibration between pipette solution and cytosole. The current capacity was measured for each cell during the 20-ms pulses from a holding potential of –80 mV to a testing potential of -85 mV. The capacity currents and residual leak currents were subtracted using P/5 protocol. The current–voltage (I–V) relationship was determined by 400 ms depolarizing pulses to potentials ranging from –50 mV to +50 mV from a holding potential of –80 mV in 10 mV increments at 0.1 Hz. The concentration- dependent relationship of drug on ICa,L was examined by measuring peak inward current for cell depolarized from –50 mV to +50 mV in the presence or absence of DS-201. The voltage-dependence of steady-state inactivation was determined by 4800 ms conditioning prepulses from –120 mV to +50 mV in 10 mV increments, followed by a test pulse of +30 mV for 300 ms. To measure the rate-dependent effect of DS-201, a 15-series depolarizing pulses with 400 ms duration from a holding potential of -80 mV to +10 mV at different stimulation frequencies  were applied without use of P/5 leak subtraction.Whole-cell voltage clamp recordings were conducted using an EPC-10 patch clamp amplifier and Pulse software . L-type Ca2+ channel current in whole-cell configuration, the bath solution was used with K+-free solution (in mM): NaCl 130, TEA-Cl 4, CsCl 1, BaCl2 10, MgCl2 1.2, D-glucose 10, and HEPES 10, pH adjusted to 7.4 with CsOH. The pipette solution contained (in mM): Cs-aspartic acid 115, CsCl 20, MgCl2 2.5, EGTA 10, HEPES 10, and Na2ATP 2, pH adjusted to 7.2 with CsOH. The presence of Cs+ instead of K+ in the solution blocks the potassium currents. All experiments were performed at room temperature (20–22°C). The results from pre-experiment showed that the ICa,L within 15–40 min after the formation of whole-cell configuration was relatively stable (rundown <10%). Therefore, we measured the effect of DS-201 during this period.Membrane currents were filtered at 1.0 kHz and sampled at 10 kHz. Data were stored in a computer for offline data analysis. Current densities (pA/pF) were obtained for each cell to normalize the whole cell currents. For recording of Cat-test for the comparisons between two-group and analysis of variance (ANOVA) for more than two groups if the evaluations of similar variances were passed. The significance between groups were determined by one-way ANOVA and student-Newman–Keuls test for the effects of DS-201 on vasorelaxation, Ca2+ transients, and L-type Ca2+ current inhibition. P < 0.05 was considered to be statistically significant (marked as ∗) and the higher significance level was set at P < 0.01 (marked as ∗∗).The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology . All datFigure 1 and the results showed that DS-201 relaxed the PE-preconstricted artery rings in a concentration-dependent manner and the effect was reversible . To investigate the role of K+ channel, artery rings were incubated with 5 mM TEA to block K+ channel . The data in Figure 1D showed that the concentration-response curve of DS-201 was shifted rightward after the blockade of K+ channel by TEA. The EC50 of DS-201 was changed from 64.2 ± 2.8 to 107.4 ± 8.6 μM (p < 0.01). Furthermore, the role of BKCa channel was also investigated for the vasorelaxing effect of DS-201 with a selective BKCa channel blocker IbTX . Results showed that IbTX also shifted the concentration–response curve of DS-201 to a rightward . The EC50 of DS-201 was changed from 62.2 ± 6.3 to 81.0 ± 8.4 μM. However, TEA could not shift the concentration-response curve of DS-201 at the same condition after the artery rings were pre-constricted in 60 mM high K+ solution . The EC50 of DS-201 on vasorelaxation was 92.1 ± 5.5 and 88.8 ± 4.2 μM, respectively, with or without TEA treatment (p > 0.05). These results indicate that the effect of DS-201 on vasorelaxation was not solely due to its effect on K+ channel because blockade of K+ channel did not completely affect its vasorelaxing effect in the precontracted artery rings by PE or high K+ solution, implying that an alternative mechanism may be existed for the effect of DS-201 on vascular tension relaxation.To measure the direct effect of DS-201 on vasorelaxation in the VSMCs, the endothelium layer of artery rings were denuded by perfusion of 0.1% triton solution before the measurement and only the arteries with less than 10% relaxation induced by 1 μM ACh were used for experiments. The artery rings were pre-constricted with 3 μM PE, and various concentrations of DS-201  were added into the bath solution when the artery rings were fully equilibrated. A typical tension recording is shown in 2+ transients. The typical recordings of high K+-induced Ca2+ transients and the effect of DS-201are shown in Figures 3, 4, respectively. The data reveal that high K+ solution did not affect Ca2+ response to the VSMCs , high K+-induced Ca2+ transients was not induced in the VSMCs incubated in Ca2+-free Tyrode’s solution with 0.2 mM EGTA , but was inhibited by the L-type Ca2+ channel inhibitor, nifedipine . The result indicated that high K+-induced Ca2+ transients were mainly induced due to the effect of extracellular Ca2+ influx. Pre-incubation of DS-201 for 10 min decreased Ca2+ response in the VSMCs in high K+ solution in a concentration-dependent manner. As shown in Figure 4A, DS-201 slightly decreased Ca2+ response to high K+ at lower concentrations (50 and 100 μM) but significantly decreased the response at higher concentration (150 μM). Furthermore, [Ca2+]i was changed from the base level of 127.4 ± 4.2 to the peak of 551.1 ± 12.6 nM in control group and 120.0 ± 4.0 to 444.3 ± 14.0 nM in 150 μM DS-201 treated group in high K+ solution . DS-201 at 50, 100 and 150 μM decreased Ca2+ response to high K+ by 10.5 ± 1.1%, 17.1 ± 2.0%, and 27.4 ± 2.0%, respectively . The results showed that the rise time of Ca2+ transients became shorter and the decay time became longer when the concentrations of DS-201 were higher than 100 μM . DS-201 also decreased the base level of [Ca2+]i . These results suggest that DS-201 did not obviously affect the [Ca2+]i level at the lower concentrations, whereas remarkably suppressed the [Ca2+]i transient at higher concentrations in the VSMCs. In addition, DS-201 also affected the time for Ca2+ reaching to the peak and removal from the cytoplasm.Freshly isolated mesenteric VSMCs were used to measure the CaCa channel in LCa channel-transfected HEK293 cells. The data in Figure 5A displayed the typical traces of ICa,L and the I-V curve, and showed that the ICa,L was elicited by Bay K 8644 (10 and 20 μM), a agonist of L-type Ca2+ channel, indicating that the recording of ICa,L was correct. The data in Figure 5B displayed the typical ICa,L traces and the I-V curve, and show that ICa,L was inhibited by nifedipine (1 μM), an antagonist of L-type Ca2+ channel. The data in Figure 5C showed that the ICa,L was stable formation in whole-cell configuration (rundown <10%) within 15–40 min. These channel properties were consistent with those of L-type Ca2+ channel in SMCs reported previously , 16.7 ± 3.3% (n = 7), 25.1 ± 3.5% (n = 8), 36.9 ± 6.1% (n = 6), and 37.2 ± 2.8% (n = 5), respectively . The normalized inhibition against DS-201 concentration and the Hill fits with the EC50 of DS-201 on L-type Ca2+ channel was 59.5 μM, the cooperative sites were 2.5 . The I–V relationship of DS-201 at 150 μM was illustrated and showed that the relationship including maximum activated potential and reversal potential has no significant changes compared to the control . The result demonstrated that DS-201 was an inhibitor of L-type Ca2+ channel and the inhibition of L-type Ca2+ channel may contribute to the vasorelaxing effect of DS-201.We also further studied the direct inhibitory effect of DS-201 on the activity of Leviously . The dat2+ channel and rate-dependent effect in LCa channel-transfected HEK293 cells . The results showed that, both of half-activation and half-inactivation voltages of ICa,L were only slightly shifted leftward after treatment of 100 μM DS-201, from - 5.4 to - 8.9 mV(p > 0.05), and -13.4 to -17.2 mV(p > 0.05), respectively . The rate-dependent effect of DS-201 (100 μM) on ICa,L in the cells was investigated at 0.1, 0.2, 0.7, and 2.0 Hz stimulated pulses . The peak amplitude of ICa,L was not changed at 0.1 and 0.2 Hz but changed at 0.7 and 2.0 Hz after 15 repetitive depolarizing pulses. Increase of the frequency of stimuli induced a progressive decline of the ICa,L amplitude at 0.7 and 2.0 Hz depolarization when the holding potential clamped to +10 mV. However, DS-201 (100 μM) did not change the suppression of ICa,L induced by increased stimuli frequency. These results suggest that DS-201 has rate-independent blockage on L-type Ca2+ channel with minimal effects on the activation and inactivation kinetics of the channel.Next, we further examined the potential effects of DS-201 on the activation and inactivation kinetics of L-type Ca2+ channel. Furthermore, we also demonstrated that DS-201 could affect Ca2+ influx and L-type Ca2+ channel in a rate-independent manner in the VSMCs of rats.The present study was aimed to investigate the new mechanisms underlying the beneficial effects of DS-201 on cardiovascular system. The results demonstrated for the first time that the effect of DS-201 on vasorelaxation was via inhibiting the L-type Ca2+ influx to initiate constriction. Change of [Ca2+]i may result in dynamic equilibrium of transmembrane transport, ER uptake and release of Ca2+. It is well known that Ca2+ transient is important in the process of excitation–contraction (E–C) coupling of VSMCs. Because of the high expression level in the VSMCs, L-type calcium channel has the greatest influence on global [Ca2+]i, and its activity largely determines the contractile state of ASMCs and ultimately the vessel diameter  effect on Ca2+ channel.We previously reported that the vasorelaxing effect of DS-201 was associated with the activation of BK channel . DS-201 guration . This di2+-mediated increase of contractility in the VSMC may be the possible target for vasorelaxation of drugs. It is well established that vascular tone can be increased by the activation of myosin with myosin light chain kinase (MLCK). MLCK is a Ca/CaM-dependent kinase and activated by