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.     

Fluorescence in situ hybridization on human metaphase chromosomes detected by near-field scanning optical microscopy

Fluorescence in situ hybridization on human metaphase chromosomes is detected by near-field scanning optical microscopy. This combination of cytochemical and scanning probe techniques enables the localization and identification of several fluorescently labelled genomic DNA fragments on a single chromosome with an unprecedented resolution. Three nucleic acid probes are used: pUC1. 77. p1–79 and the plasmid probe α-spectrin. The hybridization signals are very well resolved in the near-field fluorescence images, while the exact location of the probes can be correlated accurately with the chromosome topography as afforded by the shear force image.     

Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome

Topological analysis of the three‐dimensional (3D) chromatin nanostructure and its function in intact cell nuclei implies the use of high resolution far field light microscopy, e.g. confocal laser scanning microscopy (CLSM). However, experimental evidence indicates that, in practice, under biologically relevant conditions, the spatial resolution of CLSM is limited to about 300 nm in the lateral direction and about 700 nm in the axial direction. To overcome this shortcoming, the use of a recently developed light microscopical approach, spectral precision distance microscopy (SPDM) is established. This approach is based on the precise localization of small labelling sites of a given target in spectrally differential images. By means of quantitative image analysis, the bary centres (intensity weighted centroid analogous to the centre of mass) of these independently registered labelling sites can be used as point markers for distance and angle measurements after appropriate calibration of optical aberrations (here, polychromatic shifts). In combination with specific labelling of very small chromatin target sites with dyes of different spectral signatures by fluorescence in situ hybridization (FISH), SPDM presently allows us to analyse the nuclear topology in three‐dimensionally conserved nuclei with a ‘resolution equivalent’, many times smaller than the conventional optical resolution. Chronic myelogeneous leukaemia (CML) is genetically characterized by the fusion of parts of the BCR and ABL genes on chromosomes 22 and 9, respectively. In most cases, the fusion leads to a translocation t(9; 22) producing the Philadelphia chromosome. SPDM was applied to analyse the 3D chromatin structure of the BCR region on the intact chromosome 22 and the BCR‐ABL fusion gene on the Philadelphia chromosome (Ph) by using a new triple‐colour FISH protocol: two different DNA probes were used to detect the BCR region and the third DNA probe was used to identify the location of the ABL gene. Consistent 3D distance measurements down to values considerably smaller than 100 nm were performed. The angle distributions between the three labelled sites on the Philadelphia chromosome territory were compared to two state‐of‐the‐art computer models of nuclear chromatin structure. Significant differences between measured and simulated angle distributions were obtained, indicating a complex and non‐random angle distribution.     

Current-sensing scanning near-field optical microscopy using a metal probe for nanometre-scale observation of electrochromic films

A novel technique for scanning near‐field optical microscopy capable of point‐contact current‐sensing was developed in order to investigate the nanometre‐scale optical and electrical properties of electrochromic materials. An apertureless bent‐metal probe was fabricated in order to detect optical and current signals at a local point on the electrochromic films. The near‐field optical properties could be observed using the local field enhancement effect generated at the edge of the metal probe under p‐polarized laser illumination. With regard to electrical properties, current signal could be detected with the metal probe connected to a high‐sensitive current amplifier. Using the current‐sensing scanning near‐field optical microscopy, the surface topography, optical and current images of coloured WO₃ thin films were observed simultaneously. Furthermore, nanometre‐scale electrochromic modification of local bleaching could be performed using the current‐sensing scanning near‐field optical microscopy. The current‐sensing scanning near‐field optical microscopy has potential use in various fields of nanometre‐scale optoelectronics.     

Near‐field microscopy and fluorescence spectroscopy: application to chromosomes labelled with different fluorophores

We have coupled a spectrophotometer with a scanning near‐field optical microscope to obtain, with a single scan, simultaneously scanning near‐field optical microscope fluorescence images at different wavelengths as well as topography and transmission images. Extraction of the fluorescence spectra enabled us to decompose the different wavelengths of the fluorescence signals which normally overlap. We thus obtained images of the different fluorescence emissions of acridine orange bound to single or double stranded nucleic acids in human metaphase chromosomes before and after DNAse I or RNAse A treatment. The analysis of these images allowed us to visualize some specific chromatin areas where RNA is associated with DNA showing that such a technique could be used to identify multiple components within a cell.     

