双荧光标记生物芯片激光共聚焦检测系统

基于激光共聚焦检测原理,构建了针对荧光标记生物芯片的检测系统,对用Cy₃或Cy₅标记蛋白点样的玻片进行了扫描检测,并对采集的荧光数据信号进行了图像重建。针对实际荧光信号可能较微弱并存在较大动态范围的情况,采用形态学方法对图像进行滤波、增强处理,显著增强了图像质量。提出了标定系统信噪比和灵敏度的公式,并据此对检测实验结果进行了详细的分析计算,最终标定此激光共聚焦生物芯片检测系统的灵敏度约为0.1 fluo/μm²。     

Tubular dentin sealer penetration after different final irrigation protocols: A confocal laser scanning microscopy study

The aim of this study was to evaluate tubular dentin sealer penetration, comparing different final irrigation protocols using a conventional needle (CONV), EndoActivator system (EAS), EndoVac system (EVS), and ultrasound (PUI). Initially, fifty‐two first maxillary molars with a single canal in the palatal root, without abrupt curvatures, resorptive processes, or previous endodontic treatment were selected for this study. Then, the crowns were sectioned to obtain palatal roots 15 mm in length. The root canals were prepared with the ProTaper Universal System and irrigated with 5% NaOCl. Afterwards, the specimens were divided into four groups (n. 13), according to the final irrigation protocol: CONV, EAS, EVS, and PUI. After filling, slices at 3 mm and 5 mm from the apex were obtained for analysis by confocal laser scanning microscopy. Two‐way comparisons between the groups and the levels were performed with Games Howell's test (p < .05). Tubular dentin sealer penetration was higher at 5 mm compared with 3 mm from the apex (p < .05). The EAS group showed a higher percentage of tubular dentin sealer penetration, compared with the CONV group, at both levels. At 3 mm, there was no statistically significant difference among EAS, EVS, and PUI; however, these groups showed better performance, compared with the CONV group. At 5 mm, there was no statistically significant difference between the EAS and EVS groups, but both showed higher sealer penetration than the PUI group (p < .05). The EAS and EVS groups achieved better degrees of tubular dentin sealer penetration, compared with the other groups.     

Dentinal tubule penetration of endodontic sealers after nonthermal plasma treatment: A confocal laser scanning microscopy study

One of the factors affecting the success of endodontic treatment is to fill the root canal system hermetically. The aim of this in vitro study was to evaluate the effect of nonthermal plasma (NP) on dentinal tubule penetration of root canal sealers using confocal laser scanning microscopy. Forty mandibular premolar teeth were selected and the root canals were prepared with large‐Waveone‐Gold rotary‐files. Specimens were divided into four experimental groups according to sealer and NP treatment (n = 10). G1: AH‐Plus (AH) G2: nonthermal plasma application + AH‐Plus(AH‐P) G3: Endosequence‐BC(BC) G4: nonthermal plasma application + Endosequence‐BC(BC‐P). Cold lateral‐condensation technique was used for the obturation of root canals. The roots were sectioned horizontally and the sections were examined under confocal laser scanning microscopy. The maximum tubule penetration and percentage of penetration values were obtained from the microscopy images and were statistically analyzed with repeated measurements‐ANOVA and the Tukey (HSD) test (p < 0.05). The percentages of dentinal tubule penetration of the groups were not statistically different. The maximum tubule penetration of the AH‐P was statistically lower than that of the BC‐P (p < 0.05). Plasma application had no affect on the percentage of dentinal tubule penetration. Under the conditions of this in vitro Endosequence‐BC sealer showed higher maximum tubule penetration values than AH‐Plus after NP treatment. Percentage of dentinal tubule penetration values of experimental groups was similar.     

Isotropic high-resolution three-dimensional confocal micro-rotation imaging for non-adherent living cells

Recently, micro-rotation confocal microscopy has enabled the acquisition of a sequence of micro-rotated images of nonadherent living cells obtained during a partially controlled rotation movement of the cell through the focal plane. Although we are now able to estimate the three-dimensional position of every optical section with respect to the cell frame, the reconstruction of the cell from the positioned micro-rotated images remains a last task that this paper addresses. This is not strictly an interpolation problem since a micro-rotated image is a convoluted two-dimensional map of a three-dimensional reality. It is rather a 'reconstruction from projection' problem where the term projection is associated to the PSF of the deconvolution process. Micro-rotation microscopy has a specific difficulty. It does not yield a complete coverage of the volume. In this paper, experiments illustrate the ability of the classical EM algorithm to deconvolve efficiently cell volume despite of the incomplete coverage. This cell reconstruction method is compared to a kernel-based method of interpolation, which does not take account explicitly the point-spread-function (PSF). It is also compared to the standard volume obtained from a conventional z-stack. Our results suggest that deconvolution of micro-rotation image series opens some exciting new avenues for further analysis, ultimately laying the way towards establishing an enhanced resolution 3D light microscopy.     