increases the level of [Ca2+]i in the cytoplasm of cells . The studies confirm that inhibition of Ca2+ influx in the VSMCs is important for the vasorelaxation effect of DS-201. Our results are consistent with the reports by Lam et al. . Our results demonstrated that DS-201 did not affect the suppression of ICa,L induced by increased stimuli frequency open channel blockade, suggesting that it was a rate-independent blockage on L-type Ca2+ channel and ruled out open channel blockade by DS-201. DS-201 may either interact with open state or inactivated state, perhaps due to slowing down the recovery from inactivation to execute its effect. We will perform additional studies to confirm this hypothesis in the future. The advantage for use of the cell model with simple channel expression is that the cells can avoid the interaction between the channels to produce complex results because BKCa channel and Ca2+ channel are closely associated. We demonstrated that DS-201 was a Ca2+ channel inhibitor and the effect was concentration-dependent. Therefore, we discovered an alternative mechanism underlying the vasorelaxing effect of DS-201.In addition, we also studied the effect of DS-201 on L-type Ca2+ channel and modulated intracellular Ca2+ level through complex effects on K+ and Ca2+ channels, and finally reduced the vascular tension. Our findings may provide better understanding of the cardiovascular action of DS-201 and favor the use of DS-201 and/or Danshen in the treatment of cardiovascular diseases clinically. However, the limitation of our study is that we evaluated the effects of DS-201 in transfected HEK293 cells and the cells may not precisely reflect the actions of DS-201 on native L-type channel in VSMCs. The difference may exist between transfected HEK293 cells and VSMCs for the action of DS-201 on L-type channel because multiple factors could influence drug action including other proteins and cellular factors affecting the binding of the drug to the channel, the half-life and/or distribution of drug in VSMCs and so on.Danshen is a commonly used traditional Chinese medicinal herb, and numbers of studies have been carried out to elucidate the mechanisms . Here weThis Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigor of preclinical research recommended by funding agencies, publishers and other organizations engaged with supporting research.2+]i. NW and GL carried out the patch clamp experiments. X-RZ and J-MC participated in the protocol design and critically revised the manuscript. YY designed and directed the protocol and wrote and critically revised the manuscript.X-DZ and C-XH designed the studies, performed the experiments, acquired and analyzed the data, and drafted the manuscript. JC, JW, and P-YL carried out the measurements of arterial tension and [CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Current imaging techniques for the characterization of differentiated corneal limbal stem cells are destructive and cannot be used in eye bank for monitoring the regenerated epithelium in culture. We presented a minimally invasive, multimodal, marker-free imaging method for the investigation of epithelia regenerated with cultured human donor corneal limbal epithelial stem cells. Two-photon fluorescence and harmonic generation signals were collected from specimens in culture and used for evaluating the structure and morphology of epithelia cultured on two different bio-scaffolds; in addition, donor human corneal tissues were used as controls. The method provided reliable information on the organization of cellular and extracellular components of biomaterial substrates and was highly sensitive to determine differences between the density packing arrangement of epithelial cells of different biomaterials without relying on inferences from exogenous labels. The present minimally invasive standardized quality control methodology can be reliably translated to eye banks and used for monitoring harvested corneal limbal stem cells growth and differentiation in bioengineered materials. The development and validation of reliable means for monitoring regenerated corneal epithelial cells using entirely endogenous sources of contrast would represent a significant challenge in efficiently controlling stem cell growth and differentiation in tissue engineering and stem cell therapy for eye care.In stem cell research, there is a high demand on techniques for the minimally-invasive, marker-free observation of growth, proliferation, differentiation, and stability of living stem cells under near-physiological conditions7.Limbal stem cells deficiency (LSCD) has been defined as the destruction or dysfunction of the stem cells containing limbal epithelium, leading to failure of corneal epithelial regeneration, accompanied by chronic inflammation, stromal scarring, neovascularization and persistent epithelial defects. The disease leads to visual loss due to severe corneal opacity and is associated with high risk of corneal graft failure from rejection exceeding 35% at three years8.Current imaging techniques for the characterization of differentiated corneal limbal stem cells include immunocytochemistry, immunofluorescence microscopy and electron microscopy. The analyzed specimens, however, cannot be used for further operations due to the destructive effects of the examination procedures that require exogenous markers and cannot be used in eye bank for monitoring the regenerated epithelia during culture. The greatest advantages of two-photon optical microscopy over immunofluorescence and electron microscopy imaging include no requirements for fixation and dyes, molecular specificity, decreased out of focus photodamage, increased imaging depths and intrinsic optical sectioning, which ultimately provides the possibility of three-dimensional (3D) reconstruction of the anisotropic structural organization of tissues. The reliability of two-photon optical microscopy to image differentiated stem cells has been formerly shown by Uchugonova and Könighemicornea and fibrin gel9. The hemicornea is a human-derived bioscaffold and consists of a donor anterior corneal stromal lenticule; the fibrin gel is the golden standard for cultivating human limbal epithelial stem cells and treating patients with LSCD10. Data of regenerated epithelia and their substrates were compared with human donor eye bank corneoscleral tissues with intact epithelium and de-epithelialized anterior corneal stromal lenticules, which were used as controls.In this study, we present a minimally-invasive approach based on two-photon optical microscopy for investigating the epithelia regenerated from human corneal epithelial limbal stem cells on different bio-scaffold substrates, such as the hemicornea, the regenerated epithelium resembled the normal architecture of the human cornea; the resulting epithelium was stratified into three to five cell layers, with basal cuboidal cells differentiating upward to wing cells. In all cases, the basal epithelial plane was firmly attached to the underlying Bowman’s layer; in 50% of specimens, it showed some digital invasions of the limbal basal epithelium in the palisades of Vogt  and backward (B-SHG) directions. The collagen fibers of the hemicornea were regularly arranged and intertwined with each other, as found in the normal human cornea ; differences between the two bio-scaffolds were also statistically significant (P < 0.01). The percentage of hexagonal Voronoi tiles at the basal epithelial layer was 41 ± 7%, 37 ± 10% and 48 ± 6% respectively. The differences between the fibrin gel and control corneal tissue approached statistical significance (P = 0.05).The cell density was 4236 ± 760 cells/mmhemicornea, fibrin gel and corneoscleral tissue respectively. The difference in the results was statistically significant between the hemicornea and fibrin gel (P < 0.001) and between the fibrin gel and control corneal tissue (P < 0.01). The mean TPEF signal intensity of the keratocytes lying in the most anterior stroma beneath the Bowman’s layer was 63 ± 10 au and 50 ± 12 au in the hemicornea and control corneal tissue (P = 0.14) respectively.The mean TPEF signal intensity of the basal epithelial layer was 118 ± 29 au, 52 ± 12 au and 112 ± 47 au for the hemicornea showed relatively higher coherency values than the anterior stromal lenticules that were used as controls; the average coherency values were 0.24 ± 0.04 and 0.21 ± 0.07 (P = 0.03) respectively. Backward SHG images provided similar information as forward SHG images in terms of orientation for the stromal collagen fibers with differences within 15 degrees up to 65 µm stromal depth in the hemicornea (mean Δγ = 4.4 ± 5.3 degrees) and control anterior stromal lenticule (mean Δγ = 4.0 ± 7.0 degrees) respectively. The forward to backward second harmonic generations ratio, F/B, showed comparable values between the hemicornea and control anterior stromal lenticules, except for the most anterior 20 µm depth  of the collagen fiber orientation distribution was attributed to the highly intertwined arrangement of these fibers crossing each other at variable angles in the most anterior corneal stroma. The arrangement of collagen fibers in the hemicornea was almost comparable to the native human corneal tissue, though the preferred cell packing density arrangement of the basal layer showed values lower than controls. Data of control donor tissues were in fair accordance with previous in vivo study evaluating the corneal basal epithelial layer of adult subjects14; using confocal corneal microscopy, the size and density of the basal epithelial cells in the peripheral cornea of living healthy subjects have been found to be 10.1 ± 0.8 µm and 7120 ± 362 cells/mm2 respectively14. As far as we know, based on a thorough literature search, no data on cell density of the epithelia grown onto fibrin gels have been previously reported. Overall, the present method showed significant differences in the packing density arrangement of epithelia grown on different biomaterial substrates; it may be valuable to assess novel techniques of stem cell therapy, as they will become available.To the best of our knowledge, this is the first study reporting the feasibility and reliability of two-photon optical microscopy to investigate the