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.

Solidification process of conventional superalloy by confocal scanning laser microscope

The solidification process of a conventional superalloy, IN718, was investigated by confocal scanning laser microscope （CSLM）. The liquid fraction during solidification was obtained as a function of real time and temperature in reference with the in-situ observation. The characteristics of L→γ transformation were analyzed and the γ growing rate of each stage was also calculated. Scheil equation was employed to predict the segregation behavior, and the predict results are in consistence with the experimental results. As a result, the confocal scanning laser microscope shows a great potential for solidification process research.

A stage-scanning laser confocal microscope and protocol for DNA methylation sequencing

Recent understanding of the role of epigenetic regulation in health and disease has necessitated the development of newer and efficient methods to map the methylation pattern of target gene. In this article we report construction of a stage-scanning laser confocal microscope (SLCM) and associated protocol that determines the methylation status of target gene. We have adapted restricted Sanger’s sequencing where fluorescine labeled primers and dideoxy guanine fraction alone are used for target amplification and termination at cytosine positions. Amplified ssDNA bands are separated in 6% denaturing PAGE and scanned using SLCM to sequence the positions of methylated cytosines. We demonstrate that our me- thodology can detect < 100 femtomoles of DNA, and resolve the position of cytosine within ± 2 nucleotide. In a calibration run using a designer DNA of 99 bases, our methodology had resolved all the 11 cytosine positions of the DNA. We have further demonstrated the utility of apparatus by mapping methylation status in the Exon-1 region of a gene, E-Cadherin, in the plasma DNA sample of a healthy subject. We believe our approach constitute a low cost alternative to conventional DNA sequencers and can help develop methylation based DNA biomarkers for the diagnosis of disease and in therapeutics.

Confocal Laser Scanning Microscope Evaluation of Early Bacterial Colonization on Zirconium Oxide and Titanium Surfaces:An in vivo Study

To investigate the bacterial colonization on zirconium oxide and titanium surfaces in vivo quantitatively using a confocal laser scanning microscope （CLSM）. Ten samples of zirconium oxide ceramic and commercially pure titanium were fabricated and polished using silicon carbide abrasive paper. One sample from each group was evaluated topographic pattern under a scanning electron microscope. One sample from each group was to evaluate roughness using a profilometer. Eight volunteers were selected. The samples were cemented at the buccal surfaces of upper first molars. All samples were removed after 48 hours, immersed in SYTO-9 and propidium iodide fluorescent to stain for adherent bacteria and obseIved with CLSM. Fewer bacteria were observed in zirconia group than titanium group. However, there was no statistical difference between two groups. The experimental results demonstrate that zirconium oxide may be considered as a promising material for dental implant abutments.

Concanavalin A-induced changes in lysosomal morphology of macrophages under confocal microscope

Changes in lysosomal morphology of cultured mouse peritoneal macrophages after stimu1ation by Concanavalin A (Con A) were observed with a laser scanning confocal microscope (LSCM). A series of images were obtained including phase-contrast images, optical sectioning images, 3-dimensional reconstruction images. The changes of lysosomal fluorescence intensity and pH were measured. It was found that macrophage lysosomes were Cllstributed mainly at the periphery of the cells in resting conditions, the lysosomal area containing fluorescence probe became markedly enlarged after stimulation by Con A for 30 min, and the fluorescence intensity in the medium increased about 15 min after suggesting that Con A could induce outflow of the fluorescence probe within the macrophage lysosomes. The lysosomal pH rose from 4. 6 to 5. 7 in 7 min after Con A was added, and maintained at that level hereafter.

Real time detection of antibody-antigen interaction using a laser scanning confocal imaging-surface plasmon resonance system

A laser scanning confocal imaging-surface plasmon resonance （LSCI-SPR） instrument integrated with a wavelength-dependent surface plasmon resonance （SPR） sensor and a laser scanning confocal microscopy （LSCM） is built to detect the bonding process of human IgG and fluorescent-labeled affinity purified antibodies in real time. The shifts of resonant wavelength at different reaction time stages are obtained by SPR, corresponding well with the changes of the fluorescence intensity collected by using LSCM. The instrument shows the merits of the combination and complementation of the SPR and LSCM, with such advantages as quantificational analysis, high spatial resolution and real time monitor, which are of great importance for practical applications in biosensor and life science.

