A silanized mica substrate suitable for high-resolution fiber FISH analysis by scanning near-field optical/atomic force microscopy

We applied a novel silanized mica substrate with an extremely flat surface constructed according to Sasou et al. (Langmuir 19, 9845-9849 (2003)) to high-resolution detection of a specific gene on a DNA fiber by scanning near-field optical/atomic force microscopy (SNOM/AFM). The interaction between the substrate and fluorescence-dye conjugated peptide nucleic acid (PNA) probes, which causes fluorescence noise signal, was minimal. By using the substrate, we successfully obtained a fluorescence in situ hybridization signal from the ea47 gene on a λphage DNA labeled with an Alexa 532-conjugated 15-base PNA probe. As the results, no fluorescence noises were observed, indicating that the surface adsorbed almost none of the PNA probe. The combination of the substrate and SNOM/AFM is an effective tool for visualizing DNA sequences at nanometer-scale resolution.     

Optical microcantilever consisting of channel waveguide for scanning near-field optical microscopy controlled by atomic force

We develop a novel optical microcantilever for scanning near-field optical microscopy controlled by atomic force mode (SNOM/AFM). The optical microcantilever has the bent channel waveguide, the corner of which acts as aperture with a large tip angle. The resonance frequency of the optical microcantilever is 9 kHz, and the spring constant is estimated to be 0.59 N/m. The optical microcantilever can be operated in contact mode of SNOM/AFM and we obtain the optical resolution of about 200 nm, which is as same size as the diameter of aperture. We confirm that the throughput of optical microcantilever with an aperture of 170 nm diameter would be improved to be more than 10(-5).     

Optical microcantilever consisting of channel waveguide for scanning near-field optical microscopy controlled by atomic force

We develop a novel optical microcantilever for scanning near-field optical microscopy controlled by atomic force mode (SNOM/AFM). The optical microcantilever has the bent channel waveguide, the corner of which acts as aperture with a large tip angle. The resonance frequency of the optical microcantilever is 9 kHz, and the spring constant is estimated to be 0.59 N/m. The optical microcantilever can be operated in contact mode of SNOM/AFM and we obtain the optical resolution of about 200 nm, which is as same size as the diameter of aperture. We confirm that the throughput of optical microcantilever with an aperture of 170 nm diameter would be improved to be more than 10⁻⁵.     

Fluorescence imaging and investigations of directly labelled chromosomes using scanning near-field optical microscopy

Scanning near-field optical microscopy (SNOM) has been successfully employed to generate high resolution (<100nm) fluorescence images of directly tagged human chromosomes. Direct tagging, fluorescence in-situ hybridisation processes (with and without amplification) are investigated and their fluorescence response to near-field excitation are compared. Using the simultaneous topography mode of SNOM, chromosome morphology was seen to differ as a result of the two processes; with chromatin collapse more extensive when the amplified direct tagging procedure was used. The results are discussed in the context of developing locus specific direct tags together with high resolution SNOM imaging for the observation of chromosome aberrations.     

Near-field optical microscopy with video signal processor to apply a CCD imaging device as a variable area photo-sensor for improving operability and spectrum mode imaging

We have developed a video signal processor for improving the operability and function of scanning near-field optical microscopy (SNOM). The video signal processor applies a CCD imaging device as a variable area photo-sensor in the SNOM unit instead of conventional photo-detectors. The signal processor converts the intensity of a selected area in video frames to a numerical value with a rate of 30 Hz. Consequently, the CCD imaging device can be used as a photo-detector of variable areas and positions for detecting a small area of a optical probe position. The need for a precise optical axis alignment is relaxed due to the large sensing area of the CCD device. Using the video signal processor, near-field optical and topographic images have been obtained by SNOM/AFM system simultaneously. By adding a spectrometer between the SNOM unit and the CCD device, the spectrum signal of selected wavelength ranges has been monitored by the video signal processor to provide an optical image.     

