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。     

AFM‐ and NSOM‐based force spectroscopy and distribution analysis of CD69 molecules on human CD4+ T cell membrane

Although CD69 is well known as an early T cell‐activation marker, the possibility that CD69 are distributed as nano‐structures on membrane for immune regulation during T cell activation has not been tested. In this study, nanoscale features of CD69 expression on activated T cells were determined using the atomic force microscopy (AFM) topographic and force‐binding nanotechnology as well as near‐field scanning optical microscopy (NSOM)‐/fluorescence quantum dot (QD)‐based nanosacle imaging. Unstimulated CD4⁺ T cells showed neglectable numbers of membrane CD69 spots binding to the CD69 Ab‐functinalized AFM tip, and no detectable QD‐bound CD69 as examined by NSOM/QD‐based imaging. In contrast, Phytohemagglutinin (PHA)‐activated CD4⁺ T cells expressed CD69, and displayed many force‐binding spots binding to the CD69 Ab‐functionalized AFM tip on about 45% of cell membrane, with mean binding‐rupture forces 276 ± 71 pN. Most CD69 molecules appeared to be expressed as 100–200 nm nanoclusters on the membrane of PHA‐activated CD4⁺ T cells. Meanwhile, NSOM/QD‐based nanoscale imaging showed that CD69 were non‐uniformly distributed as 80–200 nm nanoclusters on cell‐membrane of PHA‐activated CD4⁺ T cells. This study represents the first demonstration of the nano‐biology of CD69 expression during T cell activation. Copyright © 2009 John Wiley & Sons, Ltd.     

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。     

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.     

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.     

Near-field scanning fluorescence microscopy study of ion channel clusters in cardiac myocyte membranes

Near-field scanning optical microscopy (NSOM) has been used to study the nanoscale distribution of voltage-gated L-type Ca²⁺ ion channels, which play an important role in cardiac function. NSOM fluorescence imaging of immunostained cardiac myocytes (H9C2 cells) demonstrates that the ion channel is localized in small clusters with an average diameter of 100 nm. The clusters are randomly distributed throughout the cell membrane, with some larger fluorescent patches that high-resolution images show to consist of many small closely-spaced clusters. We have imaged unstained cells to assess the contribution of topography-induced artifacts and find that the topography-induced signal is <10% of the NSOM fluorescence intensity. We have also examined the dependence of the NSOM signal intensity on the tip-sample separation to assess the contributions from fluorophores that are significantly below the cell surface. This indicates that chromophores >~200 nm below the probe will have negligible contributions to the observed signal. The ability to quantitatively measure small clusters of ion channels will facilitate future studies that examine changes in protein localization in stimulated cells and during cardiac development. Our work illustrates the potential of NSOM for studying membrane domains and protein localization/colocalization on a length scale which exceeds that available with optical microscopy.     

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.     

近场光学显微镜结合量子点标记人胃腺癌SGC-7901细胞靶点的观察

扫描近场光学显微镜突破衍射极限,具有纳米量级的空间分辨率,量子点(QD s)标记有荧光强度高且抗光漂白能力强等优点。结合上述两种技术,对人胃腺癌SGC-7901细胞膜表面特异性结合的叶酸受体(FR)进行成像探测,获得了叶酸受体在SGC-7901细胞膜表面上的分布,以及细胞内化外源性叶酸过程中叶酸受体在细胞膜表面的分布变化,成像的光学分辨率达到120 nm。实验结果表明:特异性结合的叶酸受体在SGC-7901细胞膜表面的分布,绝大部分是以聚集体的形式存在。随着SGC-7901细胞内化叶酸量的增加,叶酸受体在细胞膜表面的分布密度逐渐降低,并在经过120 m in左右趋于稳定。上述方法和手段为实现单细胞水平上靶点分布和变化的长期监测,肿瘤细胞内化受体的机制研究提供了新的技术途径。     

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.     

