Experimental Study of Metallic Elliptical Nano-Pinhole Structure-Based Plasmonic Lenses

An elliptical nano-pinhole structure-based plasmonic lens was designed and investigated experimentally by means of focused ion beam nanofabrication, atomic force microscope imaging, and scanning near-field optical microscope (NSOM). Two scan modes, tip scan and sample scan, were employed, respectively, in our NSOM measurements. Both the scan modes have their characteristics while probing the plasmonic lenses. Our experimental results demonstrated that the lens can realize subwavelength focusing with elongated depth of focus. This type of lens can be used in micro-systems such as micro-opto-electrical–mechanical systems for biosensing, subwavelength imaging, and data storage.     

Experimental Investigation of Focusing of Gold Planar Plasmonic Lenses

To experimentally demonstrate the subwavelength focusing of depth-tuned or non-depth-tuned plasmonic lenses, we first designed this type of lens using diffraction-coupling-angle based method, then fabricated the structure in gold thin film with focused ion beam, and finally characterized its focusing behavior using near-field scanning optical microscope. It is found that this type of lens has a resolution limit on the focal plane due to the field represented by angular spectrum having a cut-off frequency, while at the near field the lens has sub-diffraction limit focusing capability due to the existence of high-angular-frequency components in the field.     

Application of near-field scanning optical microscopy in photosynthesis research

Many different methods have been developed in recent years to gain insight into the structure of proteins, membranes, organelles and cells. Here we demonstrate the application of near-field scanning optical microscopy (NSOM) for analysis of the structures of typical photosynthetic membrane objects such as chloroplasts and thylakoids from spinach and chromatophores from purple bacteria. To our knowledge, this is the first report of application of NSOM to imaging chromatophores from photosynthetic bacteria and intact thylakoids from higher plants. NSOM has the ability to measure optical signals originating from the sample with a spatial resolution better than conventional optical microscopy. The main advantage of near-field optical microscopy, besides the improved lateral optical resolution, is the simultaneously acquired topography. We have applied NSOM to thylakoids obtained by osmotic shock of chloroplasts. Swollen thylakoids had average diameters of 0.8–1 micron and heights of 0.05–0.07 micron. We also describe the use of fluorescent dyes for the analysis of structures resulting from fusion of photosynthetic bacterial chromatophores with lipid impregnated collodion membranes. The structures formed after fusion of chromatophores to the collodion film have diameters ranging from 0.2 to 10 microns and heights from 0.01 to 1 micron. The dual functionality (optical and topographical), high spatial resolution, and the possibility to work with wet samples and under water, make NSOM a useful method for examining the structures, sizes, and heterogeneity of chromatophore and thylakoid preparations.     

近场扫描光学成像结合量子点标记的纳米技术在细胞生物学中的应用

近场扫描光学显微镜（NSOM）对传统的光学分辨极限产生了革命性的突破,可在超高光学分辨率下无侵人性和无破坏性地对生物样品进行观测。量子点（QDs）具有极好的光学性能,如荧光寿命长、激发谱宽、生物相容性强、光稳定性好等优点,适合先进的生物成像。NSOM结合QDs标记的纳米技术被应用在细胞生物学中。通过纳米量级NSOM免疫荧光成像（50nm）对特定蛋白分子在细胞表面的动态分布进行可视化研究和数量化分析,阐明了蛋白分子在不同细胞过程中的作用机制。因此,NSOM／QD基成像系统提供了单个蛋白分子最高分辨率的荧光图像,为可视化研究蛋白分子机制的提供了一种强有力的工具。     

Single beam optical trapping integrated in a confocal microscope for biological applications.

Confocal microscopy is very useful in biology because of its three dimensional imaging capacities and has proven to be an excellent tool to study the 3D organization of, for instance, cell structures. This property of confocal microscopy makes it also very suitable for observation during guidance of the three dimensional manipulation of single cells or cell elements. Therefore we decided to integrate a confocal microscope and a single beam optical manipulator into a single instrument. The advantage of optical manipulation over mechanical techniques is that it is non-invasive and therefore may be applied on living (micro-) organisms and cells. The creation of an effective single beam optical trap requires the use of a high numerical aperture (N.A.) objective to focus the laser beam. In this paper we briefly discuss the vertical or axial force exerted on a sphere in a single beam trap. The axial force on a sphere placed on the optical axis, caused by reflection and refraction, is calculated applying a electromagnetic vector diffraction theory to determine the field distribution in the focal region. One of the results is that the particle also experiences a vertical trapping force towards the focusing lens when it is in the strongly convergent part of the field in addition to the known negative signed trapping force in the divergent part of the field. Further we describe an instrumental approach to realize optical trapping in which the optical trap position is controlled by moving the focusing objective only.(ABSTRACT TRUNCATED AT 250 WORDS)     