Multiphoton fluorescence lifetime imaging of 3D-stem cell spheroids during differentiation

Long-term high-resolution multiphoton imaging of nonlabeled human salivary gland stem cell spheroids has been performed with submicron spatial resolution, 10.5-nm spectral resolution, and picosecond temporal resolution. In particular, the two-photon-excited coenzyme NAD(P)H and flavins have been detected by time-correlated single photon counting (TCSPC). Stem cells increased their autofluorescence lifetimes and decreased their total fluorescence intensity during the adipogenic-differentiation process. In addition, the onset of the biosynthesis of lipid vacuoles was monitored over a period of several weeks in stem-cell spheroids. Time-resolved multiphoton autofluorescence imaging microscopes may become a promising tool for marker-free stem-cell characterization and cell sorting.     

Three-dimensional analysis of cell nucleus structures visualized by confocal scanning laser microscopy

Studies of the three-dimensional (3-D) organization of cell nuclei are becoming increasingly important for the understanding of basic cellular events such as growth and differentiation. Modern methods of molecular biology, including in situ hybridization and immunofluorescence, allow the visualization of specific nuclear structures and the study of spatial arrangements of chromosome domains in interphase nuclei. Specific methods for labelling nuclear structures are used to develop computerized techniques for the automated analysis of the 3-D organization of cell nuclei. For this purpose, a coordinate system suitable for the analysis of tri-axial ellipsoidal nuclei is determined. High-resolution 3-D images are obtained using confocal scanning laser microscopy. The results demonstrate that with these methods it is possible to recognize the distribution of visualized structures and to obtain useful information regarding the 3-D organization of the nuclear structure of different cell systems.     

Real-time confocal imaging, during active air abrasion – substrate cutting

Air abrasion cutting, using particulates accelerated in a controlled compressed gas stream, is currently being re-evaluated as a precision tissue removal technique for dental cavity preparation. The minimal vibrations and heat generated during cutting commend the technique for use in the shaping of fragile or brittle materials that are vulnerable to vibrations and thermal stresses.
Traditional air abrasion studies have relied solely upon post-procedure imaging, and cutting process details have been inferred from the nature of the residual surface. In this paper, however, a real-time confocal microscopic imaging method is described, which for the first time has allowed prior target structure characterization with subsequent imaging of cutting interactions and substrate failure patterns. Using internally focusing long working distance Hill objective lenses, focusing deep to a protective microscope slide and adhesive interfaces, unhindered remote image sampling within the bulk of specimens such as tooth tissue, acrylic and brittle ceramics was possible.
Moreover, areas of active cutting and inactive regions were identified within air abraded cavities during their creation. The characteristics of the finished cut surfaces were demonstrated and confirmed the findings of previous SEM studies. The method allowed direct control over all the known variables influencing cutting with particulate streams.     

Penetration of resin‐based materials into initial erosion lesion: A confocal microscopic study

The application of resin‐based materials is an alternative of treatment for eroded lesions. Nevertheless, there are no studies about the penetration of these materials into eroded lesion, which might affect its adhesion. Therefore, this study evaluated the penetration of four resin‐based materials, with and without enamel etching. By using an in vitro protocol, types of treatment were studied at five levels (AdheSE^®, Tetric N‐Bond^®, Single Bond 2^®, Helioseal Clear^®, Icon^®) and types of enamel etching in two levels (with and without). Materials were stained with 0.02 mg/mL ethanolic solution of tetramethylrhodamine isothiocyanate. Bovine enamel samples (4 × 4 mm) were immersed in 0.01 M HCl, pH 2.3, for 30 seconds to produce initial eroded lesions. Afterward, the materials were applied on half of sample enamel surface following the manufacturer's instructions. On the other half of sample, the materials were applied without etching the enamel. Materials penetration into the enamel was assessed by Confocal Laser Scanning Microscopy on reflection and fluorescence modes. The penetration depth (PD) was measured using ImageJ software. Data were analyzed by two‐way ANOVA and Tukey test (P < 0.05). Regardless of the material, etched enamel resulted in higher PD than non‐etched (P < 0.05). Icon^® showed the highest PD in enamel followed by Helioseal Clear^® (P < 0.05), with significant difference between them (P < 0.05) and no difference was found among AdheSE^®, Tetric N‐Bond^®, and Single Bond 2^® (P > 0.05). It can be concluded that prior enamel etching increased the materials penetration into eroded enamel and the Icon^®—infiltrant presented highest penetration. Microsc. Res. Tech. 79:72–80, 2016. © 2015 Wiley Periodicals, Inc.     