Confocal scanning optical microscopy and three-dimensional imaging

Summary— Confocal scanning optical microscopy has significant advantages over conventional fluorescence microscopy: it rejects the out-of-locus light and provides a greater resolution than the wide-field microscope. In laser scanning optical microscopy, the specimen is scanned by a diffraction-limited spot of laser light and the fluorescence emission (or the reflected light) is focused onto a photodetector. The imaged point is then digitized, stored into the memory of a computer and displayed at the appropriate spatial position on a graphic device as a part of a two-dimensional image. Thus, confocal scanning optical microscopy allows accurate non-invasive optical sectioning and further three-dimensional reconstruction of biological specimens. Here we review the recent technological aspects of the principles and uses of the confocal microscope, and we introduce the different methods of three-dimensional imaging.     

Imaging of Oil/Monoglyceride Networks by Polarizing Near-Field Scanning Optical Microscopy

Polarizing near-field scanning optical microscopy (NSOM) was applied for visualization of lipid coagel structures. The technique ensures obtaining polarization contrast images at micro- and nanoscale resolution. Comparison to the polarizing light microscopy images revealed that the same fractal structural organization persists also at submicron scale, at the level of primary ordered structures creation. Many long birefringent needle-shaped primary crystallites were imaged in the corn oil:monoglyceride samples, and lower amount of smaller oval-shaped primary crystallites—in the olive oil:monoglyceride samples. Unlike atomic force microscopy, polarizing NSOM brought direct evidence on the physical state of specific features. Compared to the polarizing light microscopy, polarizing NSOM provided additional information on the structural organization of oil–monoglyceride coagels at the micro- and submicron scale.     

STED microscopy of living cells – new frontiers in membrane and neurobiology

Recent developments in fluorescence far‐field microscopy such as STED microscopy have accomplished observation of the living cell with a spatial resolution far below the diffraction limit. Here, we briefly review the current approaches to super‐resolution optical microscopy and present the implementation of STED microscopy for novel insights into live cell mechanisms, with a focus on neurobiology and plasma membrane dynamics.     

Fluorescence-topographic NSOM directly visualizes peak-valley polarities of GM1/GM3 rafts in cell membrane fluctuations

Simultaneous fluorescence-topographic nanoscale imaging of cell-surface molecules in the context of membrane ultra-structures has not been reported. Here, near-field scanning optical microscopy (NSOM)-based direct fluorescence-topographic imaging indicated that GM3 rafts/nanodomains (190.0 +/- 49.8 nm ranging 84.5-365.0 nm) were localized predominantly on the peaks of microvillus-like protrusions in the apical membrane of GM3 + Madin-Darby canine kidney cells, whereas GM1 rafts/nanodomains (159.5 +/- 63.8 nm ranging 42-360 nm) were distributed mainly on the slops of protrusions or the valleys between protrusions in the plasma membranes of GM1 + MDCK cells. The data demonstrated that gangliosides polarized not only in a well-known apical-basolateral manner but also in the more microscopic peak-valley manner, implicating unique distribution of GM1 or GM3 in cell-surface fluctuations on the apical membrane of polarized cells. The peak-valley polarities of gangliosides also implicated their different functions relevant to lipid rafts, microvilli, or cellular processes. Importantly, our study demonstrated for the first time that the NSOM-based direct fluorescence-topographic imaging is unique and powerful for elucidating nanoscale distribution of specific cell-surface molecules in membrane fluctuations.     

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.     

Near-field scanning optical microscopy in cell biology

Near-field optics has produced the highest optical resolution that has ever been achieved. The methods involved lie at the interface of far-field optical microscopy and scanned probe microscopy. This article describes the principles behind near-field scanning optical microscopy (NSOM) and highlights its potential in cell biology.     