epithelia regenerated by human corneal epithelial limbal stem cells on different biomaterials. The complementary collection of SHG and TPEF emission spectra was valuable to obtain marker-free, quantitative information on fresh regenerated epithelia of cultured human corneal limbal epithelial stem cells. The epithelial architecture of the hemicorneas; we were also able to resolve the anterior stromal nerve fibers, which have not been observed before in biomaterial substrates, entering the basal epithelial layer of the regenerated epithelium of hemicornea15. Based on current knowledge, the stroma of hemicornea could represent an active matrix for supporting, through cell-to-cell communication, all the aspects of the overlying epithelial cells’ growth and differentiation15. Although the role of the mesenchyme in the initial establishment of the histo-architecture and its instructive influence on the developing overlying epithelial structures has been well established only during embryonic development16, tissue-specific differentiation of the epithelial corneal phenotype could be probably determined by a combination of stroma-mediated mechanisms and intrinsic epithelial properties even in adult and post-mortem tissues.High-resolution TPEF images of stromal keratocytes were collected from all 18. The results were in accordance with previous studies showing the high dispersion of orientation distribution of collagen fibers through the anterior human corneal stroma20. The complementary information provided by the two parameters, i.e. the preferred orientation of collagen fibers and the coherency value, which gives a measure of the degree of order of fibers aligned along this preferential direction, may be valuable for understanding the structural integrity of the most anterior stroma22, which supports epithelial cells’ sliding movements and stratification. The present method provided information on the preferred orientation of stromal collagen fibers from the backward SHG images that were consistent with forward SHG images. This was verified both in the hemicornea and control anterior stromal lenticules. Based on these results, analysis of backward SHG images only may be valuable for monitoring the structure of the hemicornea stroma, because this will be the most likely configuration for the possible future diagnostic implementations of the technique.Analysis of second harmonic generation signals was done in order to characterize the main structural corneal stroma components, i.e., the collagen fiber, and how their organization and arrangement vary through the thickness25. It could not be excluded that tissue manipulation during epithelial stem cell cultivation may have an influence in the structural differences of the most anterior stroma25.Since collagen fibrils are the primary cause of scattering in corneal stroma, the structural differences in collagen organization and arrangement probably contributed to the F/B ratio differences in the most 20 µm anterior stroma between samples. Specifically, differences in the extrafibrillar spacing and collagen fibrils as well as in collagen fibers’ arrangement  could contribute to variable scattering between sampleshemicornea had 200 µm targeted depth and allowed us to collect more efficiently the SHG signal emitted in the backward direction. For collagenous specimens with thickness of λ/4 (λ is the excitation wavelength), whose molecular distribution possesses an axial periodicity in the vicinity of λ/4, up to 25% of the SHG power is radiated in backward directed26. In addition, the radiated SHG power in the backward direction increases when the size of the scattering structure along the optical axis is much lower than λ and also because collagen fibrils in the corneal stroma are less aligned with respect to the optical axis27. SHG microscopy can be also configured to extract more information on the collagen architecture and preferred organization throughout stromal depth through polarisation resolved methods21. It should be object of further investigation if this methodology could be used to detect the biosynthesis (fibrillogenesis) or remodelling of collagen in bio-engineered scaffolds29.The corneal stroma lenticules of 31. In addition, two-photon fluorescence lifetime imaging has been already shown to be valuable for real time monitoring of stem cell differentiation8. Collectively, both intensity-based and lifetime-based image analysis would make two-photon microscopy a powerful tool for quantitatively monitoring of epithelial stem cell differentiation during in vitro cultivation in order to optimize culture conditions, though the potential risk of photodamage for repeated scans of the sample over time should be assessed32. In this study, no change of epithelial cell morphology or other adverse effects were observed after repeated scanning.Since the main scope of this work was to thoroughly identify and characterize the structure and morphology of cellular and extracellular components of the bio-scaffolds, we could not collect information on the metabolic information of the regenerated epithelia. On the other hand, the present methodology could be enhanced, through the careful selection of combinations of excitation wavelengths  and selective emission filters , to identify the intrinsic cellular fluorophores  and achieve metabolic informationhemicornea would repair both the epithelial and stromal damage  with a “one step” surgical procedure thus saving time and improving patient care.Current management of unilateral LSCD is carried out using a complex multistage approach that requires several years for its fulfilment and definite restoration of vision, though in less than 60% of cases. This approach includes using autologous limbal stem cell transplantation grown onto fibrin gel to regenerate the corneal epithelium followed by penetrating keratoplasty to repair scarring to the anterior corneal stroma, which is often caused by caustic burns. The hemicornea, as innovative in vitro organotypic culture systems33.In conclusion, the present methodology can be helpful to gain information on growth and differentiation of regenerated epithelia under near-physiological conditions. Miniaturization of the technology  would greatly hasten the translation of two-photon optical microscopy in the eye bank as a valuable tool to establish and standardize quantitative, minimally-invasive, quality control methodology for harvested corneal limbal stem cells growth and differentiation. In addition to investigation for research and clinical purposes, the present methodology may be helpful for developing valid alternative method for assessing safety and toxicity of drugs and medical devices by imaging human corneal bio-engineered tissues, such as the 10. A donor corneoscleral tissue was placed in an artificial anterior chamber and the hemicornea was obtained by microkeratome resection of an anterior stromal lenticule with 200 µm targeted depth. Thereafter, limbal corneal keratinocytes were plated onto the hemicornea and cultured under submerged conditions for 7 days and air lifted for 14 further days; the corneal epithelial cells that were plated on fibrin gel were cultivated at the air-liquid interface for 14 days. The submerged culture technique has been shown to prevent early differentiation and retain more stem cells as opposed to the stratification and differentiation that is observed in extended airlift culture techniques, and to provide highly reproducible outcome 9. Donor corneoscleral tissues with intact epithelium and 200 µm targeted depth anterior stromal lenticules prepared by microkeratome resection were used as controls.The study was conducted in compliance with the guidelines of the Declaration of Helsinki for research involving the use of human tissues and with the guidelines for the clinical translation of stem cells of the International Society for stem cells. Written informed consent from the next of kin was obtained for the use of samples in research. The laboratory study was approved by the local ethical committee . Donors did not have history of corneal pathologies, eye surgery or any major systemic diseases. The scaffolds were prepared in the Fondazione Banca degli Occhi del Veneto , as fully described in previous workhemicorneas and three fibrin gels with cultivated epithelial limbal stem cells, three donor corneoscleral tissues with intact epithelium and three donor anterior stromal lenticules.The tissues and biomaterials used in this work included six The two-photon microscopy set-up used in this study was based on a Leica DM6000CS  upright microscope. A tunable Ti:sapphire laser  with an integrated proprietary prism-based unit designed to compensate the broadest range of Group Velocity Dispersion was used as excitation source to perform two-photon emission fluorescence (TPEF) and second harmonic generation (SHG) axial scanning measurements in all specimens. This laser presents a tunable wavelength range from 680 to 1080 nm, operating with a pulse width of 140 fs at 80 MHz of repetition rate. The laser power was attenuated by an Electro-Optical-Modulator (EOM) and then coupled into the Leica SP8-Spectral Scan-Head  where it passed through the x-y scanning module, allowing the scanning in the x-y focal plane, before being focused by a HCX IRAPO 25x/0.95 NA, water immersion objective with a working distance of 2.5 mm . Such lens was ideal for deep tissue imaging at high-resolution, with high transmission in visible and infrared ranges, and axial and lateral colour correction for multiphoton excitation. The whole system was enclosed in a black plastic box in order to assure eye-safety for any IR scattering light from the tissue samples.34. The TPEF light was collected in backward direction by a nondescan detector (NDD) for reflected light and the SHG signal was collected both in backward and forward directions by a pair of NDDs in the transmission and reflected paths of the microscope respectively. For the transmission NDD unit, the incoming light first went through a short pass filter  and then was filtered by a 10 nm FWHM band pass filter centered at 405 nm  in order to image only the SHG forward signal emitted by the corneal stroma. The reflected light coming from the sample was first