Single Molecule Genotyping by TIRF Microscopy

As part of a programme to develop a metrological framework for single molecule measurements in biology, we have investigated the applications of single molecule imaging to genomics. Specifically, we have developed a technique for measuring the frequencies of single nucleotide polymorphisms (SNPs) in complex or pooled samples of DNA. We believe that this technique has applications to statistical genotyping—the identification of correlations between SNP frequencies and particular phenotypes—and other areas where it is desirable to track the frequencies of SNPs in complex DNA populations.     

Early Membrane Responses to Magnetic Particles are Predictors of Particle Uptake in Neural Stem Cells

Magnetic particles (MPs) offer several advantages for neural cell therapy, but limited particle uptake by neural cells is a barrier to translation. It is recently proved that tailoring particle physicochemical properties (by enhancing their iron content) dramatically improves uptake in neural stem cells (NSCs)—a major transplant population. High‐throughput screening of particles with varying physicochemical properties can therefore aid in identifying particles with optimal uptake features, but research is hampered by the lack of simple methodologies for studying neural cell membrane responses to nanoparticle platforms. A high‐resolution–high throughput method has been used to study early membrane responses of primary rodent NSCs to particles of variant magnetite loading, to attempt to correlate these responses with known particle internalization profiles. Membrane imaging is enhanced through sequential staining with osmium (O) and thiocarbohydrazide (T), a method termed OTOTO, combined with field‐emission scanning electron microscopy (FESEM). A five‐point classification system was used to systematically evaluate early MP‐induced membrane responses to particles possessing distinct physicochemical properties. Significantly different profiles of membrane activation were noted that correlate with particle uptake profiles. It is suggested that our method can serve as a valuable predictor of particle internalization in neural cells for diverse particle platforms.     

Single-Fluorophore Dynamic Imaging in Living Cells

Recently, observation and tracking of single fluorophores, which we term single fluorophore dynamic imaging (SFDI) in this review, in living cells have been achieved. In particular, the recent success of SFDI of individual proteins tagged with green fluorescent protein (GFP) in live cells has opened new important possibilities for studying events occurring in living cells at the level of single molecules. Specifically, SFDI of GFP allows the tracking of movement and oligomerization levels of individual oligomers (monomers) in living cells and, thus, provides powerful means to investigate the movement, assembly, localization, and activation that signaling molecules undergo following an external stimulus. In this short account, we first review technologically important points for SFDI of GFP molecules in living cells, then give examples of its application, and, finally, propose a synergistic use of SFDI and single-particle tracking, a technique used for investigating single or small groups of molecules in live cells over the past 15 years.     

Direct Observation of Lipid Domains in Free-Standing Bilayers Using Two-Photon Excitation Fluorescence Microscopy

The direct observation of temperature-dependent lipid phase equilibria, using two-photon excitation fluorescence microscopy on giant unilamellar vesicles (GUVs) composed of different lipid mixtures, provides novel information about the physical characteristics of lipid domain coexistence. Physical characteristics such as the shape, size, and time evolution of different lipid domains are not directly accessible from the traditional experimental approaches that employ either small and large unilamellar vesicles or multilamellar vesicles. In this review article, we address the most relevant findings reported from our laboratory regarding the direct observation of lipid domain coexistence at the level of single vesicles in artificial and natural lipid mixtures. In addition, key points concerning our experimental approach will be discussed. The unique advantages of the fluorescent probe 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN) under two-photon excitation fluorescence microscopy is particularly addressed, especially, the possibility of obtaining information on the phase state of different lipid domains directly from the fluorescent images.     

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).     

Electroporative Adjustment of pH in Living Yeast Cells: Ratiometric Fluorescence pH Imaging

A number of vital cell functions including modulation of signaling pathways and regulation of the cellular transport critically depends on the cytoplasmic pH. Many pathological cellular changes are related to the abnormal cytosolic pH as well. Reliable and well-calibrated methods for quantification of the cytosolic pH are therefore of high importance. The pH calibration is particularly difficult in walled cells since standard methods fail. In this report we evaluated the new electroporative calibration method of the cytosolic pH in yeasts by the fluorescence microscopy. The calibration was done on living cells using pyranine as a ratiometric pH-sensitive probe. The probe was electroporatively delivered to the cytosol. We have shown that unlike the measurements in suspension the fluorescence microscopy reveals cell subpopulations with different sensitivity to the pH calibration. While the majority of the cells were well calibrated, there was found subpopulation of uncalibrated cell as well as singular cells exhibiting anomalous pH calibration due to the staining of acidic organelles. Resolution of cell subpopulations helps to achieve better pH calibration compared to the calibration in cuvette on a cell suspension.     