A comparison study of detecting gold nanorods in living cells with confocal reflectance microscopy and two‐photon fluorescence microscopy

Two‐photon fluorescence microscopy and confocal reflectance microscopy were compared to detect intracellular gold nanorods in rat basophilic leukaemia cells. The two‐photon photoluminescence images of gold nanorods were acquired by an 800 nm fs laser with the power of milliwatts. The advantages of the obtained two‐photon photoluminescence images are high spatial resolution and reduced background. However, a remarkable photothermal effect on cells was seen after 30 times continuous scanning of the femto‐second laser, potentially affecting the subcellular localization pattern of the nanorods. In the case of confocal reflectance microscopy the images of gold nanorods can be obtained with the power of light source as low as microwatts, thus avoiding the photothermal effect, but the resolution of such images is reduced. We have noted that confocal reflectance images of cellular gold nanorods achieved with 50 μW 800 nm fs have a relatively poor resolution, whereas the 50 μW 488 nm CW laser can acquire reasonably satisfactory 3D reflectance images with improved resolution because of its shorter wavelength. Therefore, confocal reflectance microscopy may also be a suitable means to image intracellular gold nanorods with the advantage of reduced photothermal effect.     

Multicolour analysis and local image correlation in confocal microscopy

Multiparameter fluorescence microscopy is often used to identify cell types and subcellular organelles according to their differential labelling. For thick objects, the quantitative comparison of different multiply labelled specimens requires the three-dimensional (3-D) sampling capacity of confocal laser scanning microscopy, which can be used to generate pseudocolour images. To analyse such 3-D data sets, we have created pixel fluorogram representations, which are estimates of the joint probability densities linking multiple fluorescence distributions. Such pixel fluorograms also provide a powerful means of analysing image acquisition noise, fluorescence cross-talk, fluorescence photobleaching and cell movements. To identify true fluorescence co-localization, we have developed a novel approach based on local image correlation maps. These maps discriminate the coincident fluorescence distributions from the superimposition of noncorrelated fluorescence profiles on a local basis, by correcting for contrast and local variations in background intensity in each fluorescence channel. We believe that the pixel fluorograms are best suited to the quality control of multifluorescence image acquisition. The local image correlation methods are more appropriate for identifying co-localized structures at the cellular or subcellular level. The thresholding of these correlation maps can further be used to recognize and classify biological structures according to multifluorescence attributes.     

Preliminary confocal scanning laser microscopy study of fluid inclusions in quartz

The initial results of the first dedicated confocal scanning laser microscopy (CSLM) study of fluid inclusions in quartz are presented. CSLM imaging of a large inclusion shows the quartz crystal to contain numerous small (< 1 μm), highly reflective inclusions arranged along planes in at least two directions that are not readily visible in transmitted light. The technique allows measurements to be made of the angular intersection and orientation of the planes in both two and three dimensions. Results suggest that larger inclusions (> 10 μm) occur where two planes of small inclusions intersect, and that the shape of the large inclusions is controlled by the angular relationship between intersecting planes.     

The effect of different obturation methods on sealer penetration alongside apically separated rotary nickel–titanium instruments: A confocal laser scanning microscopy study

The effects of different obturation techniques on calcium silicate‐based sealer penetration in the presence of apically separated rotary files were evaluated. Forty‐eight extracted mandibular incisors were used. ProTaper F2 rotary files were separated at the apical thirds. Samples were divided into four groups (n = 12) according to obturation technique used: (a) cold lateral compaction (CLC); (b) single cone; (c) bulk‐fill (BF) without a core material; and (d) thermoplastic injection (TI). Specimens were sectioned horizontally at 1 and 3 mm from the apex and studied using a confocal scanning laser microscope. The maximum tubule penetration depth and percentage of penetration were measured. Data were statistically analyzed using parametric and nonparametric tests with a significance level of 5%. Regarding penetration depth, a significant difference was found at 1 mm (p < .05), while no significant difference was found at 3 mm (p > .05). At the 3 mm level, all of the obturation techniques showed similar penetration depths. Regarding penetration percentage, the values of the CLC and TI groups were statistically less when compared with the BF group at 1 and 3 mm levels, respectively (p < .05). Under the limitations of this in vitro study, results suggest that the obturation technique may present a significant effect on sealer penetration.     