filtered by an IR filter SP680 and then encountered a dichroic beam splitter . Each of the transmitted and the reflected light paths from the dichroic entered to each of the two reflection NDDs; the reflected light was filtered by a band pass filter 447/60  and entered into the reflection NDD unit and the transmitted light was again filtered by FF01-405/10-25 to provide the backward SHG signal.Each specimen was placed upward on a quartz microscope slide under the microscope and illuminated by the laser tuned to 712 nm or 810 nm for collecting the TPEF or the SHG signals from cellular and extracellular components of each sample respectively. For each sample, the TPEF and SHG signals were collected consecutively in the same imaging session. The laser power was 15 mW before entering the water immersion objective in all sessions; it was measured before and during each session of measurements in order to allow direct comparison of measurements between different sessions of experiments27. Under 2-photon excitation at 700–750 nm wavelengths, tissue fluorophores are primarily derived from the aromatic amino acids such as tryptophan, tyrosine and phenylalanine which emit at 400–600 nm wavelengths. The TPEF enabled visualization of epithelial and stromal cells and nerve fibers. The SHG emission spectrum (400–430 nm) is consistent with collagen, which holds noncentrosymmetric structure, excited at wavelength between 800–860 nm31.In order to compare data between different sessions of experiments, the NDDs sensitivity settings for collecting the TPEF and SHG signals were the same for all specimens. The TPEF emission spectra collected with this configuration were consistent with the literature values for NAD(P)H and flavins, enabling visualization of cellular structures (mitochondria)z-axis, extending from above the epithelial surface to below the anterior 200 µm stroma; images with 1024 × 1024 pixels resolution (2 frames average) were recorded on several locations of the hemicornea and fibrin gel and in the central and para-central regions of the corneoscleral tissue. Image visualization were carried out using proprietary Leica software and an image processing package  and custom-written macros.Each sample was scanned twice for collecting either TPEF or SHG signals with a 2 µm or 4 µm step size in the hemicornea and control corneal tissue . Cellular fluorescence was measured as the mean intensity of all pixels within the sampling area; in TPEF images of the stroma, the average fluorescence intensity from an area devoid of cells was first measured and subtracted as background using the proprietary Leica software.32 Data were exported in a text array for statistics.A sampling area of 120 × 120 µm was used to analyse the TPEF images of the basal epithelial layer of the biomaterials and corneoscleral tissues as well as the TPEF signal of the most anterior stromal keratocytes lying at 60 ± 10 µm from the epithelial surface of both the 36; cells were selected by two expert investigators (ML and GL) and the results for each sampling window recorded . Cell spacing was calculated as the distance of the closest cell in the array for each of the cell, which is equivalent to the nearest neighbour distance 37. Cell packing arrangement was analyzed using Voronoi diagrams. This approach is commonly used in computational geometry to subdivide a plane into regions based on distance to point sites38. The Voronoi tessellation was implemented by the voronoi Matlab function from the bidimensional coordinates of labelled cells36. Each Voronoi cell was coded by a different colour corresponding to the number of their neighbouring cones: gray = tetragonal (4n) arrangement, yellow = pentagonal (5n) arrangement, green = hexagonal (6n) arrangement; blue = heptagonal (7n) arrangement and white = octagonal (8n) arrangement. The Voronoi regions containing pixels that extended beyond the bounds of each section were excluded from further analysis, thus creating a buffer zone to minimize the boundary effect.The density, spacing and preferred packing arrangements of cells were analysed on several sampling areas of 120 × 120 µm. Image cell labelling process was performed using an algorithm implemented with the image processing toolbox in Matlab http://bigwww.epfl.ch/demo/orientation/) in ImageJ was used for analysis of stromal collagen fiber orientations, based on the evaluation of the structure tensor in a local neighborhood, as previously described by Vielreicher et al.39. The Dominant Direction function was used to calculate the preferred fiber orientation and the coherency values over 120 × 120 µm sampling area for each SHG (both forward and backward) image stack from the Bowman’s layer to a maximum of 65 µm in the anterior stroma. Coherency, C, was used as a measure for the local degree of order of fibers .The plugin OrientationJ  for the forward and backward SHG images was used as a parameter for comparison between SHG signals24. Owing to its coherent nature, SHG is usually observed to be emitted in the forward (transmitted) direction. If axial scatterers are separated by a multiple of distance of λSHG/2, i.e., λ/4, an appreciable backward (coherent) directed SHG is observable. In addition, the backward SHG signal may also be produced from multiple scattered forward (incoherent) SHG. If perfect phase match occurs, i.e., ΔK = K2ϖ  − 2Kϖ = 0, the SHG emission is 100% forward directed and co-propagates along laser beam direction. If imperfect phase match  is present, it gives rise to a corresponding distribution of forward and backward emitted components and, as a result, SHG in tissues is described as quasi-coherent. In tissue imaging, the measured directionality of the SHG signal (F/B ratio) will comprise a convolution of the initially emitted directionality (FSHG/BSHG) and the subsequent scattering of these photons at λSHG41. The FSHG/BSHG is highly dependent upon the fibril diameter, the packing density and regularity relative to the size-scale of the SHG wavelength. The bulk optical properties are related to density  and organization  of the fibrillar assembly41. Specifically, structures that are ordered on the size of λSHG in the axial direction will give rise to predominantly forward SHG, whereas the emission from smaller and/or more random structures with larger Δk values will have relatively less forward directed signal and higher backward signals.The measurement of the forward and backward SHG intensities has been shown to be valuable for collecting information on the structure of fibrillar tissues, such as the corneal stromaSHG/BSHG is not the quantity measured in an experiment, which we define as F/B ratio. The first metric takes into consideration the quasi-coherent interactions associated with fibril diameter, packing density and randomness in the fibrillary matrix41. The latter metric, which was calculated as the ratio of the mean (forward and backward) SHG signal intensity for all the frames in each stack, consists of the emitted directionality and components arising from forward and backward scattering of the initially emitted photons. The F/B ratio was calculated in the region of interest (120 µm side) in order to obtain information on the structural differences between the stroma of the hemicornea and control anterior lenticules. The greater the F/B ratio, the greater the fibril packing density (if < λSHG) and/or the larger the fibril diameters 41.It should be kept in mind that F42.We chose to use sample areas of 120 μm side in the central region of each TPEF and SHG images in order to have an area big enough to evaluate the main features of the epithelia regenerated on the two different bioscaffolds and the stromal collagen fiber orientation respectively as well as to avoid any loss of information that may occur at the edges of the images due to the samples’ shape or locally due intrinsic heterogeneity of biological tissues P < 0.05 for all the tests performed.Data were expressed as mean ± standard deviation. Statistics were performed using the SPSS software . The normal data distribution was tested by using the one-sample Kolmogorov-Smirnov test. The analysis of variance and the Tukey pairwise test were used to test significance between TPEF signal intensity and cell metrics among biomaterials. Statistical significance was set at 2 between the epithelia regenerated on the two different biomaterials at a significance level of 5% and a power of 81%.Sample size  was calculated to determine a mean difference of 1200 (±600) cells/mmSupplementary infoSupplementary video 1supplementary video 2


Our results show that worms stimulated with serotonin display a pharynx [Ca2+]M oscillatory kinetics that includes both high frequency oscillations (up to about 1Hz) and very prolonged “square-wave” [Ca2+]M increases, indicative of energy depletion of the pharynx cells. Mitochondrial [Ca2+] is therefore able to follow “beat-to-beat” the fast oscillations of cytosolic [Ca2+]. The fast [Ca2+]M oscillations kept steady frequency values during the whole worm life, from 2 to 12 days old, but the height and width of the peaks was progressively reduced. [Ca2+]M oscillations were also present with similar kinetics in respiratory chain complex I nuo-6 mutant worms, although with smaller height and frequency than in the controls, and larger width. In summary, Ca2+ fluxes in and out of the mitochondria are relatively well preserved during the C. elegans life, but there is a clear progressive decrease in their magnitude during aging. Moreover, mitochondrial Ca2+ fluxes were smaller in nuo-6 mutants with respect to the controls at every age and decreased similarly during aging.Progressive decline in mitochondrial function is generally considered one of the hallmarks of aging. We have expressed a Ca C. elegans. Body wall muscle mitochondria undergo progressive fragmentation with aging, together with a decrease in mitochondrial length and area, and an increase in mitochondrial circularity  rises first in the cytosol and then enters the mitochondria through the mitochondrial Ca2+ uniporter, activating several key dehydrogenases to boost substrate oxidation and ATP production  ([Ca2+]M) dynamics in C. elegans pharynx in vivo during aging, in order to study if the progressive