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.     

Hybrid scanning ion conductance and scanning near-field optical microscopy for the study of living cells

We have developed a hybrid scanning ion conductance and scanning near-field optical microscope for the study of living cells. The technique allows quantitative, high-resolution characterization of the cell surface and the simultaneous recording of topographic and optical images. A particular feature of the method is a reliable mechanism to control the distance between the probe and the sample in physiological buffer. We demonstrate this new method by recording near-field images of living cells (cardiac myocytes).     

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.     

Picosecond multiphoton scanning near-field optical microscopy.

We have implemented simultaneous picosecond pulsed two- and three-photon excitation of near-UV and visible absorbing fluorophores in a scanning near-field optical microscope (SNOM). The 1064-nm emission from a pulsed Nd:YVO4 laser was used to excite the visible mitochondrial specific dye MitoTracker Orange CM-H2TMRos or a Cy3-labeled antibody by two-photon excitation, and the UV absorbing DNA dyes DAPI and the bisbenzimidazole BBI-342 by three-photon excitation, in a shared aperture SNOM using uncoated fiber tips. Both organelles in human breast adenocarcinoma cells (MCF 7) and specific protein bands on polytene chromosomes of Drosophila melanogaster doubly labeled with a UV and visible dye were readily imaged without photodamage to the specimens. The fluorescence intensities showed the expected nonlinear dependence on the excitation power over the range of 5-40 mW. An analysis of the dependence of fluorescence intensity on the tip-sample displacement normal to the sample surface revealed a higher-order function for the two-photon excitation compared to the one-photon mode. In addition, the sample photobleaching patterns corresponding to one- and two-photon modes revealed a greater lateral confinement of the excitation in the two-photon case. Thus, as in optical microscopy, two-photon excitation in SNOM is confined to a smaller volume.     

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.     

Continuous wave two-photon scanning near-field optical microscopy.

We have implemented continuous-wave two-photon excitation of near-UV absorbing fluorophores in a scanning near-field optical microscope (SNOM). The 647-nm emission of an Ar-Kr mixed gas laser was used to excite the UV-absorbing DNA dyes DAPI, the bisbenzimidazole Hoechst 33342, and ethidium bromide in a shared aperture SNOM with uncoated fiber tips. Polytene chromosomes of Drosophila melanogaster and the nuclei of 3T3 Balb/c cells labeled with these dyes were readily imaged. The fluorescence intensity showed the expected nonlinear (second order) dependence on the excitation power in the range of 8-180 mW. We measured the fluorescence intensity as a function of the tip-sample displacement in the direction normal to the sample surface in the single- and two-photon excitation modes (SPE, TPE). The fluorescence intensity decayed faster in TPE than in SPE.     

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.     

A scanning near-field optical microscope approach to biomolecule patterning

In view of future generations of biosensors and advanced biomaterials, photochemistry in the near field using scanning near-field optical microscopy is investigated. The potential of direct near-field-induced photoactivation is demonstrated on standard photoresist. Photoimmobilization of maleimidoaryldiazirine on silicon substrates and bovine serum albumin on glass substrates is achieved, opening the way to a controlled biopatterning of surfaces with submicrometer feature size. The obtained patterns are characterized using atomic force microscopy, time-of-flight secondary ion mass spectroscopy (ToF-SIMS), and near-field fluorescence microscopy.     

Atomic force microscopy and scanning near-field optical microscopy studies on the characterization of human metaphase chromosomes

A better knowledge of biochemical and structural properties of human chromosomes is important for cytogenetic investigations and diagnostics. Fluorescence in situ hybridization (FISH) is a commonly used technique for the visualization of chromosomal details. Localizing specific gene probes by FISH combined with conventional fluorescence microscopy has reached its limit. Also, microdissecting DNA from G-banded human metaphase chromosomes by either a glass tip or by laser capture needs further improvement. By both atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM), local information from G-bands and chromosomal probes can be obtained. The final resolution allows a more precise localization compared to standard techniques, and the extraction of very small amounts of chromosomal DNA by the scanning probe is possible. Besides new strategies towards a better G-band and fluorescent probe detection, this study is focused on the combination of biochemical and nanomanipulation techniques which enable both nanodissection and nanoextraction of chromosomal DNA.     