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.     

Upconversion fluorescence imaging of erbium-doped fluoride glass particles by apertureless SNOM

We have imaged fluorescent erbium‐doped fluoride glass particles by apertureless scanning near‐field optical microscopy. The optical excitation has been performed at λ = 780 nm whereas fluorescence emission has been collected around λ = 550 nm. This process, called upconversion by energy transfer, involves two erbium ions and is not linear. Besides an improvement of the lateral resolution, we have observed on some particles that the fluorescence is not homogeneously distributed, but is rather localized in some zones brighter than others. By making tip approach curves, we have also observed that the amount of fluorescence intensity scattered by the tip is increasing when the tip is approaching the sample surface.     

Photonic nanopatterns of gold nanostructures indicate the excitation of surface plasmon modes of a wavelength of 50–100 nm by scanning near-field optical microscopy

Scanning near‐field optical microscopy images of metal nanostructures taken with the tetrahedral tip (T‐tip) show a distribution of dark and bright spots at distances in the order of 25–50 nm. The images are interpreted as photonic nanopatterns defined as calculated scanning near‐field optical microscopy images using a dipole serving as a light‐emitting scanning near‐field optical microscopy probe. Changing from a positive to a negative value of the dielectric function of a sample leads to the partition of one spot into several spots in the photonic nanopatterns, indicating the excitation of surface plasmons of a wavelength in the order of 50–100 nm in metal nanostructures.     

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.     

Characterization and fabrication of fully metal-coated scanning near-field optical microscopy SiO2 tips

The fabrication of silicon cantilever‐based scanning near‐field optical microscope probes with fully aluminium‐coated quartz tips was optimized to increase production yield. Different cantilever designs for dynamic‐ and contact‐mode force feedback were implemented. Light transmission through the tips was investigated experimentally in terms of the metal coating and the tip cone‐angle. We found that transmittance varies with the skin depth of the metal coating and is inverse to the cone angle, meaning that slender tips showed higher transmission. Near‐field optical images of individual fluorescing molecules showed a resolution < 100 nm. Scanning electron microscopy images of tips before and after scanning near‐field optical microscope imaging, and transmission electron microscopy analysis of tips before and after illumination, together with measurements performed with a miniaturized thermocouple showed no evidence of mechanical defect or orifice formation by thermal effects.     

Harmonic demodulation and minimum enhancement factors in field‐enhanced near‐field optical microscopy

Field‐enhanced scanning optical microscopy relies on the design and fabrication of plasmonic probes which had to provide optical and chemical contrast at the nanoscale. In order to do so, the scattering containing the near‐field information recorded in a field‐enhanced scanning optical microscopy experiment, has to surpass the background light, always present due to multiple interferences between the macroscopic probe and sample. In this work, we show that when the probe–sample distance is modulated with very low amplitude, the higher the harmonic demodulation is, the better the ratio between the near‐field signal and the interferometric background results. The choice of working at a given n harmonic is dictated by the experiment when the signal at the n + 1 harmonic goes below the experimental noise. We demonstrate that the optical contrast comes from the nth derivative of the near‐field scattering, amplified by the interferometric background. By modelling the far and near field we calculate the probe–sample approach curves, which fit very well the experimental ones. After taking a great amount of experimental data for different probes and samples, we conclude with a table of the minimum enhancement factors needed to have optical contrast with field‐enhanced scanning optical microscopy.     

Nano‐optical reversible switching of organic photochromes*

Photochromic fulgides are UHV‐deposited in ultrathin films exhibiting intrinsic nanostructures. The two isomeric states (C and E) are detected in absorption via scanning near‐field optical microscopy (SNOM). The optical image contrast measured in SNOM corresponds to the nanostructure topography observed in atomic force microscopy. Nano‐optical reversible switching of individual nanostructures is demonstrated and possible applications to rewritable optical recording on the nanoscale are discussed.     