mitochondrial alteration induces changes in [Ca2+]M dynamics. We have targeted a yellow cameleon YC3.60 Ca2+ sensor M is able to undergo fast oscillations during pharynx pumping, with a frequency similar to the cytosolic [Ca2+] oscillations ([Ca2+]C) we have previously reported [2+]M elevations were also observed, similar to those we have previously described in cytosolic [Ca2+] [2+]M oscillations were observed with similar frequency along the whole life of the worms, both in control worms and in nuo-6 mutants, although both width and height decreased with age. In addition, [Ca2+]M oscillations in nuo-6 mutant worms showed reduced height and frequency, and increased width, compared with wild-type worms.We have decided to monitor mitochondrial [Ca+ sensor  to the mreported . In addic [Ca2+] , and the2+], control N2 C. elegans nematodes were microinjected with the plasmid for expression of mitochondrially-targeted YC3.60 in pharynx under the myo-2 promoter. The transformed strain, named AQ3055, showed fluorescence in the pharynx with a typical striped mitochondrial distribution when analyzed by confocal microscopy  were obtained by crossing AQ3055 worms with nuo-6 (qm200) mutants M was first monitored in the AQ3055 C. elegans strain, in live worms glued to the agar pad and subjected to stimulation of pharynx pumping with serotonin, as described previously [2+]M dynamics monitored continuously at 2.5Hz for 30 min in live worms of 2, 5, 8 and 12 days old. The left panels show typical 30 min records of [Ca2+]M dynamics obtained in different worms, two of them for every age. The middle panels show 5 min records taken each one from the insets in the corresponding left panels expanded. The right panels show 1 min records taken again each one from the insets in the corresponding middle panel expanded. The traces show that [Ca2+]M undergoes rapid and persistent oscillations similar to those we have previously described in cytosolic [Ca2+] [2+]M, that in some cases could last for many minutes until the end of the experiment (see 2nd trace of day 2) or in other cases could be shorter and reversible (see 2nd trace of day 5 and 1st trace of day 8). Similar prolonged increases in [Ca2+]M were also found in cytosolic [Ca2+], where we called them “square-wave” [Ca2+] increases [2+] dynamics behave similarly, suggesting that [Ca2+]M in the pharynx directly follows the dynamics of cytosolic [Ca2+], responding with fast [Ca2+]M oscillations when cytosolic [Ca2+] is oscillating, and with persistent increases in [Ca2+]M when cytosolic [Ca2+] keeps persistently high.[Caeviously . Figure c [Ca2+] . In addincreases . Therefo2+]M dynamics data obtained, we have made a series of experiments like those shown in Figure 2+ transient measured. As described previously [2+] peak in our experiments. Then, we can pool together all the data corresponding to every age and follow the evolution with the age of every parameter.To obtain more information on the complex Ca+M dynamieviously , our ana2+]M peaks obtained in AQ3055 worms of day 2, 5, 8 and 12 of adult life. In this plot, the prolonged “square-wave” [Ca2+]M peaks correspond to the points having larger width, those placed at the right of every graph . As previously shown for the cytosolic [Ca2+] peaks, the proportion of long “square-wave” [Ca2+]M peaks was higher in young worms (day 2) and decreased progressively as they get older (day 12). As shown before for cytosolic [Ca2+] [2+]M peaks started at some point along the experiment and stayed at high [Ca2+] until the end of the experiment . We have hypothesized that these long peaks are due to energy depletion of the pharynx cells, although paradoxically they occur more frequently in younger worms. Figure 2+]c (AQ2038 strain) or [Ca2+]M (AQ3055 strain) peaks during aging. Very similar values were obtained when either [Ca2+]c or [Ca2+]M was measured. This could be expected, because a persistent increase in [Ca2+]c should also induce a large [Ca2+] accumulation inside mitochondria. The [Ca2+]c data in this and the following panels correspond to experiments performed in the AQ2038 strain at 2.5Hz image rate.Figure c Ca2+] , many of+ , many 2+]M peaks can be seen also when we calculate the mean width of all the [Ca2+]M peaks obtained at every age. Panel f shows the evolution of the mean peak width with worm age, comparing the data obtained in pharynx cytosolic [Ca2+] in the AQ2038 strain with those obtained in pharynx mitochondrial [Ca2+] in the AQ3055 strain. The mean peak width of [Ca2+]M peaks was smaller than the corresponding one of [Ca2+]C at every age, but it followed the same decreasing trend with age. This decrease was mainly due to the disappearance of the long “square-wave” [Ca2+] peaks, because when the mean width was calculated using only the [Ca2+] peaks having a width smaller than 10s  expressing the mitochondrial [Ca2+] probe. Probably because of the slower rate of development of these mutant worms, the level of fluorescence of the probe was very small at day 2 of adult life, so that measurements were too noisy at that age. By this reason, in this strain we have performed measurements only at days 5, 8 and 12 of adult life. Figure 2+]M records obtained in this mutant strain at 5, 8 and 12 days of adult life. The traces show that mitochondrial Ca2+ oscillations are still present and are apparently very similar to the control ones . Therefore, in spite of the reduced mitochondrial respiratory chain function of the nuo-6 strain [2+]M dynamics were similar to those in the controls. However, a more detailed analysis including data from 23-36 different worms of every age uncovered significant differences.We have then performed similar experiments with ndrial Ca+ probe. 6 strain ,24, the 2+]M peaks obtained in the AQ3055  and Mnuo-6 strains . Panels a-c show the plots of peak height against peak width in Mnuo-6 mutant worms of days 5, 8, and 12 of adult life. As in the controls , it is apparent that the frequency was smaller than in the controls. Many worms were still able to keep oscillations until the end of the 30 minutes, as shown in Figure 2+]M oscillations before the end of the experiment. The same behavior was found for the [Ca2+]C oscillations in nuo-6 mutant worms (data not shown), indicating that the decay of the [Ca2+]M oscillations may be just a consequence of the decay in the [Ca2+]C oscillations. In these cases, when [Ca2+] oscillations stop, [Ca2+]C and [Ca2+]M stay at resting levels and thus it cannot be attributed to energy depletion in the pharynx, but to some other reason that affects transmission of the pumpìng signal to the pharynx.The dynamics of [Ca2+] in the pharynx of live C. elegans worms. Our data show the behavior of C. elegans pharynx [Ca2+]M dynamics for long periods of time, and how it may depend on the worm age and on the presence of a respiratory chain complex I mutation. Mitochondrial [Ca2+] has been measured previously in worm hypodermis  is able to oscillate at high frequency, reproducing the oscillations of cytosolic [Ca2+]. We have recorded frequencies of [Ca2+]M oscillation up to 1Hz. This point is important because the C. elegans pharynx is a neuromuscular organ which has been compared to the heart because it has similar electrical properties and its development is controlled by similar genes [2+] signaling in the heart is controversial because there are now two different models to explain how the [Ca2+]C oscillations are translated to mitochondria. One of the models suggests that mitochondria rapidly follow “beat-to-beat” the oscillations in [Ca2+]C, while the other model proposes that [Ca2+]M only reflects a slow integration of the [Ca2+]C transients [2+]M kinetics in response to the repetitive cytosolic [Ca2+] spikes, either repetitive [Ca2+]M spikes in the first model or a slow progressive increase in the second. Our data clearly show that the C. elegans pharynx mitochondria behave in a “beat-to-beat” mode, indicating that Ca2+ uptake and release from the mitochondria are fast enough to recover the resting levels after each Ca2+ spike. This point has several physiological implications. On the first place, energy consumption is higher with this model, because both Ca2+ uptake and release by mitochondria consume mitochondrial membrane potential. In addition, larger amounts of Ca2+ are taken up by mitochondria during each [Ca2+]c spike, so that mitochondrial Ca2+ buffering may potentially become an important regulator of cytosolic [Ca2+] and thus, of contraction.The first conclusion we can draw from our data is that mitochondrial [Caar genes . Mitochoansients . The two2+]M peaks, we find that they show a smaller width and frequency with respect to the [Ca2+]C peaks. Considering only the [Ca2+] peaks having a width smaller than 10s at all ages, the peak width of the [Ca2+]M peaks was 11.1±0.6% (mean±s.e.) smaller than that of the corresponding [Ca2+]C peaks. Similarly, pooling all the frequency data at every age together, the frequency of the [Ca2+]M peaks was 17.3±0.5% (mean±s.e.) smaller than that of the corresponding [Ca2+]C peaks. In both cases, the difference was highly significant (p < 0.001). This may be due to the low Ca2+-affinity of the mitochondrial Ca2+ uniporter [2+]c is near the peak levels, thus reducing the time mitochondrial Ca2+ uptake is active. The decrease in frequency could also be attributed to the presence of [Ca2+]C peaks too small to evoke any mitochondrial Ca2+ uptake. On the other hand, we find that the [Ca2+]M oscillation frequency hardly changes during aging, but there is however a progressive reduction in the width and amplitude of the [Ca2+]M peaks with the age. Our data therefore indicate that mitochondrial Ca2+ fluxes in C. elegans pharynx are progressively reduced during aging, although mitochondria keep able to follow “beat-to-beat” the cytosolic [Ca2+] oscillations.When we examine in detail the parameters of the M oscillations, we could also find prolonged elevations of [Ca2+]M similar to the “square-wave” [Ca2+]C increases we have recently reported [2+]M stayed high for the rest of the experiment. The proportion of worms undergoing “square-wave” [Ca2+]C or [Ca2+]M increases decayed in parallel with the age, suggesting that they are both directly linked. Our hypothesis is that these