Can scanning near‐field optical microscopy be compared with confocal laser scanning microscopy? A preliminary study on α‐sarcoglycan and β1D‐integrin in human skeletal muscle

The dystrophin–glycoprotein complex and the vinculin–talin–integrin system constitute, together a protein machinery, called costameres. The dystrophin–glycoprotein complex contains, among other proteins, also dystrophin and the sarcoglycans subcomplex, proteins playing a key role in the pathogenesis of many muscular dystrophies and linking the cytoplasmic myofibrillar contractile elements to the signal transducing molecules of the extracellular matrix, also providing structural support to the sarcolemma. The vinculin–talin–integrin system connects some components of the extracellular matrix with intermediate filaments of desmin, forming transverse bridges between Z and M lines. In our previous reports we always studied these systems by confocal laser scanning microscopy (CLSM). In this paper we report on the first applications of optical near‐field fluorescence microscopy to the spatial localization of α‐sarcoglycan and β1D‐integrin in human skeletal muscle fibres in order to better compare and test the images obtained with conventional CLSM and with scanning near‐field optical microscopy (SNOM). In addition, the analysis of the surface morphology, and the comparison with the fluorescence map is put forward and analyzed for the first time on human muscle fibres. In aperture‐SNOM the sample is excited through the nanometre‐scale aperture produced at the apex of an optical fibre after tapering and subsequent metal coating. The acquisition of the topography map, simultaneously to the optical signal, by SNOM, permits to exactly overlap the fluorescence images obtained from the two consecutive scans needed for the double localization. Besides, the differences between the topography and the optical spatial patterns permit to assess the absence of artefacts in the fluorescence maps. Although the SNOM represented a good method of analysis, this technique remains a complementary method to the CLSM and it can be accepted in order to confirm the hypothesis advanced by CLSM.     

Labelling quality and chromosome morphology after low temperature FISH analysed by scanning far-field and near-field optical microscopy

A non‐enzymatic, low temperature fluorescence in situ hybridization (LTFISH) procedure was applied to metaphase spreads and interphase cell nuclei. In this context ‘low temperature’ means that the denaturation procedure of the chromosomal target DNA usually applied by heat treatment and chaotropic agents such as formamide was completely omitted so that the complete hybridization reaction took place at 37 °C. For LTFISH, the DNA probe had to be single‐stranded, which was achieved by means of separate thermal denaturation of the DNA probe only. The DNA probe pUC1.77 was used for all LTFISH experiments. The labelling quality (number of binding sites, relative background intensity, relative intensity of major and minor binding sites) was analysed by confocal laser scanning microscopy (CLSM). An optimum in specificity and signal quality was obtained for 15 h hybridization time. For this hybridization condition of LTFISH, the chromosomal morphology was analysed by scanning near‐field optical microscopy (SNOM). The results were compared with the morphology of chromosomes after (a) labelling of all centromeres using the same chemical treatment in the FISH procedure but with the application of target denaturation, and (b) labelling of all centromeres using a standard FISH protocol including thermal denaturation of the DNA probe and the chromosomal target. Depending on the FISH‐procedure applied, SNOM images show substantial differences in the chromosome morphology. After LTFISH the chromosome morphology appeared to be much better preserved than after standard FISH. In contrast, the application of the LTFISH chemical treatment accompanied by heat denaturation had a very destructive influence on chromosomal morphology. The results indicate that, at least for certain DNA probes, specific chromosome labelling can be obtained without the usually applied heat and chemical denaturation of the DNA target, resulting in an apparently well preserved chromatin morphology as visualized by SNOM. LTFISH may be therefore a useful labelling technique whenever the chromosomal morphology had to be preserved after specific labelling of DNA regions. Binding mechanisms of single‐stranded DNA probes to double‐stranded DNA targets are discussed.     

Dynamic etching method for fabricating a variety of tip shapes in the optical fibre probe of a scanning near-field optical microscope

A novel etching method for an optical fibre probe of a scanning near-field optical microscope (SNOM) was developed to fabricate a variety of tip shapes through dynamic movement during etching. By moving the fibre in two-phase fluids of HF solution and organic solvent, the taper length and angle can be varied according to the movement of the position of the meniscus on the optical fibre. This method produces both long (sharp angle) and short (wide angle) tapered tips compared to tips made with stationary etching processes. A bent-type probe for a SNOM/AFM was fabricated by applying this technique and its throughput efficiency was examined. A wide-angle probe with a 50° angle at the tip showed a throughput efficiency of 3.3 × 10⁻⁴ at a resolution of 100 nm.     

