Scanning acoustic Doppler microscopy and scanning acoustic correlation microscopy

Acoustic microscopy with vector contrast at 100 MHz in a fluid with immersed particles is used to detect the flow profile in front of a microscopic orifice. The velocity profile concerning the component in axial direction of the focused beam is derived from the phase contrast. Possibilities to resolve the flow profile also for the components in normal direction with respect to the axis are demonstrated. The methods concerning measurement techniques and data evaluation for scanning acoustic Doppler microscopy are presented. For scanning acoustic correlation microscopy the time dependent phase and amplitude signals resulting from sound waves scattered by the immersed particles (aluminium flakes with a typical diameter of 10 microm) have been analysed by correlation procedures. From the obtained autocorrelation functions the velocity distribution can be derived. Both methods can be applied simultaneously. Data analysis is based on the information contained in the originally obtained images in vector contrast derived from temporal and spatial resolved analogue and digital processing of the acoustic signals.     

Application of pulse acoustic microscopy technique for 3D imaging bulk microstructure of carbon fiber-reinforced composites

Impulse acoustic microscopy technique is applied for 3D imaging of bulk microstructure of composite materials. Short pulses of focused high-frequency ultrasound have been employed for layer-by-layer imaging of internal microstructure of carbon fiber-reinforced composite (CFRC) laminates. The method provides spatial resolution of 60 microm and in-depth resolution of 80 microm, approximately. Echo signals reflected from structural units--plies, fiber bundles; and microflaws form acoustic images of microstructure at different depth inside samples. The images make it possible to see ply arrays, packing of bundles in plies; binding material distribution over the specimen body. They reveal failure of interply adhesion, buckling of single plies and fiber bundles, internal defoliations and disbonds, voids in the specimen body. The series of successive images offer outstanding possibilities to reconstruct the bulk structure, to estimate local variations of properties, topological and geometrical characteristics of structural components. The imaging technique has been applied to study different types of fiber packing--unidirectional, cross-ply and woven laminates. Mechanisms of ultrasonic contrast for diverse elements in acoustic images of CFRC laminate bulk microstructure and structural defects are discussed.     

Phase-sensitive acoustic microscopy of polymer thin films

The three-dimensional images obtained by scanning acoustic microscopy with vector contrast (PSAM), contain significant qualitative and quantitative information that is not easily obtainable by other methods. We employ this technique to examine homopolymer and polymer blend thin films. The complex V(z) functions derived from the images, and the results obtained by image processing and meticulous analysis are employed to render the morphology, composition and micro-mechanical properties of the polymer films. In addition, ways by which the information inherent in the phase images can be extracted are examined. This is highly desirable, as the phase images contain very useful additional information.     

Use of confocal laser scanning microscopy (CLSM) for depthwise resolved microscale-particle image velocimetry (μ-PIV)

A novel use of confocal laser scanning microscopy (CLSM) makes the truly focused field-of-view with well-defined depthwise resolution possible for microscale particle image velocimetry (μ-PIV) applications. The operating principle of the CLSM is presented using the point spread function (PSF) that describes diffracted images of extremely small particles. The implemented high-speed CLSM system using a Nipkow rotating disk is applied to measure the microscale rotating Couette flow field confined between two parallel horizontal disks that are 180-μm apart, with the bottom one stationary and the top one rotating and seeded by 200-nm fluorescent spheres. The CLSM provides much distinct particle images in comparison with the conventional wide-field microscopy (WFM) and the measured vector profiles are more concentric and accurate depicting closer to an ideal Couette flow.     

Non-invasive and low-artifact in vivo brain imaging by using a scanning acoustic-photoacoustic dual mode microscopy

Photoacoustic imaging is a potential candidate for in vivo brain imaging, whereas, its imaging performance could be degraded by inhomogeneous multi-layered media, consisted of scalp and skull. In this work, we propose a low-artifact photoacoustic microscopy (LAPAM) scheme, which combines conventional acoustic-resolution photoacoustic microscopy with scanning acoustic microscopy to suppress the reflection artifacts induced by multi-layers. Based on similar propagation characteristics of photoacoustic signals and ultrasonic echoes, the ultrasonic echoes can be employed as the filters to suppress the reflection artifacts to obtain low-artifact photoacoustic images. Phantom experiment is used to validate the effectiveness of this method. Furthermore, LAPAM is applied for in-vivo imaging mouse brain without removing the scalp and the skull. Experimental results show that the proposed method successfully achieves the low-artifact brain image, which demonstrates the practical applicability of LAPAM. This work might improve the photoacoustic imaging quality in many biomedical applications which involve tissues with complex acoustic properties, such as brain imaging through scalp and skull.     

Dark-field optical coherence microscopy

Dark-field illumination is known to enhance scattering contrast in optical microscopy. We combined this concept with Fourier domain optical coherence microscopy (OCM). The detection and illumination paths are decoupled, and only the scattered light originating from the sample generates the tomogram signal, whereas any specular reflection is highly suppressed. We analyze and discuss this dark-field OCM concept and present its superior imaging quality on live cell samples.     