long [Ca2+] transients can be attributed to energy depletion, which would arrest Ca2+ pumps and thus keep [Ca2+]C high, leading then also to mitochondrial Ca2+ accumulation. If energy depletion is transient, cells may recover and restart spiking, but if it is persistent, the increased [Ca2+]M may induce cellular damage by triggering ROS production or by activating apoptotic pathways. Energy depletion would be a consequence of the increased energy consumption during the experiment due to serotonin-induced accelerated pharynx pumping. The meaning of these “square-wave” [Ca2+]C or [Ca2+]M increases would be therefore that in these worms energy production is unable to cope with the increased energy consumption at some point during the 30 min of the experiment.Together with the fast M oscillations coupled to the [Ca2+]C dynamics also in these worms. However, [Ca2+]M oscillations in nuo-6 mutant worms were slightly wider, and had smaller amplitude and frequency than in the controls, suggesting reduced Ca2+ fluxes in and out of the mitochondria. Nuo-6 worms also had a reduced number of “square-wave” [Ca2+]C and [Ca2+]M increases at every age. This phenomenon could be due to the smaller rate of energy consumption in these mutants, which may balance the decreased rate of energy production.movement  and decrmovement . In spitmyo-2 gene (pmyo-2::YC2.1)  peaks in the ratio were only considered acceptable when inverted changes at both wavelengths were clearly observed. Fluorescence intensities and ratio changes were then analyzed with a specific algorithm designed to calculate off-line the width at mid-height expressed in seconds, the height obtained as percent of ratio change and the frequency of all the Ca2+ peaks in each experiment. The frequency was measured at each peak as 9 divided by the distance among the peak 4 positions before and the peak 4 positions after. The mean frequency was calculated as the mean of all the individual frequencies higher than 5 peaks/min obtained in worms of a given age and condition.Fluorescence was recorded and analyzed using the Metafluor program . The traces shown are the 535nm/480nm fluorescence emission ratio values of a region of interest enclosing the pharynx terminal bulb. [Ca


Escherichia coli in infections is its ability to cause attaching and effacing lesions in enterocytes, as well as to produce Shiga toxins. To clarify the pathogenic mechanism and host innate immune responses of enterohemorrhagic Escherichia coli in rabbits, experimental infections with TS and MY strains were conducted. Among the results, although the MY strain’s pathogenicity was stronger than the TS, typical symptoms were observed in both groups of bacterial-infected rabbits. Pathological changes in the heart, liver, and spleen of rabbits infected with the MY strain were more severe than those infected with the TS strain, pro-inflammatory cytokines IL-1β, IL-6, IL-8, IFN-γ, and TNF-α were induced by both strains, and α- and β-defensin were significantly upregulated at 3 d postinfection. Moreover, in the spleen, the MY strain induced greater expressions of α- and β-defensins than did the TS strain. However, in the liver, the TS strain induced greater expressions of α- and β-defensins than did the MY strain. Most likely, different replications of the MY and TS strains in the liver and spleen induced different host immune responses. Altogether, the findings provide new insights into the occurrence and development of enterohemorrhagic Escherichia coli-mediated diseases in rabbits.The major virulence factor of enterohemorrhagic  Escherichia coli (EHEC) belongs to the family of Shiga toxins (Stxs)-producing E. coli, many of whose members carry the eae gene and can cause severe disease [E. coli (EPEC), EHEC causes attaching and effacing lesions, which represent another major virulence factor in colonization of the EHEC in the colon [Escherichia coli (EHEC) was first isolated in 1977 by Konowalchuk et al [Enterohemorrhagic  disease . As A-B- disease . Similarhe colon . Enterohuk et al . In 1982uk et al . In Japauk et al . In UnitE. coli was first studied more than 30 years ago, experiments involved EHEC O157:H7 using piglets [E. coli as experimental models of human disease, rabbits orally inoculated with EPEC strain RDEC-1 emerged as a good model for human EPEC infection [The most significant sources of EHEC infection in humans are food and water -10, the  piglets , 17, and piglets . In the nfection , which snfection . Other snfection .via MyD88- and NF-κB-mediated signal pathways [Stxs can induce classical mitogen-activated protein kinases and IL-8 when co-administered with TNF-α  and flagpathways , 25 thatAntimicrobial peptides not only directly inhibit various microorganisms, but also can recruit immature dendritic cells and macrophages to reach the skin and mucosal tissues of microbial invasion through chemokine receptors, thus inducing specific immunity of pathogenic bacteria. Since their production is more than 100 times faster than that of IgM, they are also the first line of host defense . MembersE. coli content in tissues and innate immune responses in EHEC-infected rabbits.Although diseases in rabbits caused by EHEC have been reported , 15, resE. coli was bluish green with a metallic luster on the eosin-methylene blue medium. As shown in Table E. coli MY rapidly reached 1.46 × 108 CFU/mL, while that of TS was 5.99 × 107 CFU/mL (p < 0.05). After 6 h of incubation, the number of E. coli MY and TS reached 1.96 × 109 CFU/mL and 2.08 × 109 CFU/mL, respectively. During the stable phase of bacterial growth, the highest number of E. coli TS reached 6.46 × 109 CFU/mL, which was higher than that of MY 2.29 × 109 CFU/mL at 14 h (p < 0.05). MY strain grew at a faster rate than the TS stain during the logarithmic growth phase, although its greatest concentration was lower than that of the TS stain  dropped at 2 and 3 dpi, after which none of the rabbits died. By contrast, among rabbits infected with TS strain, the number of deaths peaked at 2 dpi (27.8%), and none of the rabbits died after 4 dpi. Altogether, the mortality of rabbits infected with the MY strain was 77.8%, which was greater than that of rabbits infected with TS strain (55.6%)  at 1 dpi  at 3 dpi , yet decreased at 3 dpi by 5.72- and 12.24-fold, also respectively  and 4.86-fold (p < 0.05) in rabbits infected with the MY and TS strains, respectively, to a greater extent in the former group than in the latter  and β-defensin  displayed no significant changes in rabbits infected with either the MY or TS strain. However, at 3 dpi, those genes showed greater expression in rabbits infected with the MY strain than those in the control group and those infected with the TS strain. At 3 dpi, NP5, DEFB 114, DEFB124, and DEFB128 showed a relatively low expression Figure , and 5M,p < 0.05, Figure p < 0.05, Figure p < 0.05, Figure p < 0.05) in rabbits infected with the TS strain, and its expression showed no significant changes in that group of rabbits at 3 dpi , in rabbits infected with the TS strain and to a far greater extent than that of other defensins .The EHEC TS and MY strains were isolated from intestine of clinically infected rabbits that developed acute diarrhea. Using eosin-methylene blue medium as the selective medium, and suspected colonies were subjected to staining microscopy and biochemical tests. Then bacterial strains were grown in nutrient broth medium at 37°C. Overnight cultures of bacteria were harvested for DNA extraction using the TIANamp Bacteria DNA Kit  according to the manufacturer’s instructions. Following those tests, 16S rDNA sequencing was performed with universal primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’), and the sequences were blasted in NCBI. To further amplify the classical virulence genes of EHEC, the primer pairs of eae, Stx1B, Stx2A and variants, as well as hlyA genes [9 CFU) were introduced into 100 mL nutrient broth medium. The new liquid medium were incubated at 37°C for 24 h, and calculations of E. coli content every 2 h were used to develop a growth curve.Overnight cultures of TS and MY strains in amounts of 1 mL (10n = 40) and given sufficient feed and water. Challenge tests were conducted by using intraperitoneal injections of 1.0 mL of each E. coli bacterial suspension (108 CFU/mL). Rabbits in the control group were inoculated with 1.0 mL of phosphate-buffered saline. From each group, 10 rabbits were randomly chosen to provide data used to calculate the survival rate of each group. The survival rate of the rabbits was calculated at the end of the experiment. At 1 and 3 dpi, five rabbits in each group were euthanized, the hearts, livers, spleens, brains and colons were collected, and part of the tissues were fixed with 4% paraformaldehyde solution to study histopathological changes, whereas the others parts were used for RNA extraction. The fresh and sterile heart, liver, spleen, brain, and cecal content were collected to gauge E. coli content as the following: tissue samples were weighed, suspended in PBS (1ml/g). Serial 10-fold dilutions were plated onto nutrient agar to calculate CFU per gram in each organ. All rabbits were handled according to appropriate biosecurity guidelines. All experiments were performed in a biosafety level-2 laboratory.A group of 120 healthy weaned rabbits 35 d old were randomly separated into three treatment groups . The synthesized cDNA was stored at -20°C until further use. qRT-PCR was performed using TransStartR Tip Green qPCR SuperMix (+Dye II) . Primers of IL-6 and TNF-α used for qRT-PCR have previously been reported [http://bioinfo.ut.ee/primer3-0.4.0/) based on target sequences previously reported  according to the manufacturer’s instructions. For quantitative real-time (qRT) PCR, total RNA 1 µg) was reverse transcribed with reported . Other p µg was r−ΔΔCt method. The housekeeping gene GAPDH was used as an endogenous control to normalize the expression of target genes. Data were analyzed with the non-parametric Mann-Whitney U test, and the survival rate of rabbits was analyzed using the Kaplan-Meier method. All statistical analyzes were performed with GraphPad Prism 5.0 , with statistical significance set at p < 0.05.The relative expressions of genes were calculated using the 2