The optical near-field and cell biology

A new form of scanning light microscopy is described in which the lens is replaced by a point of light that is smaller than the wavelength. Resolution is obtained that is defined not by the wavelength but by the size of the spot of light. This is the case so long as the point of light is within the dimension of a wavelength from the surface that is to imaged or within the optical near-field. This new form of light microscopy is called near-field scanning optical microscopy (NSOM). Resolutions are being obtained with NSOM that are similar to scanning electron microscopy but without the destructive effects of a vacuum or of an electron beam. In addition such a microscope is readily interfaced with fluorescent and non-fluorescent contrast enhancing stains that are commonly used in cell biology. The possibility of a near-field/far-field microscope is discussed with overlapping resolutions from a few hundred of a conventional microscope to the tens of thousand that can be obtained with NSOM.     

Experimental Study of Indirect Phase Tuning-Based Plasmonic Structures for Finely Focusing

Three types of indirect phase tuning-based plasmonic structures with subwavelength circular grooves/slits and/or central apertures corrugated on Au film supported by glass substrate: depth modulation, width modulation, and hybrid depth-width modulation, were put forth in this paper. They were investigated experimentally by means of nanofabrication and near-filed scanning optical microscope characterization. The plasmonic structures were fabricated using the technique of focused ion beam direct milling. Our experimental results demonstrated that all of the phase tuning-based structures have focusing functions. Both the width and depth modulation-based structures can realize beam focusing and produce an elongated depth of focus. Moreover, after comparison among these three structures, we found that the width modulation-based structure has the best focusing performance.     

Beam Manipulating via an Array of Nanoslits Modified by Perpendicular Cuts and Bumps

We report the observation of focusing and deflection phenomena by employing a novel technique to perform phase front profile design in nanoslit-based planar plasmonic lenses and beam deflectors. Introducing perpendicular cuts and bumps to the perforated nanoslits on a thin metallic film is utilized to change the effective depth of the nanoslits which provide the possibility of manipulating the phase front profile based on the propagation property of the surface plasmon polaritons in the metal–insulator–metal waveguides. Using the dispersive finite-difference time-domain numerical method, simulations are conducted to explore the beam focusing and deflection phenomena, and the performance parameters of the lens and beam deflector include the focal length, full-width half-maximum, depth of focus, the efficiency of focusing, and the deflection angle. The whole structure is formed on a planar thin film which is convenient for miniaturization and high density integration besides that it can be fabricated by well-known techniques such as focused ion beam milling.     

Near-Field Scanning Optical Microscopy, a Siren Call to Biology

Near-field illumination of a sample with visible light can resolve features well beyond the resolution of conventional, far-field microscopes. Near-field scanning optical microscopy (NSOM) then has the potential of extending the resolution of techniques such as fluorescent labeling, yielding images of cell structures and molecules on the nanoscale. However, major problems remain to be solved before NSOM can be easily used for wet biological samples. The most significant of these is control of the distance between near-field aperture and the sample surface. Hence, while NSOM promises much, its application to biology is about where electron microscopy was 40 or 50 years ago.     

A nanometer scale optical view on the compartmentalization of cell membranes

For many years, it was believed that the laws of diffraction set a fundamental limit to the spatial resolution of conventional light microscopy. Major developments, especially in the past few years, have demonstrated that the diffraction barrier can be overcome both in the near- and far-field regime. Together with dynamic measurements, a wealth of new information is now emerging regarding the compartmentalization of cell membranes. In this review we focus on optical methods designed to explore the nanoscale architecture of the cell membrane, with a focal point on near-field optical microscopy (NSOM) as the first developed technique to provide truly optical super-resolution beyond the diffraction limit of light. Several examples illustrate the unique capabilities offered by NSOM and highlight its usefulness on cell membrane studies, complementing the palette of biophysical techniques available nowadays.     

