Development of a portable reflectance confocal microscope and its application in the noninvasive in vivo evaluation of mesenchymal stem cell-promoted cutaneous wound healing

The process of wound healing is routinely evaluated by histological evaluation in the clinic,which may cause scarring and secondary injury.Reflectance confocal microscopy(RCM)represents a noninvasive,real-time imaging technique that allows in vivo evaluation of the skin.Traditional RCM was wide-probe-based,which limited its application on uneven and covered skin.In this study,we report the development of a portable reflectance confocal microscope(PRCM)in which all components were assembled in a handheld shell.Although the size and weight of the PRCM were reduced based on the use of a microelectromechanical system,the resolution was kept at 0.91μm,and the field of view of the system was 343μm×532μm.When used in vivo,the PRCM was able to visualize cellular and nuclear morphology for both mouse and human skin.PRCM evaluations were then performed on wounds after topically applied mesenchymal stem cells(MSCs)or saline treatment.The PRCM allowed visualization of the formation of collagen bundles,re-epithelization from the wound edge to the wound bed,and hair follicle regeneration,which were consistent with histological evaluations.Therefore,we offer new insights into monitoring the effects of topically applied MSCs on the process of wound healing by using PRCM.This study illustrates that the newly developed PRCM represents a promising device for real-time,noninvasive monitoring of the dynamic process of wound healing,which demonstrates its potential to diagnose,monitor,or predict disease in clinical wound therapy.

In situ observation of the dissolution kinetics of Al_(2)O_(3) particles in CaO–Al_(2)O_(3)–SiO_(2) slags using laser confocal scanning microscopy

The dissolution kinetics of Al_(2)O_(3) in CaO-Al_(2)O_(3) SiOslags was studied using a high-temperature confocal scanning laser microscope at 1773 to 1873 K.The results show that the controlling step during the Al_(2)O_(3) dissolution was the diffusionin molten slag.It was found that the dissolution curves of Al_(2)O_(3) particles were hardly agreed with the traditional boundary layer diffusion model with the increase of the CaO/Al_(2)O_(3) ratio of slag.A modified diffusion equation considering slag viscosity was developed to study the dissolution mechanism of Al_(2)O_(3) in slag.Diffusion coefficients of Al_(2)O_(3) in slag were calculated as 2.8×10to 4.1×10m~2/s at the temperature of 1773-1873 K.The dissolution rate of Al_(2)O_(3) increased with higher temperature,CaO/Al_(2)O_(3),and particle size.A new model was shown to be v_(Al_(2)O_(3))=0.16×r_(0)^(1.58)×x^(3.52)×(T-T_(mp))^(1.11)to predict the dissolution rate and the total dissolution time of Al_(2)O_(3) inclusions with various sizes,where vAl_(2)O_(3) is the dissolution rate of Al_(2)O_(3) in volume,μm^(3)/s;x is the value of CaO/Al_(2)O_(3) mass ratio;R_(0) is the initial radius of Al_(2)O_(3),μm;T is the temperature,K;T_(mp) is the melting point of slag,K.

Highly specific characterization and discrimination of monosodium urate crystals in gouty arthritis based on aggregation-induced emission luminogens

Existing technologies used to detect monosodium urate(MSU)crystals for gout diagnosis are not ideal due to their low sensitivity and complexity of operation.The purpose of this study was to explore whether aggregation-induced emission luminogens(AIEgens)can be used for highly specific imaging of MSU crystals to assist in the diagnosis of gout.First,we developed a series of luminogens(i.e.,tetraphenyl ethylene(TPE)-NH_(2),TPE-2NH_(2),TPE-4NH_(2),TPE-COOH,TPE-2COOH,TPE-4COOH,and TPE-Ketoalkyne),each of which was then evenly mixed with MSU crystals.Next,optimal fluorescence imaging of each of the luminogens was characterized by a confocal laser scanning microscope(CLSM).This approach was used for imaging standard samples of MSU,hydroxyapatite(HAP)crystals,and mixed samples with 1:1 mass ratio of MSU/HAP.We also imaged samples from mouse models of acute gouty arthritis,HAP deposition disease,and comorbidities of interest.Subsequently,CLSM imaging results were compared with those of compensated polarized light microscopy,and we assessed the biosafety of TPE-Ketoalkyne in the RAW264.7 cell line.Finally,CLSM time series and three-dimensional imaging were performed on MSU crystal samples from human gouty synovial fluid and tophi.As a promising candidate for MSU crystal labeling,TPE-Ketoalkyne was found to detect MSU crystals accurately and rapidly in standard samples,animal samples,and human samples,and could precisely distinguish gout from HAP deposition disease.This work demonstrates that TPE-Ketoalkyne is suitable for highly specific and timely imaging of MSU crystals in gouty arthritis and may facilitate future research on MSU crystal-related diseases.