The intracellular glutathione (GSH) and catalase activity were determined after the incubation; cell viability was evaluated 24 h later. TBBPA concentration-dependently increased [Ca2+]i and ROS production, and reduced GSH content, catalase activity, ∆Ψm and neuronal viability. The combination of NMDA and ryanodine receptor antagonists, MK-801 and bastadin 12 with ryanodine, respectively, prevented Ca2+ transients and partially reduced cytotoxicity induced by TBBPA at both concentrations. The antagonists also completely inhibited oxidative stress and depolarization of mitochondria evoked by 10 µM TBBPA, whereas these effects were only partially reduced in the 25 µM TBBPA treatment. Free radical scavengers prevented TBBPA-induced development of oxidative stress and improved CGC viability without having any effect on the rises in Ca2+ and drop in ∆Ψm. The co-administration of scavengers with NMDA and ryanodine receptor antagonists provided almost complete neuroprotection. These results indicate that Ca2+ imbalance and oxidative stress both mediate acute toxicity of TBBPA in CGC. At 10 µM TBBPA Ca2+ imbalance is a primary event, inducing oxidative stress, depolarization of mitochondria and cytotoxicity, whilst at a concentration of 25 µM TBBPA an additional Ca2+-independent portion of oxidative stress and cytotoxicity emerges.Using primary cultures of rat cerebellar granule cells (CGC) we examined the role of calcium transients induced by tetrabromobisphenol A (TBBPA) in triggering oxidative stress and cytotoxicity. CGC were exposed for 30 min to 10 or 25 µM TBBPA. Changes in intracellular calcium concentration ([CaThe online version of this article (doi:10.1007/s11064-016-2075-x) contains supplementary material, which is available to authorized users. Tetrabromobisphenol A  is one of the main representatives of brominated flame retardants, substances that are additives to industrial products reducing their flammability. The mass production and use of TBBPA results in its leakage to the environment –3. TBBPA2+ ATPase (SERCA) [2+ from the intracellular ryanodine-sensitive stores via ryanodine receptors (RyR) [2+ concentration ([Ca2+]i) in cultured cells, including neurons, challenged with TBBPA i i . We, the2+]i in the oxidative stress and cytotoxicity in neurons. The cultures were acutely challenged with TBBPA and we measured changes in [Ca2+]i, ROS production and selected indicators of oxidative stress, in the mitochondrial membrane potential (∆Ψm) and in the viability of neurons. The combination of NMDAR and RyR antagonists was applied to verify the hypothesis on the primary role of the TBBPA-induced increases in [Ca2+]i, while free radical scavengers were used to prevent oxidative stress and cyclosporin A was used to test the role of mitochondrial permeability transition pores (MPTP).In the present study, primary cultures of rat CGC were used to determine the role of TBBPA-induced increases in ·cyclohepten-5,10-imine hydrogen maleate (MK-801), butylated hydroxyanisole (BHA), reduced glutathione (GSH), ascorbic acid (vit. C), cyclosporin A (CsA), dimethyl sulphoxide (DMSO), fetal calf serum and other materials for cell culturing, were purchased from Sigma Chemical Poland . TBBPA (99.8 % purity) was synthesized and delivered commercially by the Institute of Industrial Organic Chemistry, Analytical Department, in Warsaw, Poland. Bastadin 12 synthesized in the Laboratory of Natural Products Synthesis and Bioorganic Chemistry, Institute of Bioorganic Chemistry NCRS “Demokritos” in Athens, Greece, was kindly provided by Dr. Emmanuel N. Pitsinos. Fluorescent probes fluo-3/AM, rhodamine123 (Rh123) and 2′7′-dichlorodihydrofluorescein-diacetate (DCFH-DA) were produced by Molecular Probes Inc. . All other chemicals were of analytical grade.ad libitum and kept on a 12:12 h dark-light cycle, at room temperature with a constant humidity of approximately 60 %.Primary CGC cultures were prepared from 7 day-old Wistar rats of the outbred stock CmD:(WI)WU. The animals were bred in the animal house of the Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw under standard conditions i.e. fed and watered 6 per well. The replication of non-neuronal cells was prevented by the application of 7.5 µM cytosine arabinofuranoside. The CGC cultures were used for experiments after 7 days in vitro.The cells were isolated and cultured according to a standard method  with sli2+ concentration ([Ca2+]i) in CGC were monitored using the fluorescent calcium-sensitive probe fluo-3. Its acetoxymethyl ester derivative, fluo-3 AM, easily penetrates plasma membranes, and inside the cells esterases cleave it to fluo-3, which becomes highly fluorescent after binding Ca2+ i concentration in CGC untreated with TBBPA and on its increase evoked by 10 µM TBBPA , had no significant effect on the production of ROS in CGC challenged with 10 and 25 µM TBBPA  reduced catalase activity in a concentration-dependent manner, by 28, 32 and 41, respectively  in CGC cultures treated with TBBPA were evaluated using the fluorescent probe rhodamine 123 (Rh123). As shown in Fig. Figure 2+]i, induces oxidative stress and depolarizes mitochondria, and that all these effects are completely (at 10 µM TBBPA) or very strongly (at 25 µM TBBPA) inhibited by a combination of NMDAR and RyR antagonists. These substances also provide significant, but incomplete protection of neuronal viability against TBBPA cytotoxicity. The administration of free radical scavengers does not inhibit TBBPA-induced calcium imbalance and mitochondrial depolarization, but the scavengers do reduce oxidative stress and provide significant neuroprotective effects. Co-administration of free radical scavengers with NMDAR and RyR antagonists almost completely prevents TBBPA cytotoxicity. The inhibitor of MPTP formation, cyclosporin A (5 µM), has no significant effect on TBBPA-induced ROS production and decrease in ∆Ψm, but it does reduce the cytotoxic effect of 10 µM TBBPA. These results demonstrate that both calcium imbalance and oxidative stress are involved in the mechanism(s) of TBBPA cytotoxicity in CGC. In the 10 µM TBBPA treatment they are indicative for a primary role of increases in [Ca2+]i in triggering oxidative stress, depolarization of mitochondria and cytotoxicity; however, calcium-independent mechanisms emerge with a higher TBBPA concentration of 25 µM.This study shows that in CGC TBBPA concentration-dependently increases [Ca2+]i, ROS production and ∆Ψm, respectively. Reports criticizing the low resolution and sensitivity of plate readers in measuring highly dynamic and transient changes in [Ca2+]i with the fluorescent probes [2+]i in CGC, including the concentration-effect relation and pharmacological modulation i in CGC are dependent on TBBPA concentration and may be totally prevented by co-application of the NMDAR antagonist 0.5 µM MK-801 with ligands of RyR, 200 µM ryanodine and 2.5 µM bastadin 12  into H2O2, whilst catalase and GSH-Px are responsible for converting this potentially toxic product into water i, and that incubation of cells in Ca2+-free medium reduces ROS production, suggesting the role of calcium in inducing oxidative stress, although MK-801 failed to significantly inhibit ROS production in CGC treated with 10 µM TBBPA [2+]i in triggering TBBPA-evoked oxidative stress in CGC. Also, under these conditions, the free radical scavengers GSH and BHA prevented accumulation of ROS and a decrease in the antiradical potential of the cells. In contrast, with 25 µM TBBPA, despite the almost complete inhibition of the increase in [Ca2+]i by the combination of NMDAR and RyR antagonists, these compounds only partially reversed an increase in the production of ROS and a decrease in GSH level . Our prBelow is the link to the electronic supplementary material.Supplementary material 1 (PDF 300 KB)Supplementary material 2 (PDF 436 KB)


The hydrophobic pattern of the developed Ca2+ analysis microarray could largely suppress the “coffee-ring” effects to facilitate the better distribution density of testing microspots toward the high-throughput detections, and especially prevent the cross-contamination of the multiple samples between adjacent microspots. Moreover, the use of APS matrix could endow the CCA probe the enhanced environmental stability and fluorescence intensity, which is about 2.3-fold higher than that of free CCA. The interactions between APS-CCA and Ca2+ ions were systematically characterized by UV-vis and fluorescence measurements including microscopy imaging. It was demonstrated that the fluorimetric microarray could display the strong capacity of specifically sensing Ca2+ ions with the minimal interferences from blood backgrounds. Such an APS-CCA-based fluorimetric microarray can allow for the analysis of Ca2+ ions down to 0.0050 mM in blood, promising a highly sensitive and selective detection candidate for Ca2+ ions to be applied in the clinical laboratory.A rapid, ultrasensitive, and high-throughput fluorimetric microarray method has been developed using hydrophobic pattern as the microarray substrate and 3-aminopropyltriethoxysilane-coupled carboxylic acid calcium (APS-CCA) as the fluorescent probes for sensing Ca As a result, the changes in Ca2+ ions levels in human body have an inseparable relation with the cell function, signal transduction and cell apoptosis, especially the occurrence and development of the specific diseases. Therefore, it is of particular importance to detect the Ca2+ ions in human body fluids (such as blood) for the revelation of cellular activities and clinical diagnosis.Calcium ions Ca+, a kind2+ ions, such as ion-selective-electrode1823et al. developed an alternative analytical procedure with the silanized microelectrodes for the accurate measurement of Ca2+ and Mg2+ ionset al. depicted a profile by using atomic absorption spectrophotometer to probe co-existing metals  in some important herbal plants2526211819202120212+ ions with high sensitivity, ideal portability, and high throughput is of considerable interest2829Contemporarily, many classic analytical technologies have been excogitated to detect Ca2+ ions, most known as calconcarboxylic acid -3-naphtolic acid). For example, Mondal2+ ions by measuring the absorbance changes. Unfortunately, it is still difficult to