A method for micrometer resolution patterning of primary culture neurons for SPM analysis

In this work we present a method for ultra-fine patterning of primary culture neuron cell growth, which is compatible for scanning near-field optical atomic force microscopy (SNOAM) analysis. SNOAM uses near-field optics to break the fundamental diffraction limit imposed on normal microscopy. SNOAM can achieve sub-100 nm optical resolutions, but requires transparent, open substrates. The ability to do physiological measurements on patterns of neurons, combined with ultra high resolution optical and fluorescent analysis, is useful in the study of long-term potentiation. The patterning method consists of chemical guidance with an element of physical confinement and allows for ultra-fine patterning of neural growth on transparent glass substrates. Substrates consist of microfabricated perfluoropolymer barrier structures on glass. Poly-L-lysine was selectively deposited using a silicone-based microfluidic stencil aligned to the perfluoropolymer/glass substrate. Primary culture neurons were extracted from 8-day-old chicks and grown for 3 days to form good networks. This patterning system shows very specific growth with patterning separations down to the level of individual neurites. Fluorescent imaging was carried out on both cell viability during growth and immuno-tagged microtubule-associated proteins on the neurites. Neurons inside the patterned structures were imaged and analyzed with a tapping mode SNOAM.     

Fabrication and characterization of patterned immobilization of quantum dots on metallic nano-gratings

Surfaces featuring nano-structures and biochemical patterns are increasingly developed as novel and superior substrates for biosensors and assays. Metallic periodic nano-structures have been studied for their unique optical properties and in particular their ability to support surface plasmon waves. Here we present a new nano-structuring approach based on gentle metal lift-off process coupled with self-assembled surface chemistry for the fabrication of a zeroth-order 400nm period metallic grating with differentiated surface chemistries on the mesas and troughs. The approach, using terminated self-assembled monolayers, creates versatile functionalized substrates allowing the precise deposition of complex biomolecular structures. We use this technique to perform the guided deposition of a three-dimensional polyelectrolyte multilayer structure and the patterned adsorption of quantum dots. Finally, we demonstrate that scanning near-field optical microscopy, used in conjuncture with atomic force microscopy and scanning electron microscopy, is an ideal tool for the characterization of this nano-structured surface as it provides a complete chemical, topographical and optical image of the surface. This ability to pattern and locally measure the surface properties is likely to have an important impact on the design of novel and optimized biointerfaces and transducers for biosensors.     

Near-field optical imaging of abasic sites on a single DNA molecule

Scanning near-field optical microscopy (SNOM) imaging was performed to allow for the direct visualization of damaged sites on individual DNA molecules to a scale of a few tens of nanometers. Fluorescence in situ hybridization on extended DNA molecules was modified to detect a single abasic site. Abasic sites were specifically labelled with a biotinlylated aldehyde-reactive probe and fluorochrome-conjugated streptavidin. By optimizing the performance of the SNOM technique, we could obtain high contrast near-field optical images that enabled high-resolution near-field fluorescence imaging using optical fiber probes with small aperture sizes. High-resolution near-field fluorescence imaging demonstrated that two abasic sites within a distance of 120 nm are clearly obtainable, something which is not possible using conventional fluorescence in situ hybridization combined with far-field fluorescence microscopy.     

Simultaneous detection of near-field topographic and fluorescence images of human chromosomes via scanning near-field optical/atomic-force microscopy (SNOAM).

Scanning near-field optical/atomic-force microscopy (SNOAM) provided us with simultaneous topographical and optical images of human chromosomes using a sharp and bent optical fiber as a near-field optical probe. Native chromosomes were spread out onto a coverslip using the surface-spreading whole-mount method. The SNOAM system does not need pretreatment of samples such as metal coating or chemical immobilization. Near-field topographic and fluorescence images provided useful information on native chromosome structure.     

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.     

Plasmon Polariton Imaging Characteristics of Near-Field Optical Fiber Probes

The influence of different near-field optical (near-field scanning optical microscopy) probes on the imaging of surface plasmon polaritons propagating on thin metal films is investigated. Metal-coated fiber probes exhibit a suppression of the measured plasmon signal close to the metal film surface and increased local scattering of the plasmon field. Purely dielectric fiber probes are shown to be largely free of these effects.     