Internal reflection mode scanning near-field optical microscopy with the tetrahedral tip on metallic samples

An internal reflection mode is introduced for scanning near-field optical microscopy (SNOM) with the tetrahedral tip. A beam of light is coupled into the tip and the light specularly reflected out of the tip is detected as a photosignal for SNOM. An auxiliary STM mode is used to control the distance during the scanning process and to record the topography of the sample simultaneously with the SNOM image. Images were obtained of different metallic samples which show a contrast in the order of 10% of the total reflected photosignal. In images of metallic samples an inverted contrast is consistently obtained compared to images previously obtained of comparable samples in a transmission mode. The contrast shows a pronounced dependence on the polarization of the incident beam with respect to the orientation of the edges of the tip. In the case of gold surfaces, the photosignal as a function of distance between the tip and the surface shows a pronounced peak in the near-field range of 0–20 nm which is tentatively attributed to the excitation of surface plasmons on the gold surface. The pronounced near-field effects and the strong contrast in the near-field images and the resolution well below 50 nm are an indication of a highly efficient coupling of the incident beam to a local excitation of the tip apex which is essential for the function of the tip as a probe for SNOM. Received: 17 May 1999 / Accepted: 18 May 1999 / Published online: 21 October 1999     

基于双螺旋点扩散函数工程的多焦点图像扫描显微

在传统共聚焦显微技术的基础上,图像扫描显微技术使用面阵探测器来代替单点探测器,结合虚拟数字针孔并利用像素重定位和解卷积图像重构算法将传统宽场显微镜的分辨率提高一倍,实现了高信噪比的超分辨共焦成像.但是,由于采用逐点扫描的方式,三维成像速度相对较慢,限制了其在活体样品成像中的应用.为了进一步提高图像扫描显微术的成像速度,本文提出了一种基于双螺旋点扩散函数工程的多焦点图像扫描显微成像方法和系统.在照明光路中,利用高速数字微镜器件产生周期分布的聚焦点阵对样品进行并行激发和快速二维扫描;在探测光路中,利用双螺旋相位片将激发点荧光信号的强度分布转换为双螺旋的形式;最终,利用后期数字重聚焦处理,从单次样品扫描数据中重构出多个样品层的超分辨宽场图像.在此基础上,利用搭建的系统分别对纤维状肌动蛋白和海拉细胞线粒体进行成像实验,证明了该方法的超分辨能力和快速三维成像能力.     

Scanning ion conductance microscopy for imaging biological samples in liquid: a comparative study with atomic force microscopy and scanning electron microscopy

The present study was designed to show the applicability of scanning ion conductance microscopy (SICM) for imaging different types of biological samples. For this purpose, we first applied SICM to image collagen fibrils and showed the usefulness of the approach-retract scanning (ARS)/hopping mode for such samples with steep slopes. Comparison of SICM images with those obtained by AFM revealed that the ARS/hopping SICM mode can probe the surface topography of collagen fibrils and chromosomes at nanoscale resolution under liquid conditions. In addition, we successfully imaged cultured HeLa cells, with 15 μm in height by ARS/hopping SICM mode. Because SICM can obtain non-contact (or force-free) images, delicate cellular projections were visualized on the surface of the fixed cell. SICM imaging of live HeLa cells further demonstrated its applicability to study the morphological dynamics associated with biological processes on the time scale of minutes under liquid conditions. We further applied SICM for imaging the luminal surface of the trachea and succeeded in visualizing the surface of both ciliated and non-ciliated cells. These SICM images were comparable with those obtained by scanning electron microscopy. Although the dynamic mode of AFM provides better resolution than the ARS/hopping mode of SICM in some samples, only the latter can obtain contact-free images of samples with steep slopes, rendering it an important tool for observing live cells as well as unfixed or fixed soft samples with complicated shapes. Taken together, we demonstrate that SICM imaging, especially using an ARS/hopping mode, is a useful technique with unique capabilities for imaging the three-dimensional topography of a range of biological samples under physiologically relevant aqueous conditions.     

Photoacoustic microscopy

Photoacoustic microscopy (PAM) is a hybrid in vivo imaging technique that acoustically detects optical contrast via the photoacoustic effect. Unlike pure optical microscopic techniques, PAM takes advantage of the weak acoustic scattering in tissue and thus breaks through the optical diffusion limit (∼1 mm in soft tissue). With its excellent scalability, PAM can provide high‐resolution images at desired maximum imaging depths up to a few millimeters. Compared with backscattering‐based confocal microscopy and optical coherence tomography, PAM provides absorption contrast instead of scattering contrast. Furthermore, PAM can image more molecules, endogenous or exogenous, at their absorbing wavelengths than fluorescence‐based methods, such as wide‐field, confocal, and multi‐photon microscopy. Most importantly, PAM can simultaneously image anatomical, functional, molecular, flow dynamic and metabolic contrasts in vivo. Focusing on state‐of‐the‐art developments in PAM, this Review discusses the key features of PAM implementations and their applications in biomedical studies.     