同时应用近场光学显微镜的透射和反射模式研究单个细胞的光学性质

通常认为．在近场光学显微技术的光收集模式中,观察透光性好的样品时采用透射模式．研究不透明样品时采用反射模式。本文同时采用透射和反射两种模式观察透明性较好的PCI2细胞和淋巴细胞样品．初步研究单个细胞的反射、吸收、透射和荧光等光学性质,以促进组织光学和激光生物医学等领域的进一步发展。细胞光学的时代就要到来。     

Ultra‐structural analysis of human spermatozoa by aperture scanning near‐field optical microscopy

Scanning near‐field optical microscopy (SNOM) represents a potential candidate for investigation of ultrastructure in human spermatozoa. It is a noninvasive optical technique that offers two main advantages: minimal sample preparation and simultaneous topographical and optical images acquisition with a spatial resolution beyond the diffraction limit. This enables the combination of surface characterization and information from the inner cellular organization in a single acquisition providing an immediate and comprehensive analysis of the cellular portions. In this work spermatozoa are immobilized on poly‐L‐lysine coated coverslips, fixed according to a standard protocol and imaged by aperture‐SNOM in air. In the SNOM images, all peculiar sperm portions show well‐resolved optical features, which exhibit good similarities with the structures revealed in transmission electron microscopy images, as compared with literature data. The optical features of anomalous spermatozoa are clearly different as respect with those observed for healthy ones. This analysis reveals the potentialities of SNOM and opens to its application to high‐resolution analysis of sperm morphological alterations, which might be relevant in reproductive medicine.     

The lipid raft hypothesis revisited - New insights on raft composition and function from super-resolution fluorescence microscopy

Recently developed super‐resolution microscopy techniques are changing our understanding of lipid rafts and membrane organisation in general. The lipid raft hypothesis postulates that cholesterol can drive the formation of ordered domains within the plasma membrane of cells, which may serve as platforms for cell signalling and membrane trafficking. There is now a wealth of evidence for these domains. However, their study has hitherto been hampered by the resolution limit of optical microscopy, making the definition of their properties problematic and contentious. New microscopy techniques circumvent the resolution limit and, for the first time, allow the fluorescence imaging of structures on length scales below 200 nm. This review describes such techniques, particularly as applied to the study of membrane organisation, synthesising newly emerging facets of lipid raft biology into a state‐of‐the art model. Editor's suggested further reading in BioEssays: Super‐resolution imaging prompts re‐thinking of cell biology mechanisms Abstract and Quantitative analysis of photoactivated localization microscopy (PALM) datasets using pair‐correlation analysis Abstract     

Nanoscale fluorescence correlation spectroscopy on intact living cell membranes with NSOM probes

Characterization of molecular dynamics on living cell membranes at the nanoscale is fundamental to unravel the mechanisms of membrane organization and compartmentalization. Here we demonstrate the feasibility of fluorescence correlation spectroscopy (FCS) based on the nanometric illumination of near-field scanning optical microscopy (NSOM) probes on intact living cells. NSOM-FCS applied to fluorescent lipid analogs allowed us to reveal details of the diffusion hidden by larger illumination areas. Moreover, the technique offers the unique advantages of evanescent axial illumination and straightforward implementation of multiple color excitation. As such, NSOM-FCS represents a powerful tool to study a variety of dynamic processes occurring at the nanometer scale on cell membranes.     

Integrating photoacoustic microscopy with other imaging technologies for multimodal imaging

As a hybrid optical microscopic imaging technology, photoacoustic microscopy images the optical absorption contrasts and takes advantage of low acoustic scattering of biological tissues to achieve high-resolution anatomical and functional imaging. When combined with other imaging modalities, photoacoustic microscopy-based multimodal technologies can provide complementary contrast mechanisms to reveal complementary information of biological tissues. To achieve intrinsically and precisely registered images in a multimodal photoacoustic microscopy imaging system, either the ultrasonic transducer or the light source can be shared among the different imaging modalities. These technologies are the major focus of this minireview. It also covered the progress of the recently developed penta-modal photoacoustic microscopy imaging system featuring a novel dynamic focusing technique enabled by OCT contour scan.