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.     

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.     

Chemically resolved imaging of biological cells and thin films by infrared scanning near-field optical microscopy

The infrared (IR) absorption of a biological system can potentially report on fundamentally important microchemical properties. For example, molecular IR profiles are known to change during increases in metabolic flux, protein phosphorylation, or proteolytic cleavage. However, practical implementation of intracellular IR imaging has been problematic because the diffraction limit of conventional infrared microscopy results in low spatial resolution. We have overcome this limitation by using an IR spectroscopic version of scanning near-field optical microscopy (SNOM), in conjunction with a tunable free-electron laser source. The results presented here clearly reveal different chemical constituents in thin films and biological cells. The space distribution of specific chemical species was obtained by taking SNOM images at IR wavelengths (lambda) corresponding to stretch absorption bands of common biochemical bonds, such as the amide bond. In our SNOM implementation, this chemical sensitivity is combined with a lateral resolution of 0.1 micro m ( approximately lambda/70), well below the diffraction limit of standard infrared microscopy. The potential applications of this approach touch virtually every aspect of the life sciences and medical research, as well as problems in materials science, chemistry, physics, and environmental research.     

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°.     

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.     

High-speed near-field fluorescence microscopy combined with high-speed atomic force microscopy for biological studies

BackgroundHigh-speed atomic force microscopy (HS-AFM) has successfully visualized a variety of protein molecules during their functional activity. However, it cannot visualize small molecules interacting with proteins and even protein molecules when they are encapsulated. Thus, it has been desired to achieve techniques enabling simultaneous optical/AFM imaging at high spatiotemporal resolution with high correlation accuracy.MethodsScanning near-field optical microscopy (SNOM) is a candidate for the combination with HS-AFM. However, the imaging rate of SNOM has been far below that of HS-AFM. We here developed HS-SNOM and metal tip-enhanced total internal reflection fluorescence microscopy (TIRFM) by exploiting tip-scan HS-AFM and exploring methods to fabricate a metallic tip on a tiny HS-AFM cantilever.ResultsIn tip-enhanced TIRFM/HS-AFM, simultaneous video recording of the two modalities of images was demonstrated in the presence of fluorescent molecules in the bulk solution at relatively high concentration. By using fabricated metal-tip cantilevers together with our tip-scan HS-AFM setup equipped with SNOM optics, we could perform simultaneous HS-SNOM/HS-AFM imaging, with correlation analysis between the two overlaid images being facilitated.ConclusionsThis study materialized simultaneous tip-enhanced TIRFM/HS-AFM and HS-SNOM/HS-AFM imaging at high spatiotemporal resolution. Although some issues remain to be solved in the future, these correlative microscopy methods have a potential to increase the versatility of HS-AFM in biological research.General significanceWe achieved an imaging rate of ~3 s/frame for SNOM imaging, more than 100-times higher than the typical SNOM imaging rate. We also demonstrated ~39 nm resolution in HS-SNOM imaging of fluorescently labeled DNA in solution.     

Fine structure in near-field and far-field laser diffraction patterns from skeletal muscle fibers.