Wavelength considerations in confocal microscopy of botanical specimens

We have used a multiple-laser confocal microscope with lines at 325, 442, 488, 514 and 633 nm to investigate optical sectioning of botanical specimens over a wide range of wavelengths. The 442-nm line allowed efficient excitation of Chromomycin A3, with minimal background autofluorescence, to visualize GC-rich heterochromatin as an aid to chromosome identification. Sequential excitation with 442- and 488-nm light enabled ratio imaging of cytosolic pH using BCECF. The red HeNe laser penetrated deep into intact plant tissues, being less prone to scattering than shorter blue lines, and was also used to image fluorescent samples in reflection, prior to fluorescence measurements, to reduce photobleaching. Chromatic corrections are more important in confocal microscope optics than in conventional microscopy. Measured focus differences between blue, green and red wavelengths, for commonly used objectives, were up to half the optical section thickness for both our multi-laser system and a multi-line single-laser instrument. This limited high-resolution sectioning at visible wavelengths caused a loss in signal. For ultraviolet excitation the focus shift was much larger and had to be corrected by pre-focusing the illumination. With this system we have imaged DAPI-stained nuclei, callose in pollen tubes using Aniline Blue and the calcium probe Indo-1.     

Effects of specimen refractive index on confocal imaging

The aberrations introduced when focusing within a specimen with a refractive index equal to that of water using an oil-immersion objective are investigated theoretically. The peak intensity in the confocal point spread function drops by a factor of two for focusing less than 10 μm into the specimen. The effects on scaling of dimensions in the resulting images are discussed. The image exhibits an axial stretching by a factor of about 1.12.     

Simultaneous degradation estimation and restoration of confocal images and performance evaluation by colocalization analysis

A novel method for joint restoration and estimation of the degradation of confocal microscope images is presented. The observed images are degraded due to two sources: blurring due to the band-limited nature of the optical system [modelled by the point spread function (PSF)], and Poisson noise contaminates the observations due to the discrete nature of the photon detection process. The proposed method iterates noise reduction, blur estimation and deblurring, and applies these steps in two phases, i.e. a training phase and a restoration phase. In the first phase, these three steps are iterated until the blur estimation converges. Noise reduction and blur estimation are performed using steerable pyramids, and the deblurring is performed by the Richardson–Lucy algorithm. The second phase is the actual restoration. From then on, the blur estimation is used as a criterion to measure the image quality. The iterations are stopped when this measure converges, a result that is guaranteed. The integrated method is completely automatic, and no prior information on the image is required. The method has been given the name SPERRIL (Steerable Pyramid-based Estimation and Regularized Richardson–Lucy restoration). Compared with existing techniques by both objective measures and visual observation, in the SPERRIL-restored images noise is better suppressed.     

Axial super resolution topography of focal adhesion by confocal microscopy

The protein organization within focal adhesions has been studied by state‐of‐the‐art super resolution methods because of its thin structure, well below diffraction limit. However, to achieve high axial resolution, most of the current approaches rely on either sophisticated optics or diligent sample preparation, limiting their application. In this report we present a phasor‐based method that can be applied to fluorescent samples to determine the precise axial position of proteins using a conventional confocal microscope. We demonstrate that with about 4,000 photon counts collected along a z‐scan, axial localization precision close to 10 nm is achievable. We show that, with within 10 nm, the axial location of paxillin, FAK, and talin is similar at focal adhesion sites, while F‐actin shows a sharp increase in height towards the cell center. We further demonstrated the live imaging capability of this method. With the advantage of simple data acquisition and no special instrument requirement, this approach could have wide dissemination and application potentials. Microsc. Res. Tech., 76:1070–1078, 2013. © 2013 Wiley Periodicals, Inc.     

Cosmetic assessment of the human hair by confocal microscopy

The optical sectioning property of the confocal microscope offers a breakthrough from the classic observation of the hair in a scanning electron microscope (SEM). Confocal microscopy requires minimal sampling preparation, and the hair can be observed in its natural environment with less damage than by other microscopic methods such as SEM. While used in the reflection mode, the true morphology of the cuticle and the various exogenous deposits at the surface can be identified and quantified. This relatively noninvasive, nondestructive technique is routinely used by us to monitor the efficiency of cleansing shampoos, to assess the homogeneity of layering polymers, and to evaluate the changes they induce in the optical properties of the hair surface in terms of opacity, transparency, and brilliancy. A second important field of investigation uses the fluorescence channel which reveals the internal structure of the hair. Fluorescent probes (rhodamine and its derivatives) demonstrate the routes of penetration and outline the geometry of cortical cells and of the medulla according to their lipophilic or hydrophilic properties. A volume rendering of a hair cylinder provides a better understanding of the interrelationships between cuticle cells, cortical cells, and the medullar channel. This recent technology is becoming an invaluable tool for the cosmetic assessment of the hair.     

