荧光共焦显微术与多光子显微术的差别

共焦显微术和多光子显微术因其可以对厚的生物样品实现光学断层成像，因而在生物医学等领域具有广泛的应用前景，本文详细综述了共焦显微术和多光子显微术在成像原理及特性等方面的差别，在此基础上，讨论了每种显微术各自的优点、局限性及应用前景。     

Intracellular localization of the antitumour drug adriamycin in living cultured cells: A confocal microscopy study

The intracellular distribution of the anthracyclinic antibiotic adriamycin in living cultured cells has been investigated by confocal microscopy. In human melanoma cells (M14), adriamycin was localized inside the nuclei. When adriamycin-treated M14 cells were allowed to recover in drug-free medium, a complete efflux of the drug from the nucleus was revealed. In recovered cells, a weakly fluorescent signal was observed in the perinuclear region. When M14 cells were recovered in a medium containing colcemid, a microtubule depolymerizing agent, the drug transport from the nucleus to the cell periphery appeared to be inhibited, suggesting that the microtubule network is strongly involved in drug transport mechanisms. In multidrug-resistant (MDR) cells the intracellular location of adriamycin was shown to be noticeably different from that of the parental wild-type cells. In particular, in resistant human breast carcinoma cells (MCF-7), adriamycin appeared to be exclusively located within the cytoplasm whereas the nuclei were shown to be completely negative. When adriamycin treatment was performed in association with MDR revertants, such as Lonidamine (inhibitor of the energy metabolism) or verapamil (inhibitor of the P-glycoprotein efflux pump), a marked enhancement of the cytoplasmic signal was observed in resistant cells. Under these conditions, adriamycin appeared concentrated in the perinuclear region, but the nuclei were still negative. Confocal microscopy proved to be a very useful method for the study of the intracellular transport of fluorescent substances, such as anthracyclinic antibiotics, and for the investigation of the multidrug resistance phenomenon in tumour cells.     

自聚焦共焦探测系统轴向分辨率的影响因素

从微小内尺度测量角度出发，研究了针孔尺寸、探测器位置、透镜的数值孔径、放大倍率和杂散光等对自聚焦共焦式探测系统轴向分辨率的影响。结果表明，为提高系统的轴向分辨率，需将有效针孔尺寸控制在≤2.5；探测器需配以精密微调整装置以实现横向精确定位；选用差动共焦光路及大数值孔径和大放大倍率的自聚焦透镜，同时，偏振光路的使用将有效的提高系统的信噪比。     

Bright-field scanning confocal electron microscopy using a double aberration-corrected transmission electron microscope

Scanning confocal electron microscopy (SCEM) offers a mechanism for three-dimensional imaging of materials, which makes use of the reduced depth of field in an aberration-corrected transmission electron microscope. The simplest configuration of SCEM is the bright-field mode. In this paper we present experimental data and simulations showing the form of bright-field SCEM images. We show that the depth dependence of the three-dimensional image can be explained in terms of two-dimensional images formed in the detector plane. For a crystalline sample, this so-called probe image is shown to be similar to a conventional diffraction pattern. Experimental results and simulations show how the diffracted probes in this image are elongated in thicker crystals and the use of this elongation to estimate sample thickness is explored.     

Electron beam lithography-assisted fabrication of Au nano-dot array as a substrate of a correlated AFM and confocal Raman spectroscopy

This paper documents a study of an Au nano-dot array that was fabricated by electron beam lithography on a glass wafer. The patterns that had features of 100nm dots in diameter with a 2-mum pitch comprised a total area of 200x200mum(2). The dot-shaped Cr underlayer was open to the air after developing Poly(methyl methacrylate) (PMMA). When dipped into the Cr etchant, the exposed Cr layer was eliminated from the glass wafer in a short period of time. In order to ultimately fabricate the Ti/Au dot arrays, Ti and Au were deposited onto the arrays with a thickness of 2 and 40nm, respectively. The lift-off procedure was carried out in the Cr etchant using sonication in order to completely remove the residual Cr/PMMA layer. The fabricated Au nano-dot array was then immersed in an Ag enhancing solution and then into an ethanol solution containing (N-(6-(Biotinamido)hexyl)-3'-(2'-pyridyldithio)-propionamide (Biotin-HPDP). The substrate was analyzed using a correlated atomic force microscopy (AFM) and confocal Raman spectroscopy. Through this procedure, position-dependent surface-enhanced Raman spectroscopy (SERS) signals could be obtained.     

Development of an image-based model for capillary vasculature of retina

The paper presents a method of development of a detailed network model to represent retinal capillary vasculature. The capillary model is a circular mesh consisting of concentric rings with an increasing diameter. Each of the rings has uniformly distributed bifurcation nodes to represent capillary vessels. The model is customized using the data that has been measured from confocal microscopic images of a mouse retina. The capillary model developed can be connected to networks of larger vessels of the vasculature such as arterial and venous networks to form a complete model of the retinal network. A method to automate such interface connections between capillary and other vascular networks using connecting vessels (i.e., pre-capillary and post-capillary) is also presented in the paper. Such a detailed image-based capillary model together with the arterial and venular networks can be used for various circulation simulations to obtain accurate information on hemodynamic quantities such as the spatial distribution of pressure and flow in the vasculature for both physiological and pathological conditions. The method presented for the development of the capillary model can also be adopted for vasculatures of other organs.     

The influence of specimen refractive index,detector signal integration,and non-uniform scan speed on the imaging properties in confocal microscopy

Refraction of light in a specimen volume may cause aberrations that influence the imaging properties in confocal microscopy. In this paper the influence on three-dimensional resolution and geometry is experimentally investigated for a uniform specimen volume. It is found that the depth resolution is more severely affected than the lateral resolution. This is unfortunate, because even under ideal conditions the depth resolution is lower than the lateral resolution. Lateral image geometry is little affected by the specimen refractive index, whereas the depth scale can be considerably elongated or compressed. The influence of a finite detector integration time is also considered. This can give a noticeable, but not particularly severe effect on the image resolution in the line-scan direction. Because the integration time can be accurately controlled, a shorter integration time can be used when maximum resolution is essential, albeit at the price of a higher noise level. In scanning fluorescence microscopy a non-uniform scan speed may give large variations in bleaching over the specimen surface. Experiments illustrate how serious such non-uniform bleaching effects can be when a specimen area is repeatedly scanned, for example when recording optical serial sections.     

Estimation of individual feature surface area with the vertical spatial grid

The area of an individual bounded surface (e.g. the boundary of a properly sampled cell) can be estimated from an isotropic uniform random stack of parallel sections, or of non-invasive planar scans, using the well-known spatial grid. A standing problem was to estimate the area of an individual bounded surface with an arbitrary degree of accuracy from a vertical (i.e. not isotropic) stack of sections or scans. A new tool to do this, called the ‘vertical spatial grid’, is presented.     

Radon track imaging in CR-39 plastic detectors using confocal scanning laser microscopy

Confocal scanning laser microscopy (CSLM) has been used to provide the first images of radon track populations in two external CR-39 plastic detectors. Measurements of variables including track area distribution and estimates of the angle of track inclination (dip) derived from surface CSLM sections are presented. CSLM depth slices, combined with three-dimensional (3D) visualization techniques, provide a new, non-destructive way of examining the 2D and 3D geometry of the etched tracks within solid-state nuclear track detectors that may prove useful in complementing existing optical microscopy methods.