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。     

Near-Field Optical Imaging of a Porous Au Film: Influences of Topographic Artifacts and Surface Plasmons

In this work, near-field scanning optical microscopy is employed to study a porous Au film and the direct observation of topographic artifacts and surface plasmon influences is revealed. Under tip illumination, topographic artifacts are found to be present in a reflection mode optical image but not in a transmission mode image. A simple algorithm is used for filtering the topographic artifacts and extracting a correct near-field optical image approximately. On the other hand, surface plasmon influences are present in both modes. By using three exciting wavelengths of 488, 647.1, and 520.8 nm, it is confirmed that a suitable wavelength should be chosen for avoiding the surface plasmon interference in a near-field optical investigation of morphological or material dielectric contrast. Finally, plasmonic or nonplasmonic regions on the porous Au film can be identified from the observed optical intensity variation in the optical images obtained at incident polarizations of 0°, 90°, and 45°.     

Extraction of near-field fluorescence from composite signals to provide high resolution images of glial cells

The subdiffraction optical resolution that can be achieved using near-field optical microscopy has the potential to permit new approaches and insights into subcellular function and molecular dynamics. Despite the potential of this technology, it has been difficult to apply to cellular samples. One significant problem is that sample thickness causes the optical information to be comprised of a composite signal containing both near- and far-field fluorescence. To overcome this issue we have developed an approach in which a near-field optical fiber is translated toward the cell surface. The increase in fluorescence intensity during z-translation contains two components: a far-field fluorescence signal when the tip of the fiber is distant from the labeled cell, and combined near- and far-field fluorescence when the tip interacts with the cell surface. By fitting a regression curve to the far-field fluorescence intensity as the illumination aperture approaches the cell, it is possible to isolate near-field from far-field fluorescent signals. We demonstrate the ability to resolve actin filaments in chemically fixed, hydrated glial cells. A comparison of composite fluorescence signals with extracted near-field fluorescence demonstrates that this approach significantly increases the ability to detect subcellular structures at subdiffraction resolution.     

Rapid Patterning of 1-D Collagenous Topography as an ECM Protein Fibril Platform for Image Cytometry

Cellular behavior is strongly influenced by the architecture and pattern of its interfacing extracellular matrix (ECM). For an artificial culture system which could eventually benefit the translation of scientific findings into therapeutic development, the system should capture the key characteristics of a physiological microenvironment. At the same time, it should also enable standardized, high throughput data acquisition. Since an ECM is composed of different fibrous proteins, studying cellular interaction with individual fibrils will be of physiological relevance. In this study, we employ near-field electrospinning to create ordered patterns of collagenous fibrils of gelatin, based on an acetic acid and ethyl acetate aqueous co-solvent system. Tunable conformations of micro-fibrils were directly deposited onto soft polymeric substrates in a single step. We observe that global topographical features of straight lines, beads-on-strings, and curls are dictated by solution conductivity; whereas the finer details such as the fiber cross-sectional profile are tuned by solution viscosity. Using these fibril constructs as cellular assays, we study EA.hy926 endothelial cells'' response to ROCK inhibition, because of ROCK''s key role in the regulation of cell shape. The fibril array was shown to modulate the cellular morphology towards a pre-capillary cord-like phenotype, which was otherwise not observed on a flat 2-D substrate. Further facilitated by quantitative analysis of morphological parameters, the fibril platform also provides better dissection in the cells'' response to a H1152 ROCK inhibitor. In conclusion, the near-field electrospun fibril constructs provide a more physiologically-relevant platform compared to a featureless 2-D surface, and simultaneously permit statistical single-cell image cytometry using conventional microscopy systems. The patterning approach described here is also expected to form the basics for depositing other protein fibrils, seen among potential applications as culture platforms for drug screening.     

Variations in cell surfaces of estrogen treated breast cancer cells detected by a combined instrument for far-field and near-field microscopy.