Formation of a Plasmonic Surface Optical Vortex by Evanescent Bessel Light

The total internal reflection of an optical mode with a phase singularity, such as a Bessel beam, can generate evanescent light that displays a rotational property. Notably, using a metallic layer surface, field components extending into the vacuum region have vortex properties besides surface plasmonic features. This vortex retains the phase singularity of the original light, and also maps its associated orbital angular momentum of incident Bessel light of the order ?? >?0. Additionally to a two-dimensional patterning on the metallic surface, the strongly restricted intensity distribution decays with distance vertical to the metallic surface. The detailed characteristics of this vortex structure depend on the input light parameters and the dielectric mismatch of the media. As well as this, they can be controlled by varying the incident angle and the order of Bessel light.     

Near-field fluorescence microscopy

The ability to study the structure and function of cell membranes and membrane components is fundamental to understanding cellular processes. This requires the use of methods capable of resolving structures with nanometer-scale resolution in intact or living cells. Although fluorescence microscopy has proven to be an extremely versatile tool in cell biology, its diffraction-limited resolution prevents the investigation of membrane compartmentalization at the nanometer scale. Near-field scanning optical microscopy (NSOM) is a relatively unexplored technique that combines both enhanced spatial resolution of probing microscopes and simultaneous measurement of topographic and optical signals. Because of the very small nearfield excitation volume, background fluorescence from the cytoplasm is effectively reduced, enabling the visualization of nano-scale domains on the cell membrane with single molecule detection sensitivity at physiologically relevant packing densities. In this article we discuss technological aspects concerning the implementation of NSOM for cell membrane studies and illustrate its unique advantages in terms of spatial resolution, background suppression, sensitivity, and surface specificity for the study of protein clustering at the cell membrane. Furthermore, we demonstrate reliable operation under physiological conditions, without compromising resolution or sensitivity, opening the road toward truly live cell imaging with unprecedented detail and accuracy.     

近场扫描光学显微镜及其在生物学领域的应用

评述了近场扫描光学显微镜（near-field scanning optical microscopy,NSOM）的仪器构造、工作原理及其在生物学领域的应用成果。对NSOM目前存在的主要问题进行了讨论,并展望了NSOM在该领域的发展潜力。     

Scanning near-field fluorescence resonance energy transfer microscopy

A new microscopic technique is demonstrated that combines attributes from both near-field scanning optical microscopy (NSOM) and fluorescence resonance energy transfer (FRET). The method relies on attaching the acceptor dye of a FRET pair to the end of a near-field fiber optic probe. Light exiting the NSOM probe, which is nonresonant with the acceptor dye, excites the donor dye introduced into a sample. As the tip approaches the sample containing the donor dye, energy transfer from the excited donor to the tip-bound acceptor produces a red-shifted fluorescence. By monitoring this red-shifted acceptor emission, a dramatic reduction in the sample volume probed by the uncoated NSOM tip is observed. This technique is demonstrated by imaging the fluorescence from a multilayer film created using the Langmuir-Blodgett (LB) technique. The film consists of L-alpha-dipalmitoylphosphatidylcholine (DPPC) monolayers containing the donor dye, fluorescein, separated by a spacer group of three arachidic acid layers. A DPPC monolayer containing the acceptor dye, rhodamine, was also transferred onto an NSOM tip using the LB technique. Using this modified probe, fluorescence images of the multilayer film reveal distinct differences between images collected monitoring either the donor or acceptor emission. The latter results from energy transfer from the sample to the NSOM probe. This method is shown to provide enhanced depth sensitivity in fluorescence measurements, which may be particularly informative in studies on thick specimens such as cells. The technique also provides a mechanism for obtaining high spatial resolution without the need for a metal coating around the NSOM probe and should work equally well with nonwaveguide probes such as atomic force microscopy tips. This may lead to dramatically improved spatial resolution in fluorescence imaging.     

用于全眼段成像和视轴参数测量的四焦点开关光学相干断层扫描

本文演示了四焦点开关傅里叶域光学相干断层扫描系统用于全眼段成像和体内视轴参数(VAP)测量的可行性.使用包括不同厚度平行玻璃板的两个同步转盘来将探测光束的焦点位置从角膜以及晶状体的前部和后部切换到视网膜.该过程同时增加了参考光束的深度范围.这种多级聚焦的方法可以使探测光束完全聚焦在人眼的每个部分.初步实验表明,该方法可...     