High-speed scanning by dual feedback control in SNOM/AFM

We have developed a high-speed scanning near-field optical microscope (SNOM)/atomic force microscope (AFM) system including dual feedback controllers. The system includes an additional piezoelectric actuator with fast response in the z direction and a correction circuit to eliminate unnecessary components from the feedback signal. From the measurement of a patterned chromium layer of 2 x 2 microm2 checks on a quartz glass plate, we confirmed that our system had more effective feedback control and faster scanning than current SNOM/AFM systems that use only a piezo-tube. The scanning speed of the present system was estimated to be about five times faster than that of current SNOM/AFM systems.     

Dynamic etching method for fabricating a variety of tip shapes in the optical fibre probe of a scanning near-field optical microscope

A novel etching method for an optical fibre probe of a scanning near-field optical microscope (SNOM) was developed to fabricate a variety of tip shapes through dynamic movement during etching. By moving the fibre in two-phase fluids of HF solution and organic solvent, the taper length and angle can be varied according to the movement of the position of the meniscus on the optical fibre. This method produces both long (sharp angle) and short (wide angle) tapered tips compared to tips made with stationary etching processes. A bent-type probe for a SNOM/AFM was fabricated by applying this technique and its throughput efficiency was examined. A wide-angle probe with a 50 degrees angle at the tip showed a throughput efficiency of 3.3 x 10(-4) at a resolution of 100 nm.     

High-resolution constant-height imaging with apertured silicon cantilever probes

We present high-resolution aperture probes based on non-contact silicon atomic force microscopy (AFM) cantilevers for simultaneous AFM and near-infrared scanning near-field optical microscopy (SNOM). For use in near-field optical microscopy, conventional AFM cantilevers are modified by covering their tip side with an opaque aluminium layer. To fabricate an aperture, this metal layer is opened at the end of the polyhedral probe using focused ion beams (FIB). Here we show that apertures of less than 50 nm can be obtained using this technique, which actually yield a resolution of about 50 nm, corresponding to λ/20 at the wavelength used. To exclude artefacts induced by distance control, we work in constant-height mode. Our attention is particularly focused on the distance dependence of resolution and to the influence of slight cantilever bending on the optical images when scanning at such low scan heights, where first small attractive forces exerted on the cantilever become detectable.     

近场光学显微镜及其在生物膜方面的应用

本文首先介绍近场光学显微镜的基本原理,然后介绍近场光学显微镜与传统光学显微镜、原子力显微镜、扫描隧道显微镜相比,在生物膜研究方面的优势。并在此基础上着重介绍近场光学显微镜在生物膜方面的应用。     

High-speed scanning by dual feedback control in SNOM/AFM

We have developed a high-speed scanning near-field optical microscope (SNOM)/atomic force microscope (AFM) system including dual feedback controllers. The system includes an additional piezoelectric actuator with fast response in the z direction and a correction circuit to eliminate unnecessary components from the feedback signal. From the measurement of a patterned chromium layer of 2 × 2 μm² checks on a quartz glass plate, we confirmed that our system had more effective feedback control and faster scanning than current SNOM/AFM systems that use only a piezo-tube. The scanning speed of the present system was estimated to be about five times faster than that of current SNOM/AFM systems.     

Scanning near-field optical microscopy using semiconductor nanocrystals as a local fluorescence and fluorescence resonance energy transfer source

Local fluorescence probes based on CdSe semiconductor nanocrystals were prepared and tested by recording scanning near‐field optical microscopy (SNOM) images of calibration samples and fluorescence resonance energy transfer SNOM (FRET SNOM) images of acceptor dye molecules inhomogeneously deposited onto a glass substrate. Thousands of nanocrystals contribute to the signal when this probe is used as a local fluorescence source while only tens of those (the most apical) are involved in imaging for the FRET SNOM operation mode. The dip‐coating method used to make the probe enables diminishing the number of active fluorescent nanocrystals easily. Prospects to realize FRET SNOM based on a single fluorescence centre using such an approach are briefly described.     