Regions of muscle fibers that are many sarcomeres in length and uniform with regard to striation spacing, curvature, and tilt have been observed by light microscopy. We have investigated the possibility that these sarcomere domains can explain the fine structure in optical diffraction patterns of skeletal muscle fibers. We studied near-field and far-field diffraction patterns with respect to fiber translation and to masking of the laser beam. The position of diffracted light in the near-field pattern depends on sarcomere length and position of the diffracting regions within the laser beam. When a muscle fiber was translated longitudinally through a fixed laser beam, the fine structural lines in the near-field diffraction pattern moved in the same direction and by the same amount as the fiber movement. Translation of the muscle fiber did not result in fine structure movement in the far-field pattern. As the laser beam was incrementally masked from one side, some fine structural lines in both the near-field and far-field diffraction patterns changed in intensity while others remained the same. Eventually, all the fine structural lines broadened and decreased in intensity. Often a fine structural line increased in intensity or a dark area in the diffraction pattern became brighter as the laser beam was restricted. From these results we conclude that the fine structure in the laser diffraction pattern is due to localized and relatively uniform regions of sarcomeres (domains) and to cross interference among light rays scattered by different domains.     

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.     

Imaging brain tissue slices with terahertz near-field microscopy

Photoconductive antenna microprobe (PCAM)-based terahertz (THz) near-field imaging technique is promising for biomedical detection due to its excellent biocompatibility and high resolution; yet it is limited by its imaging speed and the difficulty in the control of the PCAM tip-sample separation. In this work, we successfully realized imaging of mouse brain tissue slices using an improved home-built PCAM-based THz near-field microscope. In this system, the imaging speed was enhanced by designing and applying a voice coil motor-based delay-line. The tip-sample separation control was implemented by developing an image analysis-based technique. Compared with conventional PCAM-based THz near-field systems, our improved system is 100 times faster in imaging speed and the tip-sample separation can be controlled to a few micrometers (e.g., 3 μm), satisfying the requirements of THz near-field imaging of biological samples. It took about ~30 min (not the tens of hours it took to acquire the same kind of image previously) to collect a THz near-field image of brain tissue slices of BALb/c mice (500 μm × 500 μm) with pixel size of 20 μm × 20 μm. The results show that the mouse brain slices can be properly imaged and different regions in the slices (i.e., the corpus callosum region and the cerebrum region) can be identified unambiguously. Evidently, the work demonstrated here provides not only a convincing example but a useful technique for imaging biological samples with THz near-field microscopy. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2741, 2019.     

Electromagnetic absorption in a multilayered slab model of tissue under near-field exposure conditions

The electromagnetic energy deposited in a semi-infinite slab model consisting of skin, fat, and muscle layers is calculated for both plane-wave and near-field exposures. The plane-wave spectrum (PWS) approach is used to calculate the energy deposited in the model by fields present due to leakage from equipment using electromagnetic energy. This analysis applies to near-field exposures where coupling of the target to the leakage source can be neglected. Calculations were made for 2,450 MHz, at which frequency the layered slab adequately models flat regions of the human body. Resonant absorption due to layering is examined as a function of the skin and fat thicknesses for plane-wave exposure and as a function of the physical extent of the near-field distribution. Calculations show that for fields that are nearly constant over at least a free-space wavelength, the energy deposition (for the skin, fat, and muscle combination that gives resonant absorption) is equal to or less than that resulting from plane-wave exposure, but is appreciably greater than that obtained for a homogeneous muscle slab model.     

Mode Controlling of Surface Plasmon Polaritons by Geometric Phases

Metasurfaces are made of two-dimensional arrays of subwavelength nanostructures that form a spatially varying optical response, to control the wave fronts of optical waves. As the feature size of its constituent materials is nanoscale, investigation of the light-nanostructure interactions in the near field is critical for understanding the novel properties of metasurfaces. Here, we used a scanning near-field optical microscope (SNOM) to observe the near-field distribution of surface plasmon polaritons (SPPs) from a ring-shaped metasurface under illumination of circularly polarized light. It was found that with an additional degree of freedom of the geometric phase provided by the regularly arranged metamolecules, control over the near-field interference of the SPPs can be achieved, which is governed by the metasurface geometric symmetry that can be tuned by its topological charge. Meanwhile, the planar chiral character of the metamolecules exerts a deep influence on the near-field interference patterns. Our results can pave the way for active control of SPP propagation in near fields and have potential applications in highly integrated optical communication systems.