Micro-Scanning Error Correction Technique for an Optical Micro-Scanning Thermal Microscope Imaging System

An error correction technique for the micro-scanning instrument of the optical micro-scanning thermal microscope imaging system is proposed. The technique is based on micro-scanning technology combined with the proposed second-order oversampling reconstruction algorithm and local gradient image reconstruction algorithm. In this paper, we describe the local gradient image reconstruction model, the error correction technique, down-sampling model and the error correction principle. In this paper, we use a Lena original image and four low-resolution images obtained from the standard half-pixel displacement to simulate and verify the effectiveness of the proposed technique. In order to verify the effectiveness of the proposed technique, two groups of low-resolution thermal microscope images are collected by the actual thermal microscope imaging system for experimental study. Simulations and experiments show that the proposed technique can reduce the optical micro-scanning errors, improve the imaging effect of the system and improve the system's spatial resolution. It can be applied to other electro-optical imaging systems to improve their resolution.

Quantum Dots as Fluorescent Labels for Detection of Heat Shock Protein in Tumor Tissue Using Laser Scanning Confocal Microscope

A new Quantum Dots（Qdots） nanocrystal composed of semiconductor core and zinc sulfide shell, and its feasibility as labels in immunofluorescence analysis for the imaging of tumor biomarkers by laser scanning confocal microscope（LSCM） was investigated. Qdots taged by mercaptoacetic acid were conjugated with second antibody, then imaging differences of Heat Shock Proteins 70（HSP70） in renal carcinoma tissure sections with immunofluorescence analysis method using Qdots bioconjugates and conventional organic dye FITC were observed by LSCM to assess the brightness and opticalstability of Qdots. The experimental results showed Qdots bioconjugates achieved the better results in demonstrating HSP70 with more brighter color and more clear picture than FITC labels. Moreover, the label signals of Qdots did not fade clearly after continued exposure to a 488 nm laser for 1 h. The Qdots bioconjugates have good feasibility in immunofluorescence analysis for the bioimaging by LSCM.

Optical micro-scanning location calibration of thermal microscope imaging system

A method of micro-scanning location adaptive calibration was proposed, which was real- ized by the digital image micro-displacement estimation. With geometric calculation, this calibration method used the displacement estimation of two thermal microscope images to get the size and direc- tion of each scanning location calibration angle. And each location calibration process was repeated according to the offset given by the system beforehand. The comparison experiments of sequence oversampling reconstruction before and after the micro-scanning location calibration were done. The results showed that the calibration method effectively improved the thermal microscope imaging qual- ity.

Qualitative analysis of re mineralized carious lesions subjected to fluoride supplement through confocal laser scanning microscope

Aim: 1] Comparative evaluation of the linear depth of induced remineralized lesions after subjecting to fluoride supplements and 2] To assess the average fluorescence at both the demineralized and the remi-neralized zones in all the three study groups under confocal laser scanning microscope. Method: Forty five sound human premolars extracted for orthodon-tic reasons were decoronated 1 mm below the ce-mento-enamel junction and coated with nail varnish except for a 3 × 3 mm window on the buccal surface. The samples were placed in 50 ml of de mineralizing solution at pH 4.6 for 96 hours. Following deminera-lization, the lower half of the 3 × 3 mm window in all the samples were covered with nail varnish to serve as control. The samples were randomly divided into three groups of fifteen teeth each (n = 15) and speci-mens in group A[Nfd] were remineralized using non-fluoridated dentifrice [control], those in groups B [Fd5] and group C [Fd10] using 500 ppm and 1000 ppm of fluoride containing dentifrice, respectively. The specimens were subjected to a 20 day reminera-lization treatment regimen and were sectioned into 100 μm thick sections and two images were captured on the buccal surface from either side of the midpoint of occluso-cervical length using confocal laser scan-ning microscope [CLSM]. Results: were tabulated and statistically analyzed by Anova. Study concluded that 1000 ppm fluoridated dentifrice showed a greater degree of remineralization than other groups and confocal laser scanning microscopes gives promising results in the diagnosis of early enamel lesions over the conventional methods.