Fluorescence response and sensitivity determination for ATC 3000 flow cytometer

The fluorescence emitted by labeled particles after interaction with exciting light is conditioned by laser beam geometry and by the mode of fluorescence collection and filtration. A laser elliptic focusing mode is described, and the fluorescence characteristics of the sample cell flow are calculated. Fluorescence collection and detection through optical filters were analyzed, and efficiency was calculated for the ATC 3000 flow cytometer (Odam-Bruker, Wissembourg, France). A mathematical model is proposed for calculation of the fluorescence signal and its fluctuations. The background noise for the ATC 3000 was quantified experimentally using fluorescent microspheres of a known number of bound equivalent fluorescein isothiocyanate (FITC) molecules. These experimental measurements were found to fit the theoretical predictions, thus validating the proposed model.     

Lateral diffusion of membrane proteins in the presence of static and dynamic corrals: suggestions for appropriate observables

We consider the possibility of inferring the nature of cytoskeletal interaction with transmembrane proteins via optical experiments such as single-particle tracking (SPT) and near-field scanning optical microscopy (NSOM). In particular, we demonstrate that it may be possible to differentiate between static and dynamic barriers to diffusion by examining the time-dependent variance and higher moments of protein population inside cytoskeletal "corrals." Simulations modeling Band 3 diffusion on the surface of erythrocytes provide a concrete demonstration that these statistical tools might prove useful in the study of biological systems.     

Submicron structure in L-alpha-dipalmitoylphosphatidylcholine monolayers and bilayers probed with confocal, atomic force, and near-field microscopy.

Langmuir-Blodgett (LB) monolayers and bilayers of L-alpha-dipalmitoylphosphatidylcholine (DPPC), fluorescently doped with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diIC18), are studied by confocal microscopy, atomic force microscopy (AFM), and near-field scanning optical microscopy (NSOM). Beyond the resolution limit of confocal microscopy, both AFM and NSOM measurements of mica-supported lipid monolayers reveal small domains on the submicron scale. In the NSOM studies, simultaneous high-resolution fluorescence and topography measurements of these structures confirm that they arise from coexisting liquid condensed (LC) and liquid expanded (LE) lipid phases, and not defects in the monolayer. AFM studies of bilayers formed by a combination of LB dipping and Langmuir-Schaefer monolayer transfer exhibit complex surface topographies that reflect a convolution of the phase structure present in each of the individual monolayers. NSOM fluorescence measurements, however, are able to resolve the underlying lipid domains from each side of the bilayer and show that they are qualitatively similar to those observed in the monolayers. The observation of the small lipid domains in these bilayers is beyond the spatial resolving power of confocal microscopy and is complicated in the topography measurements taken with AFM, illustrating the utility of NSOM for these types of studies. The data suggest that the small LC and LE lipid domains are formed after lipid transfer to the substrate through a dewetting mechanism. The possible extension of these measurements to probing for lipid phase domains in natural biomembranes is discussed.     

Beam-Scanning Planar Lens Based on Metal–Dielectric–Metal Waveguide Arrays

Under specific illumination conditions, periodic arrays of metal–dielectric–metal (MDM) waveguides act as uniform optical phased-array antenna where the phase of the radiating optical wave can be controlled by modifying the refractive index distribution of the dielectric material. Based on this property, we propose a planar gradient index MDM-based lens which can transform spherical waves of the transverse-magnetic surface plasmon polariton waves to plane waves with specific beam deflections by adjusting the refractive index configurations. Using numerical simulations based on two-dimensional finite-difference time-domain method, it is confirmed that beam focusing and splitting with multiple dflection angles can also be achieved.     

Near-field optical analysis of living cells in vitro

Near-field optical analysis (NOA) provides morphological nanoscale mappings of living cells in liquid cell culture media and nondestructive insight into cell functionality. Here we show for the first time the performance of NOA in imaging living cells. Unlabeled human endothelial cells attached to polished titanium disks were analyzed with hydrophobically coated optical biosensors mounted to a near-field scanning optical microscope (NSOM). Biosensors and titanium substrates could be simply implemented in standard NSOM and high-throughput NOA.