Static and dynamic experiments in cryo-electron microscopy: comparative observations using high-vacuum, low-voltage and low-vacuum SEM

We carried out a unique comparative study between three modes of cryo‐scanning electron imaging: high‐vacuum, low‐voltage and low‐vacuum, using ice cream as a model system. Specimens were investigated both with and without a conductive coating (Au/Pd) and at temperatures for which ice either remains fully frozen (< ?110 °C) or undergoes sublimation (?110 to ?90 °C). At high magnification, high‐vacuum imaging of coated specimens gave the best results for ‘static’ specimens (i.e. containing fully frozen ice). Low voltages, such as 1 kV, could be used for imaging uncoated specimens at high vacuum, although slight ‘classical’ charging artefacts remained an issue, and the reduced electron beam penetration tended to decrease the definition between different microstructural features. However, this mode was useful for observing in situ sublimation from uncoated specimens. Low‐vacuum mode, involving small partial pressures of nitrogen gas, was particularly suited to in situ sublimation work: when sublimation was carried out in low vacuum in the absence of an anti‐contaminator plate, sublimation rates were significantly reduced. This is attributed to a small partial pressure of sublimated water vapour remaining near the specimen surface, enhancing thermodynamic stability.     

Large-area topography analysis and near-field Raman spectroscopy using bent fibre probes

We present a method for combined far‐field Raman imaging, topography analysis and near‐field spectroscopy. Surface‐enhanced Raman spectra of Rhodamine 6G (R6G) deposited on silver nanoparticles were recorded using a bent fibre aperture‐type near‐field scanning optical microscope (NSOM) operated in illumination mode. Special measures were taken to enable optical normal‐force detection for control of the tip–sample distance. Comparisons between far‐field Raman images of R6G‐covered Ag particle aggregates with topographic images recorded using atomic force microscopy (AFM) indicate saturation effects due to resonance excitation.     

Apertureless scanning near-field optical microscopy for ion exchange channel waveguide characterization

We report the characterization of an integrated Ag⁺/Na⁺ ion exchange waveguide realized in a silicate glass substrate using apertureless scanning near‐field optical microscopy. Our experimental set‐up is based on the combination of a commercial atomic force microscope with an optical confocal detection system. Thanks to this system, the topography and evanescent optical field at the waveguide top surface are mapped simultaneously. Also, the process of apertureless scanning near‐field optical microscopy image formation is analysed. In particular, fringe patterns appearing in the image reveal the intrinsic interferometric nature of the collected signal, due to interference between the field scattered by the tip end and background fields related to guide losses. We give a quantitative interpretation of these fringes. Evanescent intensity mapping on the sample surface allowed us to extract physical waveguide parameters. In particular, it shows an unambiguous multimode beat along the waveguide propagation axis. Furthermore, we show that analysis of this intensity profile reveals back‐reflection effects from the waveguide exit facet. The resulting standing waves pattern allows us to evaluate the eigenmode propagation constants.     

Human teeth biobank: Microbiological analysis of the teeth storage solution

The cross‐infections may occur during handling of dental elements, affecting the health of dental practitioners and researchers. This study aimed to analyze the influence of the storage medium temperature on the bacterial contingent of the human teeth used for research purposes. Thirty human teeth were donated to the Human Teeth Biobank immediately after extraction. The teeth were cleaned with tap water and neutral soap. The teeth were randomly distributed according to the temperature of the storage solution (deionized water): at 4 °C (refrigerator) or at ?10 °C (freezer) and were stored individually in sterile vials during 60 days. After this period, a microbiological analysis (CFU/mL) of the storage solutions was performed and teeth were submitted to SEM analysis. Data were analyzed by Kruskal–Wallis test followed by Dunn's post‐test (p ≤ .05). Total aerobic bacteria ranged from 5.8 to 8.4 log₁₀ CFU/mL for refrigerated solution and from 1.9 to 8.5 log₁₀ CFU/mL for frozen solution. No statistical differences were found between the storage solutions (p > .05). The counts of Streptococcus spp., Lactobacillus spp., and Staphylococcus spp. were similar for both storage solutions (p > .05). SEM analysis showed spiral‐ and rod‐shaped bacteria attached on teeth stored under 4 °C, which may suggest the presence of Treponema spp. and Lactobacillus spp. Similar morphological forms were found on teeth stored under ?10 °C. A biofilm organized in honeycomb‐like form was found in the frozen teeth. Cocci were eventually found in all the samples. It was concluded that bacterial growth and survival were not influenced by the temperature of the teeth storage solution.     