X-ray phase-sensitive microscope imaging with a grating interferometer:Theory and simulation

A general theoretical framework is presented to explain the formation of the phase signal in an x-ray microscope integrated with a grating interferometer,which simultaneously enables the high spatial resolution imaging and the improved image contrast.By using this theory,several key parameters of phase contrast imaging can be predicted,for instance,the fringe visibility and period,and the conversion condition from the differential phase imaging(DPI)to the phase difference imaging(PDI).Additionally,numerical simulations are performed with certain x-ray optical components and imaging geometry.Comparison with the available experimental measurement[Appl.Phys.Lett.113063105(2018)]demonstrates the accuracy of this developed quantitative analysis method of x-ray phase-sensitive microscope imaging.

Oversample Reconstruction Based on a Strong Inter-Diagonal Matrix for an Optical Microscanning Thermal Microscope Imaging System

Based on a strong inter-diagonal matrix and Taylor series expansions,an oversample reconstruction method was proposed to calibrate the optical micro-scanning error. The technique can obtain regular 2 ×2 microscanning undersampling images from the real irregular undersampling images,and can then obtain a high spatial oversample resolution image. Simulations and experiments show that the proposed technique can reduce optical micro-scanning error and improve the system＇s spatial resolution. The algorithm is simple,fast and has low computational complexity. It can also be applied to other electro-optical imaging systems to improve their spatial resolution and has a widespread application prospect.

Deep learning method for cell count from transmitted-light microscope

Automatic cell counting provides an effective tool for medical research and diagnosis.Currently,cell counting can be completed by transmitted-light microscope,however,it requires expert knowledge and the counting accuracy which is unsatisfied for overlapped cells.Further,the image-translation-based detection method has been proposed and the potential has been shown to accomplish cell counting from transmitted-light microscope,automatically and effectively.In this work,a new deep-learning(DL)-based two-stage detection method(cGAN-YOLO)is designed to further enhance the performance of cell counting,which is achieved by combining a DL-based fluorescent image translation model and a DL-based cell detection model.The various results show that cGAN-YOLO can effectively detect and count some different types of cells from the acquired transmitted-light microscope images.Compared with the previously reported YOLO-based one-stage detection method,high recognition accuracy(RA)is achieved by the cGAN-YOLO method,with an improvement of 29.80%.Furthermore,we can also observe that cGAN-YOLO obtains an improvement of 12.11%in RA compared with the previously reported image-translation-based detection method.In a word,cGAN-YOLO makes it possible to implement cell counting directly from the experimental acquired transmitted-light microscopy images with high flexibility and performance,which extends the applicability in clinical research.

Axial intensity in a fiber-optic confocal microscope

A fiber-optic confocal microscope has been analyzed by Fourier optics.It is found that the detected light intensity has three parts,each of which is depennted on the coupled lens,the detective lens,and the part comprised of the fiber and the microprobe.The simulated results show that the less the value of the parameter A is,which is dependent on the fiber and microprobe,the higher the axial resolution of the system is. For the case,as A→∞, the axial resolution is zero,which is corresponding to the conventional microscope.as A≤1,the axial resolution changes slightly,and is close to the optimal value,which is corresponding to the perfect confocal microscope.when the reflective loss takes place at the end of fiber,the contrast of axial intensity will decrease.All that will help the design of endoscope with confocal microscope at cellular level.

Effect of Hypericin on Confocal Imaging of Ca^(2+) Signaling in Cultured Human Retinal Pigment Epithelium

Purpose: To investigate the mechanism of the Ca2 + signaling in cultured human retinal pigment epithelial(RPE) cells with the protein kinase C(PKC) specific inhibitor-hypericin stimulation.Methods: Cultured human RPE cells were analyzed using the fluorescence Ca2+ dye fluo-3 AM and laser scanning confocal microscope(LSCM) after stimulation with 100nM phorbol 12-myristate 13-acetate(PMA) and (or)5 concentrations of hypericin(1, 2, 3, 4 and 5 μM).Results: The normal fluorescence in RPE cells was strong and distributed throughout the cells. The nucleus appeared to be more fluorescent than the cytoplasm. After stimulation with PMA alone or 5 concentrations of hypericin, a rapid decrease in flurescence intensity was observed. There was no obvious difference in decreased curve among 5concentrations. However, after stimulation with a 24 hr preincubation of PMA and 5 concentrations of hypericin, a further decrease was not observed.Conclusion: Fluo-3 AM appears to be a good indicator of the change in Ca2+ occurring in RPE cells and hypericin is a strong inhibitor of Ca2 + influx channel. Hypericin has potential as a therapeutic drug for proliferative vitreoretinopathy(PVR), the inhibitory effect on PVR might be caused by blocking the PKC activity and inhibiting Ca2+ influxpathway.