achieve the high-throughput detection for the medical analysis of Ca2+ ions in biological media like blood. Moreover, in modern detection measurements, lots of analysis methods have been developed for the high-throughput detections of targets, of which the typical one is the fluorimetric microarray. Nowadays, the high-throughput fluorimetric technologies have sparked the increasing interests in practical research applications for the analysis of multiple samples32333639et al. designed a photoluminescent array for exploring multiplex metal ions such as Cd2+, Mg2+, Ag+, and Hg2+ ionsRecent years have witnessed the rapid development of optical functional materials or fluorescent dyes used as the probes to detect Ca2+ ions in human body fluids  by using hydrophobic hexadecyltrimethoxysilane (HDS) to pattern the microarray substrate and 3-aminopropyltriethoxysilane-coupled carboxylic acid calcium (APS-CCA) as the fluorescent Ca2+ probe. The fabrication procedure of the APS-CCA fluorimetric microarray was schematically illustrated in 2+ detections, where the addition of Ca2+ ions could induce the increased fluorescence intensity of APS-CCA. Moreover, the HDS patterns are transparent enough to be tailored for the optical observations and measurements. Importantly, the HDS-patterned hydrophobic surfaces of microarray would suppress the “coffee-ring” effects so as to improve the distribution of APS-CCA microspots on the microarray deposited with the highly uniform density for the high-throughput detections. Also, the HDS-patterned microarray could functionalize with the lotus-like “self-cleaning” for removing the fouling or cross-pollution of the samples between the adjacent APS-CCA microspots. In addition, the APS-CCA droplets on microarray could display the different colors changing from navy to pale yellow depending on the different concentrations of Ca2+ ions, so as to expect the naked-eye observations. Subsequently, the application feasibility of the APS-CCA fluorimetric microarray for the detections of Ca2+ ions in human body fluids  was demonstrated.Inspired by these pioneering research studies, in this work, we tried to develop a new high-throughput fluorimetric microarray method for Ca2+ ions solution was prepared by diluting a calcium chloride (CaCl2) aqueous solution . Blood samples were provided by clinical laboratory of Xiangya hospital. All the other chemicals were of analytical grade and were used as received.Hexadecyltrimethoxysilane (HDS), 3-aminopropyltriethoxysilane (APS), dimethylcarbinol, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide sodium salt (NHS) were obtained from Aladdin reagent (Shanghai) co., LTD. Concentrated sulfuric acid, hydrochloric acid and sodium hydroxide were supplied by Lai Yang city tower chemical products factory. Calconcarboxylic acid (CCA) was purchased from Shanghai flute cypress chemical technology co., LTD. A standard CaUV-visible absorption spectra were obtained by Lambda 750 spectrophotometer . Fluorescence spectra were collected on a FluoroMax-4 spectrometer . Contact angles were measured by JY-PHb contact angle measuring instrument  Ultraviolet transilluminator CUV-10 and Inverted fluorescence Microscope  were used to take fluorescent photos.Hydrophobic glasses were prepared in a typical method2+ ions in water and real blood were conducted by the following procedure. Firstly, after the preparation of array, a series of 1.0 μL of Ca2+ ions solutions with different concentrations from 0.010 mM to 2.0 mM diluted by double-distilled water were separately added onto the microspots of APS-CCA. After 6 min at room temperature to ensure the completed reaction, the changes of fluorescence intensities of the resulting solutions were recorded under the ultraviolet transilluminator. Secondly, the control tests for 2.0 mM metal ions  were conducted accordingly. Thirdly, by following the same procedure above, the developed fluorimetric microarray assays were applied to detect Ca2+ ions of different concentrations ranging from 0.020 mM to 1.8 mM that were prepared by being separately spiked in the fresh blood samples. Herein, the enhancing efficiencies of APS-CCA by Ca2+ ions were calculated according to the relative fluorescence (F − F0)/F0, where F0 and F refer to the fluorescence intensities of APS-CCA (λem 335 nm) in the absence and presence of Ca2+ ions, respectively. Finally, the results of probing Ca2+ ions in real blood samples obtained by the developed method were compared with the classic inductively coupled plasma-mass spectrometry (ICP-MS) method used in the clinical laboratory.The selective detections for Ca2+ ions. Also, the so developed Ca2+ microarray could exhibit the APS-enhanced environmental stability and fluorescence intensity of CCA. More importantly, the hydrophobic HDS pattern would help to achieve the highly uniform and dense deposition of testing dots on the microarrays for the high-throughput detections, resulting from that the hydrophobic surfaces could influence the evaporation of droplets both in the constant contact radius and contact angle modes towards the large suppression of the “coffee-ring” effects29442+ ions with the sensitive and reproducible signal outputs.It is generally recognized that the common detection microarrays can suffer from the low distribution density of testing microspots and the risk of undesirable fouling or cross-contamination of multiple samples, which can be resulted from the “coffee ring” effects of the sensing substances or probes to be immobilized. Moreover, it was well demonstrated that the design of hydrophobic surfaces could be expected to suppress the “coffee-ring” effects29442+ ions. The unique optical properties of the obtained APS-CCA were characterized by fluorescence measurements , and reaction time /F0, where F and F0 refer to the fluorescence intensities of APS-CCA (λem 335 nm) with and without Ca2+ ions, respectively. As is shown in 2+ ions. In the presence of Ca2+ ions, the relative fluorescence intensities of APS-CCA increased gradually with the increasing NaCl concentrations till 0.15 M, which is close to the concentration of human physiological saline. As the concentrations of NaCl increasing beyond 0.15 M, the relative fluorescence intensities of APS-CCA decreased gradually. Thus, the concentration of NaCl of 0.15 M was chosen for the Ca2+ detections. The effects of pH values on the fluorimetric responses of APS-CCA to Ca2+ ions were explored with the results shown in 2+ ions could induce the increase in fluorescence intensities with the different degrees. According to the increased fluorescence efficiencies, pH 7.1 was chosen as the optimal one for the analysis experiments for Ca2+ ions. 2+ ions. Obviously, the relative fluorescence values could attain the maximum at the reaction temperature of 37 °C.The main conditions for the developed fluorimetric microarray assays were optimized for the detection of Caion time . Herein,2+ ions were studied, taking CCA as the comparison , suggesting a straightforward fluorescence-enhancement protocol was built up. Furthermore, 2+ ions separately. As the time changes (1–6 months), the relative fluorescence intensities of the APS-CCA remained about the same. In contrast, the relative fluorescence intensity of CCA was weakened gradually towards being quenched after about 6 months. Such an experimental phenomenon implies that the APS could enhance the fluorescence stability of CCA in the presence of Ca2+ ions to promise the significance of practical detection applications.Moreover, the influences of response time on the fluorimetric detections of APS-CCA for Camparison . Obvious2+ sensing selectivity, including 17 kinds of common metal ions of Zn2+, Cd3+, Ca2+, K+, Cu2+, Ni2+, Fe2+, Mg2+, Ba2+, Al3+, Co2+, Ag+, Pb2+, Hg2+, Cr3+, Na+, Mn2+ ions , with a limit of detection (LOD) of 0.0050 mM, which is much lower than the normal value in the human body of about 0.94–1.26 mM2+ ions of different concentrations could also be observed by naked eyes under ultraviolet transilluminator , as shown in 2+ ions in real blood samples. The regression equation for the detection results for Ca2+ ions was obtained with correlation coefficient of 0.9751. Obviously, there is no significant difference between the results obtained from the two methods for analyzing Ca2+ ions in blood. Therefore, the developed fluorimetric microarray strategy with APS-CCA probe promises the potential feasibility of serving as a rapid and reliable candidate for the selective and sensitive detection of Ca2+ ions in blood.The application feasibility of the APS-CCA based on fluorimetric microarray method was investigated for Ca2+ ions in blood. The so fabricated hydrophobic microarray substrate could suppress the “coffee-ring” effects to facilitate the better distribution density of testing microspots and prevent the cross-contamination of the multiple samples between adjacent microspots toward the high-throughput detections. Moreover, the use of APS could endow the APS-CCA probes the enhanced fluorescence intensity and environmental stability for specifically sensing Ca2+ ions. The developed Ca2+ fluorimetric microarray was fabricated by the efficient “dip and dry” procedure, without the need for any complicated treatment. The detection results could also be observed by naked eyes if using ultraviolet transilluminator. It should be pointed out that the denser testing microspots would be created on a microarray by a mechanical sample spotter to achieve an even higher throughput of sample analysis. The developed APS-CCA-based fluorimetric microarray strategy is simple, rapid, selective, and highly sensitive, thus holding great promise for the high throughput detection of Ca2+ ions in the clinical, food hygiene, and environmental monitoring fields.In summary, a high-throughput fluorimetric microarray has been successfully developed using hydrophobic HDS pattern as the microarray substrate and APS-CCA as the fluorescent probes for sensing CaHow to cite this article: Ding, Y. et al. A high-throughput fluorimetric microarray with enhanced fluorescence and suppressed “coffee-ring” effects for the detection of calcium ions in blood. Sci. Rep.6, 38602; doi: 10.1038/srep38602 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.