全内反射荧光显微术

全内反射荧光显微技术是当今世界上最具前途的新型生物光学显微技术之一，可以用来实现对单个荧光分子的直接探测。它利用全内反射产生的隐失波照明样品，使照明区域限定在样品表面的一薄层范围内，因此具有其它光学成像技术无法比拟的高的信噪比和对比度。近上来，已被生物物理学家们广泛应用于单分子的荧光成像中。本文系统的介绍了全内反射荧光显微技术的原理。国内外的发展和现状及其在生物学上的应用，并对其未来做了展望。     

全内反射荧光显微术

全内反射荧光显微技术是当今世界上最具前途的新型生物光学显微技术之一 ,可以用来实现对单个荧光分子的直接探测。它利用全内反射产生的隐失波照明样品 ,使照明区域限定在样品表面的一薄层范围内 ,因此具有其它光学成像技术无法比拟的高的信噪比和对比度。近年来 ,已被生物物理学家们广泛应用于单分子的荧光成像中。本文系统的介绍了全内反射荧光显微技术的原理、国内外的发展和现状及其在生物学上的应用 ,并对其未来做了展望。     

DNA Vector Polyethyleneimine Affects Cell pH and Membrane Potential: A Time-Resolved Fluorescence Microscopy Study

Polyethyleneimine (PEI) is one of the very efficient nonviral vectors being developed and tested for artificial gene transfer into target cells. One of its serious limitations is the significant cytotoxicity of the large amounts of free PEI in the mixtures of DNA and PEI used for transfection. To further investigate the cellular effects of free PEI, we have analyzed the PEI-induced alterations of various cell parameters such as membrane heterogeneity and fluidity, cytoplasmic pH, and plasma membrane potential in a variety of cells such as Swiss 3T3 fibroblast, Chinese hamster ovary, insect cells SF9, plant cell line BY2, and Saccharomyces cerevisae. Fluorescence probes such as Nile red, SNARF-1, and cyanine dye DiSC₂(3), coupled with the technique of picosecond time-resolved fluorescence microscopy, were used in estimating the above-mentioned cell parameters. It was found that the cell membranes were largely unperturbed by PEI. However, the cytoplasmic pH showed an increase of 0.1–0.4 units when the cells were treated with PEI. The plasma membrane potential was found to be depolarized in S. cerevisae and Swiss 3T3 cells. These results suggest that the cytotoxic effects of PEI may partly originate from inhibition of regulation of cytoplasmic pH and plasma membrane potential. Further, it is proposed that the resultant cell alterations favors the transfection process.     

10 nm Spatial Resolution Fluorescence Imaging of Single Molecules by Near-Field Scanning Optical Microscopy Using a Tiny Aperture Probe

We demonstrate high-resolution fluorescence imaging of single molecules using near-field scanning optical microscopy (NSOM) with a tiny aperture probe for two different wavelengths in visible range in the illumination mode of operation. The spatial resolutions obtained at both excitation wavelengths were almost the same and the highest resolution realized was about 10 nm. To discuss the achievable resolution in aperture NSOM, we also employed a computer simulation by the finite-difference time-domain method for various aperture sizes and wavelengths. The resolution of 10 nm is predicted to be contributed by the single peak of localized near-field light around the rim of the aperture.     

In Situ Self-Assembly of Quantum Dots at the Plasma Membrane Mediates Energy Transfer-Based Activation of Channelrhodopsin

The use of nanoparticle (NP) bioconjugates to control the activity of membrane ion channels has recently emerged as a new paradigm for the activation of electrically excitable cells. An NP-based strategy is reported for the specific activation of channelrhodopsin C1V1 (ChR-C1V1) expressed in the plasma membrane of HEK 293T/17 cells. Hydrophilic CdSe/ZnS core–shell semiconductor quantum dots (QDs) are self-assembled to the exofacial face of recombinantly expressed ChR-C1V1 by metal affinity-driven interaction of the QD ZnS shell with an N-terminal hexahistidine tag displayed on ChR-C1V1. This configuration enables the Förster resonance energy transfer (FRET)-based excitation and activation of the 11-cis-retinal moiety of ChR-C1V1 using the QD as a light harvesting transducer/energy donor. It is shown that the specific laser-induced opening of the ChR-C1V1 channel wherein the photoexcited QD (405 nm excitation, 530 nm emission) iteratively activates ChR-C1V1 channels as confirmed using the voltage-sensitive dye (VSD) bis-(1,3-diethylthiobarbituric acid)trimethine oxonol (DiSBAC ₂ (3)). In the absence of the QD transducer, excitation of ChR-C1V1-expressing cells at 405 nm results in no activation of ChR-C1V1. The results demonstrate the ability to controllably interface QDs with living cells for the activation of ChR membrane proteins and detail a new NP-bioconjugate hybrid system for the specific activation of ion channels.     