MicroScope高分辨率成像测井技术及应用

MicroScope随钻高分辨率成像测井技术是斯伦贝谢公司最新推出,能记录超高分辨率电阻率图像UHRI和多个探测深度深钮电阻率成像图,介绍了超高分辨率电阻率成像图UHRI识别岩性及地层结构、识别沉积构造、划分沉积微相、分析古水流与砂体展布等的方法,在南海西部沉积相研究方面发挥了重要作用,是值得推广的新型成像测井技术。

A Simple,Compact and Rigid Scanning Tunneling Microscope

We present a homebuilt scanning tunneling microscope(STM)which employs an inner-wall polished sapphire guiding tube as a rail for the scanner to form a short tip-sample mechanical loop.The scanner is mounted on a square rod which is housed in the guiding tube and held by a spring strip.The stiff sapphire guiding tube allows the STM body to be made in a simple,compact and rigid form.Also the material of sapphire improves the thermal stability of the STM for its good thermal conductivity.To demonstrate the performance of the STM,high quality atomic-resolution STM images of high oriented pyrolytic graphite were given.

Retrieving analog images from a scanning electron microscopewith a synchronous data acquisition method

In this work,an old scanning electron microscope(SEM)is refurbished to enhance its image processing capability.How to digitally sample and process an analog image is also presented.An NI PCI-6259 multiple input/output data acquisition(DAQ)board is used to acquire signals originally being sent to an analog display,and then convert the signals into a digital image.Two output channels are used for raster scan of the horizontal and verticle axes of the image buffer,while one input channel is used to read the brightness signals at various coordinate points.Synchronous method is used to maximize the DAQ speed.Finally,the digitally buffered images are read out to display and saved in a hard drive.The hardware and software designs of this work are explained in great detail,which can serve as a very good example for fast synchronous DAQ,advanced virtual instrument design and structural driver programming with LabVIEW.

Molecular Axis Orientation in Charge Transfer Reactions Determined with a Reaction Microscope

Based on the reaction microscope at the institute of modern physics, the reaction mechanism in molecular ion-atom collisions is investigated experimentally. The features of this system is illustrated by a kinematically complete experhnent performed for the collision process. Using the so-called list-mode data recording technique and the coincidence measurement, the momentum vector of each fragment from the molecular ion were recorded event by event. The orientation of the molecular axis for H2^＋ dissociation reactions could be determined for each event in the off-line analysis. The measured orientation of the molecular ion is believed the same as the one at the instance of collision under axial recoil approximation. The polar angle resolution of the molecular orientation of ±8° was obtained.

Dark-field line confocal imaging with point confocality and extended line field for bulk defects detection

Fabrication of high-quality optics puts a strong demand on high-throughput detection of macroscopic bulk defects in optical components.A dark-field line confocal imaging method is proposed with two distinct advantages:(ⅰ)a point-to-line confocal scheme formed by a columnar elliptical mirror and an optical fiber bundle breaks through the constraint on light collection angle and field of view in the traditional line confocal microscopy using an objective,allowing for an extended confocal line field of more than 100 mm while maintaining a light collection angle of 27°;(ⅱ)the bulk defects are independently illuminated as a function of time to eliminate the cross talk in the direction of the confocal slit,thus preserving point confocality and showing the optical section thicknesses to be 162μm in the axial direction,and 19 and 22μm in the orthogonal transverse directions.The experimental results verify that the method has a minimum detectable bulk defect of less than 5μm and an imaging efficiency of 400 mm2/s.The method shows great potential in high-throughput and highsensitivity bulk defects detection.