SNOM and AFM microscopy techniques to study the effect of non-ionizing radiation on the morphological and biochemical properties of human keratinocytes cell line (HaCaT)

In this study we have employed atomic force microscopy (AFM) and scanning near‐field optical microscopy (SNOM) techniques to study the effect of the interaction between human keratinocytes (HaCaT) and electromagnetic fields at low frequency. HaCaT cells were exposed to a sinusoidal magnetic field at a density of 50 Hz, 1 mT. AFM analysis revealed modification in shape and morphology in exposed cells with an increase in the areas of adhesion between cells. This latter finding was confirmed by SNOM indirect immunofluorescence analysis performed with a fluorescent antibody against the adhesion marker β4 integrin, which revealed an increase of β4 integrin segregation in the cell membrane of 50‐Hz exposed cells, suggesting that a higher percentage of these cells shows a modified pattern of this adhesion marker.     

Numerical chromosomal abnormalities detected by atomic force microscopy.

The numerical abnormalities of human metaphase chromosomes, fixed according to standard procedures for optical microscopy but not treated for banding, were detected by atomic force microscopy (AFM). High-resolution AFM imaging of chromosomes in trisomy 13, 21, and Klinefelter syndrome can be compared directly with the traditional optical image. The unbanded metaphase chromosomes, including the extra ones in trisomic patients showed a structural pattern very similar to G-banding. Comparison of AFM images with light microscopic data allows the identification of specific chromosomes, and images of chromosomes showing numerical and structural abnormalities can then be analysed.     

Mechanisms of cell-cell adhesion identified by immunofluorescent labelling with quantum dots: A scanning near-field optical microscopy approach

Scanning near-field optical microscopy (SNOM) has been employed to simultaneously acquire high-resolution fluorescence images along with shear-force atomic force microscopy from cell membranes. Implementing such a technique overcomes the limits of optical diffraction found in standard fluorescence microscopy and also yields vital topographic information. The application of the technique to investigate cell-cell adhesion has revealed the interactions of filopodia and their functional relationship in establishing adherens junctions. This has been achieved via the selective tagging of the cell adhesion protein, E-cadherin, by immunofluorescence labelling. Two labelling routes were explored; Alexa Fluor 488 and semiconductor quantum dots. The quantum dots demonstrated significantly enhanced photostability and high quantum yield making them a versatile alternative to the conventional organic fluorophores often used in such a study. Analysis of individual cells revealed that E-cadherin is predominantly located along the cell periphery but is also found to extend throughout their filopodia. We have demonstrated that with a fully optimised sample preparation methodology, quantum dot labelling in conjunction with SNOM imaging can be successfully applied to interrogate biomolecular localisation within delicate cellular membranes.     

High-Resolution Lithography with Near-Field Optical Microscopy

Scanning near-field optical microscopy (SNOM) is used for lithography to avoid the resolution limiting diffraction of conventional optical methods. We have expanded a commercial SNOM for writing even complex structures on the nanometer scale. Scanning near-field optical lithography (SNOL) has been applied to conventional resists to explore its potential and the possible combination with conventional optical lithography (mix and match technique).     

Combining AFM and FRET for high resolution fluorescence microscopy

Here we demonstrate a new microscopic method that combines atomic force microscopy (AFM) with fluorescence resonance energy transfer (FRET). This method takes advantage of the strong distance dependence in Förster energy transfer between dyes with the appropriate donor/acceptor properties to couple an optical dimension with conventional AFM. This is achieved by attaching an acceptor dye to the end of an AFM tip and exciting a sample bound donor dye through far-field illumination. Energy transfer from the excited donor to the tip immobilized acceptor dye leads to emission in the red whenever there is sufficient overlap between the two dyes. Because of the highly exponential distance dependence in this process, only those dyes located at the apex of the AFM tip, nearest the sample, interact strongly. This limited and highly specific interaction provides a mechanism for obtaining fluorescence contrast with high spatial resolution. Initial results in which 400 nm resolution is obtained through this AFM/FRET imaging technique are reported. Future modifications in the probe design are discussed to further improve both the fluorescence resolution and imaging capabilities of this new technique.