不同荧光波长的双光子共焦成像分析

研究了双光子共焦显微镜中不同荧光波长对成像特性的影响,导出了不同荧光波长的三维脉冲响应函数和三维光学传递函数并进行数值计算.研究结果表明：不同荧光波长对双光子共焦显微镜的三维光学传递函数、三维脉冲响应函数和空间截止频率产生明显的影响,随着荧光波长的增大,分辨率明显下降,但不会出现单光子共焦显微镜中的失锥现象,选取适当的荧光波长进行成像,有利于进一步改善图像分辨率和成像质量.     

单分子荧光检测在生命科学中的应用

文章对单分子荧光检测在分子马达、离子通道、信号分子、蛋白折叠、蛋白构象变化动力学、酶活性反应、细胞过程实时观察等生命科学领域中的应用进行了介绍.这些研究结果表明,单分子荧光检测在研究生物大分子的活动规律与机制方面不但有着无法替代的优越性,而且有着广阔的发展空间.     

Three-Dimensional Fluorescence Microscopy of Endothelial Cells Labeled with Coumarins

Fluorescent coumarins were synthesized with the objective of introducing a glucosamine part in the chemical structure, with either hydroxyl or acetyl functions. The photophysical behavior was studied in organic solvents with different polarity and viscosity. The location of the fluorescent coumarins in endothelial cells was studied using fluorescence microscopy imaging, especially with a 3-D CELLscan instrument.     

Scanning Near-Field Optical Microscopy and Microspectroscopy of Green Fluorescent Protein in Intact Escherichia coli Bacteria

Scanning near-field optical microscopy (SNOM) yields high-resolution topographic and optical information and constitutes an important new technique for visualizing biological systems. By coupling a spectrograph to a near-field microscope, we have been able to perform microspectroscopic measurements with a spatial resolution greatly exceeding that of the conventional optical microscope. Here we present SNOM images of Escherichia coli bacteria expressing a mutant green fluorescent protein (GFP), an important reporter molecule in cell, developmental, and molecular biology. Near-field emission spectra confirm that the fluorescence detected by SNOM arises from bacterially expressed GFP molecules.     

Determining the elemental composition of Calotropis procera using X‐ray Analytical Microscopy

Caloptropis procera (Oshar) is a desert plant that did not receive much attention from the science community. The objective of this study was to investigate the elemental composition of the different parts of the plant using an X‐ray analytical microscope, to identify the elements naturally present in the plant and in the future detect the presence of any contaminants absorbed by the plants from the surrounding environment. Stalks, leaves and flowers from three Oshar plants were qualitatively and quantitatively analyzed. Leaves were scanned to establish the elemental spatial distribution within individual leaves. Subsequently, parts of the plants were dried, crushed and pulverized, then analyzed to determine elemental concentrations. The major elements present throughout the plant were Cl, K and Ca with varied concentrations. Other elements (Mg, Si, P, Fe, Sr, Mn and Br) are present in the leaf with various low concentrations of <5%. Major, minor and trace elements present in the various plant parts were determined. The outcome of this study will be used as a pilot for further investigations pertaining to the utilization of the Oshar plant for environmental cleaning purposes. Copyright © 2012 John Wiley & Sons, Ltd.     

纳米光学和生物单分子探测

纳米光学技术展示了纳米级探测本领,同时生物单分子探测所需要分辨尺度也是纳米数量级的,因此在生物单分子探测过程中,纳米光学发挥了巨大的作用.文章介绍了与生物单分子探测技术相关的纳米光学技术,包括量子近场光学探针技术、近场光学成像技术（包括扫描近场光学显微术及全内反射荧光显微术）和激光光钳测控技术及它们在生物单分子探测上的进展,从而在染色、成像、测控三个方面展示了纳米光学技术在生物方面的应用,并对其未来的发展方向进行了展望.     

反射式数字全息显微术对细胞的研究

根据全息理论和光的衍射理论,理论分析了数字全息显微术原理,并依据四步相移和最小二乘相位展开技术,研究了重构细胞相位的方法.设计了用球面光波作为参考光的反射式数字全息显微光路,通过反射式数字全息显微术的方法分析研究了新鲜洋葱表皮细胞的形貌结构.实验以新鲜洋葱表皮细胞为样本,完成了实验检测和相位重构,得到了细胞的相位和三维信息.分析表明,系统理论分辨率应达到0.8 μm.