Overexpression of Myocardin-related transcription factor-A attenuated middle cerebral artery occlusion/reperfusion-induced apoptosis via the Mcl-1/Cyt C/cleaved caspase 3 pathway

To investigate the effect of Myocardin related transcription factor A(MRTF-A)on apoptosis induced by ischemic/reperfusion(I/R),middle cerebral artery occlusion/reperfusion(MCAO/R)in rats were applied to mimic I/R.The neurological deficit score,cerebral infarct size,cortical neuron apoptosis and cleaved caspase 3 level were evaluated to determine the effect and the level of apoptosis by TTC straining,terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)straining,Western blot and immunofuorescence staining.The myeloid cell leukemia-1(Mcl-1)expression,release of cytochrome C(Cyt C)and its colocalization with apoptotic pro-tease activating factor-1(Apaf-1)were analyzed by quantitative real-time PCR(qRT-PCR),Western blot,and immunofuorescence staining.The results showed that MRTF-A over-expression could decrease the neurological deficit score and reduce cerebral infarct size(P<0.01 versus Sham).In the MRTF-A-I/R group,TUNEL-positive cells and apoptosis ratio(%)(51.61±6.17%)were significantly decreased compared to the Neg-I/R group(76.45±8.77%)at 24 h reperfusion.Meanwhile,the cleaved caspase 3 expression revealed a similar trend while the expression of Mcl-1 was the opposite.Moreover,MRTF-A overexpression significantly enhanced Mcl-1 fAuorescence intensity,which up-regulated the mRNA and protein level(P<0.05or P<0.01 versus Neg-I/R).Furthermore,MRTF-A overexpression markedly inhibited the release of Cyt C,and decreased the colocalization with Apaf-1 in the cytoplasm(P<0.05 or P<0.01.versus Neg-I/R).All the data indicated that MRTF-A overexpression could improve the neu-rological function against cerebral I/R-induced apoptosis since underlying mechanism might be involved in the Mc-1/Cyt C/cleaved caspase 3 signaling pathway.

THE COCHlEAR RIBBON SYNAPTIC RESPONSE TO AMINOGIYCOSIDE OTOTOXICITY IN C57BL/6J MICE

Objective To investigate the early change of cochlear ribbon synapses on inner hair cells in response to aminoglycoside ototoxicity. Methods C57BL/6J mice received intraperitoneal injection of gentamicin(100mg/kg/day), and the apical coil organ of Corti was examined on the 4th, 7th and 10 th day(n=10). Littermates without gentamicin treatment served as controls(n=10). RIBEYE on the presynaptic membrane and AMPA receptors on the postsynaptic membrane were labeled with CtBP2 or GluR2/3 respectively. Three dimension reconstruction was conducted using the 3DS MAX 8.0 software. Results There were no disruptions of outer or inner hair cells in all groups. However, the number of ribbon synapses on cochlear inner hair cells increased significantly within 7 days after gentamicin exposure(P<0.01), followed by a significant decrease after 7 days.Conclusion During the early stage of aminoglycoside ototoxicity, increased population of cochlear ribbon synapses may indicate a significant down-regulation of synaptic function.

Optimizing signal collection efficiency of the VIPA-based Brillouin spectrometer

Brillouin spectroscopy is an emerging tool for microscopic optical imaging as it allows for non-invasive and direct assessment of the viscoelastic properties of materials.Recent advances of background-free confocal Brillouin spectrometer allows investigators to acquire the Brillouin spectra for turbid samples as well as transparent ones.However,due to strong signal loss induced by the imperfect optical setup,the Brillouin photons are usually immersed in background noise.In this report,we proposed and experimentally demonstrated multiple approaches to enhance the signal collction eficiency.A signal enhancement by>4 times can be observed,enabling ob-servation of ultra-weak signals.

A Protocol for Fractal Studies on Porosity of Porous Media: High Quality Soil Porosity Images

A protocol for obtaining digital images from natural porous media with a wide range of pore sizes, intended for fractal studies of the porosity, is proposed. Soil porosity is used as paradigm of complex natural porous media in this study. The use of several imaging devices and fluorescent compounds to enhance the contrast between the solid and the pore phase is tested. Finally a protocol is reached using a photo camera and a confocal microscope. It is the first time that confocal microscopy is used for this purpose. Artificial porous images are created through random Sierpinski carpet fractals and the statistical information of real soil images. These ground truth images are used in an objective comparison of automatic segmentation algorithms for the obtained images. A statistical classification on the performance of several automatic segmentation algorithms for this type of images is reached.

Constructing zwitterionic nanofiber film for anti-adhesion of marine corrosive microorganisms

A zwitterionic nanofiber film was constructed through combining zwitterionic polymer with anodic aluminum oxide template for anti-adhering typical marine corrosive microorganisms,i.e.Pseudomonas aeruginosa,Desulfovibrio vulgaris and Chaetoceros muelleri.Results showed that the fabricated zwitterionic polymers film has fibrous nanostructure with uniform distribution and super hydrophilia.This film has wide anti-adhering properties,and it can effectively reduce the attachment of these three microorganisms by more than 99%.Moreover,the adhesion of extracellular polymeric substances secreted from these three microorganisms are also inhibited,which is one reason for the fabricated nanofiber film with antiadhesion characteristic of microorganisms.This research provides valuable information for solving the problem of microbial adhesion on metal surfaces in the marine environment.

Effect of high temperature cooking on the quality of rice porridge

In order to explore the effect of high-temperature cooking instant porridge on the taste and quality of porridge,this study explored the changes in the taste quality of rice porridge under different heating times(15 min,20 min,25 min and 30 min)at a higher initial temperature(100°C),and compared with that cooked at room temperature(25°C),and explained the reasons for the change.From the perspective of migration rate in moisture content,the rice porridge with special treatment has a more rapid moisture change,which makes texture,whiteness,smell and other factors change in a shorter time and increases the taste quality.It was concluded that under special treatment condition of starting with 100°C water and heating for 15 min,the kinds of rice flavor increased 2.2 times,the white of rice decreased by 10.04%,hardness decreased by 3.54 times,and the elasticity decreased by 35.48%respectively.In general,cooking with higher initial temperature(100°C)is more valuable than cooking from 25°C,in terms of the texture,flavor,saving time,and stable quality during the household and industrial production of instant porridge.