激光差动共焦曲率半径测量系统的研制

针对国内高精度曲率半径计量需求,研制一套激光差动共焦曲率半径测量系统.该系统采用差动共焦定焦技术,利用轴向光强响应曲线的过零点精确对应物镜聚焦焦点这一特性,借助过零点对被测件的猫眼和共焦位置进行精密瞄准定位,通过干涉测长技术获取两点间的距离,继而实现曲率半径的高精度测量.该测量系统的机电控制由主控软件完成,可实现机电扫描、数据采集及数据处理,自动化程度高.实验证明,该系统定焦灵敏度高,受环境波动影响小,测量精度可达3×10-6,满足了高精度曲率半径的计量需求.     

In-vivo analysis of angiogenesis and revascularization of transplanted pancreatic islets using confocal microscopy

A technique to measure angiogenesis and revascularization in pancreatic islets transplanted at the renal subcapsular site in the rat has been developed. In-vivo imaging of the microcirculation of transplanted pancreatic islets was conducted using a confocal scanning laser microscope (CSLM) to achieve optical sectioning through the graft in order to perform a computer reconstruction of the three-dimensional neovascular morphology. Individual islets were harvested by enzymatic digestion of excised pancreas from Fischer 344 rats. Isolated islets were cultured for 24 h, and approximately 300–350 islets were transplanted at the renal subcapsular site of the left kidney in an anaesthetized rat. Six to 14 days post-transplantation, the animal was anaesthetized and prepared for in-vivo imaging of the microvasculature on a Zeiss LSM-10. Optical contrast of the microvasculature was enhanced by the administration of fluorescein-labelled dextran into the circulating blood. The transplant site was identified and serial sections were obtained through the vascular bed at varying z-intervals. Complementary fluorescence video images were also obtained via a silicon intensifier tube camera mounted on the CSLM. At completion of the imaging procedure, the kidney was returned into the body cavity, the area was sutured and the animal was allowed to recuperate for subsequent examinations. Image processing algorithms, such as grey-level thresholding, median filtering, skeletonization and template matching, were applied to compute the vessel density and diameters and extrapolated to measure 3-D vessel lengths and the tortousity index of the neovasculature.     

Dual-mode reflectance and fluorescence near-video-rate confocal microscope for architectural, morphological and molecular imaging of tissue

We have developed a near‐video‐rate dual‐mode reflectance and fluorescence confocal microscope for the purpose of imaging ex vivo human specimens and in vivo animal models. The dual‐mode confocal microscope (DCM) has light sources at 488, 664 and 784 nm, a frame rate of 15 frames per second, a maximum field of view of 300 × 250 μm and a resolution limit of 0.31 μm laterally and 1.37 μm axially. The DCM can image tissue architecture and cellular morphology, as well as molecular properties of tissue, using reflective and fluorescent molecular‐specific optical contrast agents. Images acquired with the DCM demonstrate that the system has the sub‐cellular resolution needed to visualize the morphological and molecular changes associated with cancer progression and has the capability to image animal models of disease in vivo. In the hamster cheek pouch model of oral carcinogenesis, the DCM was used to image the epithelium and stroma of the cheek pouch; blood flow was visible and areas of dysplasia could be distinguished from normal epithelium using 6% acetic acid contrast. In human oral cavity tissue slices, DCM reflectance images showed an increase in the nuclear‐to‐cytoplasmic ratio and density of nuclei in neoplastic tissues as compared to normal tissue. After labelling tissue slices with fluorescent contrast agents targeting the epidermal growth factor receptor, an increase in epidermal growth factor receptor expression was detected in cancerous tissue as compared to normal tissue. The combination of reflectance and fluorescence imaging in a single system allowed imaging of two different parameters involved in neoplastic progression, providing information about both the morphological and molecular expression changes that occur with cancer progression. The dual‐mode imaging capabilities of the DCM allow investigation of both morphological changes as well as molecular changes that occur in disease processes. Analyzing both factors simultaneously may be advantageous when trying to detect and diagnose disease. The DCM's high resolution and near‐video‐rate image acquisition and the growing inventory of molecular‐specific contrast agents and disease‐specific molecular markers holds significant promise for in vivo studies of disease processes such as carcinogenesis.