The response of single breast cancer cells (cell line T-47D) to 17beta-estradiol (E(2)) under different concentrations was studied by using an instrument that allows to combine far-field light microscopy with high resolution scanning near-field (AFM/SNOM) microscopy on the same cell. Different concentrations of E(2) induce clearly different effects as well on cellular shape (in classical bright-field imaging) as on surface topography (atomic force imaging) and absorbance (near-field light transmission imaging). The differences range from a polygonal shape at zero via a roughly spherical shape at physiological up to a spindle-like shape at un-physiologically high concentrations. The surface topography of untreated control cells was found to be regular and smooth with small overall height modulations. At physiological E(2) concentrations the surfaces became increasingly jagged as detected by an increase in membrane height. After application of the un-physiological high E(2) concentration the cell surface structures appeared to be smoother again with an irregular fine structure. The general behaviour of dose dependent differences was also found in the near-field light transmission images. In order to quantify the treatment effects, line scans through the normalised topography images were drawn and a rate of co-localisation between high topography and high transmission areas was calculated. The cell biological aspects of these observations are, so far, not studied in detail but measurements on single cells offer new perspectives to be empirically used in diagnosis and therapy control of breast cancers.     

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。     

Antenna-Attached Parabolic Probe for Excitation and Collection Modes in SNOM

A novel optical fiber probe with a parabola-like shape and a nano-antenna mounted on the center of its endface is proposed for simultaneous excitation and collection modes in scanning near-field optical microscopy. The working principles of the probe are demonstrated, and its optical properties are theoretically investigated and compared with the conventional tip-on-aperture probe. It shows that the probe can greatly boost both the enhancement factor for the excitation mode and the collection efficiency for the collection mode. The proposed probe is a promising tool to realize low-cost and high resolution for a wide variety of near-field measurements in biology, physics, and chemistry.     

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.     

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.     

Confined detection volume of fluorescence correlation spectroscopy by bare fiber probes

A fiber-tip-based near-field fluorescence correlation spectroscopy (FCS) has been developed for confining the detection volume to sub-diffraction-limited dimensions. This near-field FCS is based on near-field illumination by coupling a scanning near-field optical microscope (SNOM) to a conventional confocal FCS. Single-molecule FCS analysis at 100 nM Rhodamine 6G has been achieved by using bare chemically etched, tapered fiber tips. The detection volume under control of the SNOM system has been reduced over one order of magnitude compared to that of the conventional confocal FCS. Related factors influencing the near-field FCS performance are investigated and discussed in detail. In this proof-of-principle study, the preliminary experimental results suggest that the fiber-tip-based near-field FCS might be a good alternative to realize localized analysis at the single-molecule level.     

Optical chemical imaging of tobacco mosaic virus in solution at 60-nm resolution.

Scanning near-field optical microscopy can provide images with a resolution less than the wavelength of light, and therefore ought in principle to be of great value in studies of biological structures. In this work we show how for the first time images have been obtained of tobacco mosaic virus particles at 60-nm resolution, combined with chemical imaging using monoclonal antibodies under in vitro conditions.     

Specific fluorescent tracers. Imaging and applications for photodynamic therapy

Our main objective is to enlarge the fluorescence use in biosciences, with especially the photodynamic therapy (PDT) used for cancer treatment as one of the target applications. Meta-tetra(hydroxyphenyl)chlorin (m-THPC) is a second-generation photosensitiser, applied in photodynamic therapy. The localisation of this sensitiser as well as its induced cell death mechanisms in human breast cancer cells (MCF-7 and its resistant subline MCF-7DXR, DXR: doxorubicin) were evaluated using fluorescence microscopy. In addition, we will present two additional routes, whose aims are to create new features to respond to the PDT questioning: firstly, the synthesis of fluorescent tracers, with a particular attention to the presence of hydrophilic groups (glucosamine ring) on the basic fluorophore structure to orientate the localisation of the probe and, secondly, the use of scanning near-field optical microscopy to reach a better resolution for the fluorescence microscopy analysis.     

Near-field optics: from subwavelength illumination to nanometric shadowing

Near-field optics uniquely addresses problems of x, y and z resolution by spatially confining the effect of a light source to nanometric domains. The problems in using far-field optics (conventional optical imaging through a lens) to achieve nanometric spatial resolution are formidable. Near-field optics serves a bridging role in biology between optical imaging and scanned probe microscopy. The integration of near-field and scanned probe imaging with far-field optics thus holds promise for solving the so-called inverse problem of optical imaging.     

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.