Phase separation in polyfluorene – polymethylmethacrylate blends studied using UV near-field microscopy

In this paper we present a near‐field microscopy study of thin films of a phase‐separated blend of the fluorescent conjugated‐polymer poly(9,9‐dioctylfluorene) [PFO] with the non‐fluorescent polymer polymethylmethacrylate [PMMA]. A scanning near‐field optical microscope (NSOM) was used to generate (blue) fluorescence from the PFO following UV excitation at 362 nm. A range of different concentrations of PFO in PMMA were studied ranging from 1 to 50% PFO in PMMA by mass. By studying both the shear force and fluorescence images we were able accurately to determine the distribution of PFO in the PMMA. We found that phase separation occurs over a number of different length‐scales between 5 µm and 250 nm. We show that at PFO concentrations of 1%, the PFO lies on top of the PMMA. At a PFO relative concentration of 50%, the PMMA phase extends through the whole thickness of the film to the underlying substrate. We use such samples to discuss the resolution of NSOM when imaging thick organic films. Furthermore, we confirm that the length‐scales of phase separation can be modified via control over spin‐casting protocols.     

Mapping cholesteryl ester analogue uptake and intracellular flow in Paramecium by confocal fluorescence microscopy

In Paramecium primaurelia the uptake and intracellular flow of cholesteryl ester was studied by fluorescence confocal laser scanning optical microscopy and by the fluorescent analogue cholesteryl‐BODIPY^® FL C₁₂ (BODIPY‐CE). The BODIPY FL fluorophore has the characteristic of emitting green fluorescence, which is red‐shifted as the probe concentrates. In cells incubated with 25 µm BODIPY‐CE for 30 s, fluorescence is found in vesicles located around the cytopharynx in the posterior half of the cell. Successively, the lipid is internalized by food vacuoles, the fluorescent vesicles are distributed throughout the cell and the intracellular membranes are labelled. The food vacuole number is maximum after 10–15 min of continuous labelling, then it decreases until no food vacuoles are found in 30‐min fed cells. BODIPY‐CE accumulates in red‐labelled cytoplasmic droplets located in the anterior half of the cell. When food vacuole formation is inhibited by trifluoperazine, fluorescence is found on cellular membranes and in small green‐labelled vesicles at the apical pole. The inhibition of clathrin‐mediated endocytosis does not interfere in P. primaurelia with BODIPY‐CE intracellular flow: intracellular membranes and storage droplets in the cell anterior part are dyed. Conversely, the use of sterol‐binding drugs prevents the lipid accumulation in droplets, stopping the lipid within the cytoplasmic membranes. Furthermore, the cells treated with monensin and cytochalasin B show a labelling of the cellular membranes and lipid droplets, whereas NH₄Cl reduces the lipid storage. Low temperature (4 °C) does not prevent the internalization of BODIPY‐CE that, however, is localized at the cytoplasmic membrane level and does not accumulate in storage droplets. In addition, BODIPY‐CE inhibits phagocytosis, as evidenced by comparing the kinetics of food vacuole formation of control cells, only fed with latex particles, with that of cells fed with latex particles and BODIPY‐CE. In conclusion, this study points out that in P. primaurelia the cholesteryl ester enters the cell via food vacuoles and through the plasma membrane and, inside the cell, it alters cell functions.     

Near-field imaging of surface plasmon on gold nano-dots fabricated by scanning probe lithography

We obtained scanning near‐field optical microscopy images to study the excitation of surface plasmons on metallic dots fabricated using scanning probe lithography. Gold nano‐dots were fabricated by applying electric voltages to conducting probes installed in an atomic force microscope using the mechanism of field‐induced diffusion and nano‐oxidation plus Au‐coating. High spatial resolution of scanning near‐field optical microscopy revealed a ‘bifold’ pattern of surface plasmon mode on fabricated Au dots in the polarization direction of incident light. We found that scanning near‐field optical microscopy imaging combined with scanning probe lithography is able to provide a systematic study of surface plasmon excitation on nano‐metallic structures.