共焦显微扫描探测技术的发展

传统的共焦显微探测多是采用单点机械扫描方式完成的,存在着扫描效率低的缺点,针对于此,一种多光束共焦显微系统成为当前的研究热点,通过不同方式对光束进行分割,使单点检测变为多路并行检测,提高了测量速度,减少了扫描过程中光源和振动噪声的影响,实现多光束的同步测量。介绍了几种典型的单光束和多光束共焦显微测量系统的原理、研究进展、发展方向及作者在该方面的研究。     

Laser applications and system considerations in ocular imaging

We review laser applications for primarily in‐vivo ocular imaging techniques, describing their constraints based on biological tissue properties, safety, and the performance of the imaging system. We discuss the need for cost‐effective sources with practical wavelength‐tuning capabilities for spectral studies. Techniques to probe the pathological changes of layers beneath the highly scattering retina and diagnose the onset of various eye diseases are described. The recent development of several optical‐coherence‐tomography‐based systems for functional ocular imaging is reviewed, as well as linear and nonlinear ocular‐imaging techniques performed with ultrafast lasers, emphasizing recent source developments and methods to enhance imaging contrast.     

Experimental studies of an unstable confocal resonator alexandrite laser

The effects of excited converging wave in an unstable confocal resonator onthe properties of an alexandrite laser are investigated experimentally.The results show thatexciting the converging wave inside the resonator is an effective method to reduce the diver-gence of the laser beam for low gain laser medium.     

Determination of Resting Membrane Potential of Individual Neuroblastoma Cells (IMR-32) Using a Potentiometric Dye (TMRM) and Confocal Microscopy

The potentiometric dye, Tetramethylrhodamine methyl ester (TMRM) has been extensively used with fluorometry or optical microscopy to evaluate the electric potential across plasma or mitochondrial membranes. We present here a TMRM confocal microscopy-based potential measurement technique. Corrections are introduced to minimize nonspecific dye binding and insensitivity to low background levels. We have used this technique to compare the resting membrane potential of proliferating and differentiated human neuroblastoma cells (IMR-32).     

Effective combinatorial strategy to increase affinity of carbohydrate binding by peptides

The Thomsen-Friedenreich antigen, a carcinoma-associated disaccharide involved in carcinoma cell homotypic aggregation and increased metastatic potential, has clinical value as a prognostic indicator and a marker of metastasized cells. Hence, it can reasonably be predicted that antigen-binding macromolecules are valuable clinical in vivo diagnostic/therapeutic targeting agents. Recently, we have selected first-generation antigen-binding peptides from a random peptide bacteriophage display library and have applied combinatorial affinity maturation to select functionally-maturated peptides, which target cultured carcinoma cells and inhibit carcinoma cell aggregation. In the current study we hypothesize that a targeted search of sequence space surrounding the antigen-binding consensus sequence will select unpredictable amino acid sequences in the non-consensus portions of the peptides, leading to increased affinity for the carbohydrate and greater solubility in physiological buffers. This comprehensive in vitro analysis demonstrates that preferential evolution of the amino-terminal sequence of the peptides occurred, which correlated, in structure/function studies, with the acquisition of maturated function. The maturated peptides are more soluble than the earlier peptides. Studies of peptide binding to the disaccharide indicate that two maturated peptides (P-30-1, F03) have higher affinity for the antigen and bind with higher intensity to the surface of cultured human carcinoma cells than the first-generation peptides. The results support our hypothesis that affinity maturation can improve carbohydrate binding by peptides and have theoretical importance as the first report of maturation of carbohydrate-binding affinity in a small, soluble peptide.     

Edge-preserving restoration in 2-D fluorescence microscopy

Procedures for software image restoration can be useful for studying biological events. This is especially true in the case of low light-intensity fluorescence images, obtained with both wide-field and confocal microscopy. This review describes most of the algorithms for image restoration, with particular emphasis on edge-preserving procedures, and shows the results of their application to synthetic and fluorescence 2-D images.     

Assessment of murine neuroblastoma (N1E-115) resting membrane potential by confocal microscopy

Digital imaging (confocal microscopy) and a slow potentiometric dye (tetramethylrhodamine methyl ester) were used to assess the resting membrane potential (V _m) of murine neuroblastoma cells (N1E-115). The averageV _m was found to be –64.0±2.0 mV. The difference between this and the previously reported higher values was attributed to the use of glass microelectrode techniques that probably caused mechanical injury to the cell membranes: Digital imaging of N1E-115V _m was found to be sensitive, reproducible, fast, and simple.     

Photon Counting Histogram Analysis for Two‐Dimensional Systems

Photon counting statistics in 3D photon counting histogram analysis for one‐photon excitation is a function of the number of molecules of particular brightness in the excitation‐detection volume of a confocal microscope. In mathematical form that volume is approximated by a three‐dimensional Gaussian function which is embedded in the PCH theoretical equations. PCH theory assumes that a molecule can be found anywhere inside the excitation‐detection volume with equal probability. However, one can easily imagine systems in which this assumption is violated because molecules are constrained by the geometry of the sample. For example, molecules on a surface or in a membrane would be constrained to two dimensions. To enable the analysis of such systems by PCH, the theoretical framework requires modification. Herein, we present an extension of the PCH analysis to systems where molecules exist in thin structures that are effectively two‐dimensional. The method, aptly called two‐dimensional photon counting histogram (2D PCH), recovers the number of fluorescent particles per unit area and their molecular brightness. Both theoretical background and experimental results are presented. The theory was tested using computer‐simulated and experimental 2D PCHs obtained from confocal experiments. We demonstrate that this modification of the theoretical framework provides a tool to extract data that reveal states of aggregation, surface photophysics, and reactivity.     

自成像共焦腔相干合成空间滤波器设计

理论分析了利用自成像共焦腔实现激光列阵相干合成的物理模型,对自成像共焦腔的一维相干合成和二维相干合成进行了模拟仿真计算,得到腔内场的强度分布图样.在此基础上,研究了空间滤波器在相干合成中实现模式选择的机理,并讨论滤波器的设计对相干合成及模式选择的影响,分别对一维相干合成和二维相干合成的情况,仿真计算了输出功率与滤波器位置和宽度的关系,为实验提供了理论依据.     

Fast mapping of inhomogeneities in the popular metallic perovskite Nb:SrTiO3 by confocal Raman microscopy

Confocal Raman microscopy was applied in order to investigate the homogeneity of donor doping in Nb:SrTiO₃ single crystals. Measurements of local Raman spectra revealed a systematic relation between the intensity of the Raman signal and the donor content of the crystals. We successfully elaborated a correspondence between the electronic structure and the intensity of the Raman lines using a crystal with macroscopic inhomogeneity as a demonstration sample. By mapping the distribution of the intensity of the Raman signal, we identified a characteristic inhomogeneous structure related to the presence of clusters with sizes of 5 µm to 20 µm, indicating inhomogeneous donor distribution caused by flaws introduced during crystal growth. Hence, we propose confocal Raman microscopy as a convenient technique for investigating the homogeneity and quality of doped perovskite surfaces, which are needed for various technological applications. (© 2014 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)