Confocal laser scanning microscopy and scanning electron microscopy of tissue Ti-implant interfaces.

Microscopic inspection of heterogenous three-dimensional (3D) objects such as oral implants, or implants in general, is conventionally performed either on ground sections of methyl-metacrylate-embedded material, at the cellular level by histologic analysis of the peri-implant tissue by light microscopy (LM), or at the supramolecular level by transmission electron microscopy (TEM). Alternatively, the architecture of the tissue/implant interface is visualized by scanning electron microscopy (SEM). The two approaches exclude each other because of the sample preparation.We elaborate conditions for the non-invasive analysis of tissue/implant interfaces by confocal laser scanning microscopy (CLSM) in buffer, hoping to obtain a 3D view of fluorescently labeled tissue constituents at the tissue implant interface and, through subsequent SEM, of the metal surface. The use of water-immersion objectives, originally developed for high LM under physiological conditions is essential.In an exploratory approach, the tissue/Ti-interfaces of two retrieved dental implants were analyzed. One was a step-cylinder used for orthodontic anchoring and the other was an endosseous step-screw implant retrieved after infection-related loosening prior to load. The adhering tissue fragments were fluorescently triple-labeled for actin, fibronectin, and sm-alpha-actin. Optical sections for fluorescent images and for the laser reflection map were registered concomitantly. This approach allowed the labeled structures to be located on the metal surface. Subsequently, the same implants were prepared for SEM of the tissue/implant interface, and upon removal of the adhering structures, of the underlying metal surface. Thus, specific proteins can be identified and their spatial architecture as well as that of the underlying metal surface can be visualized for one and the same implant. The immediate visualization after fluorescence labeling in buffer by means of water immersion objective lenses proved most critical.     

Studies of imaging experiment for photon scanning tunneling microscopy

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Zernike phase contrast in scanning microscopy with X-rays

Scanning X-ray microscopy focuses radiation to a small spot and probes the sample by raster scanning. It allows information to be obtained from secondary signals such as X-ray fluorescence, which yields an elemental mapping of the sample not available in full-field imaging. The analysis and interpretation from these secondary signals can be considerably enhanced if these data are coupled with structural information from transmission imaging. However, absorption often is negligible and phase contrast has not been easily available. Originally introduced with visible light, Zernike phase contrast(1) is a well-established technique in full-field X-ray microscopes for visualization of weakly absorbing samples(2-7). On the basis of reciprocity, we demonstrate the implementation of Zernike phase contrast in scanning X-ray microscopy, revealing structural detail simultaneously with hard-X-ray trace-element measurements. The method is straightforward to implement without significant influence on the resolution of the fluorescence images and delivers complementary information. We show images of biological specimens that clearly demonstrate the advantage of correlating morphology with elemental information.     

Quantitative optical phase microscopy

We present a new method for the extraction of quantitative phase data from microscopic phase samples by use of partially coherent illumination and an ordinary transmission microscope. The technique produces quantitative images of the phase profile of the sample without phase unwrapping. The technique is able to recover phase even in the presence of amplitude modulation, making it significantly more powerful than existing methods of phase microscopy. We demonstrate the technique by providing quantitatively correct phase images of well-characterized test samples and show that the results obtained for more-complex samples correlate with structures observed with Nomarski differential interference contrast techniques.     

Visualization of human umbilical vein endothelial cells by acoustic microscopy

The morphology and acoustic properties of the human umbilical vein endothelial cells (HUVECs) were evaluated using a scanning acoustic microscope system. HUVECs were cultured for 4 days and exposed to the endotoxin for 4 h. The frequency of the scanning acoustic microscope was variable between 100 and 210 MHz. By changing the measuring frequency, ultrasonic amplitude and phase were measured and the quantitative value of attenuation was calculated. Before and after endotoxin stimuli, HUVECs were observed by scanning acoustic microscopy and the attenuation was measured. The acoustic images were successfully obtained to identify the outer shape of the HUVEC and the location of the nucleus in the cell. The attenuation of the nucleus is higher than that of the cytoplasm. The attenuation of the cytoplasm was increased and became inhomogeneous after endotoxin exposure. This finding would be related to the change of F-actin filaments, which is the main component of the cytoskeleton. Scanning acoustic microscopy is useful for assessing the cellular viscoelastic properties since it can detect both the morphological and acoustic changes without contacting the cellular surface.     

Multicontrast nonlinear optical microscopy with a compact and rapid pulse shaper

Homodyne detection can dramatically enhance measurement sensitivity for weak signals. In nonlinear optical microscopy it can make accessible a range of novel, intrinsic, contrast like nonlinear absorption and nonlinear phase contrast. Here a compact and rapid pulse shaper is developed, implemented, and demonstrated for homodyne detection in nonlinear microscopy with high-repetition rate mode-locked femtosecond lasers. With this method we generate two-photon absorption (TPA) and self-phase modulation images of gold nanostars in biological samples. Simultaneous imaging of two-photon luminescence and TPA also enables us to produce two-photon quantum yield images.     

Reflection and mode conversion of surface acoustic waves studied by scanning acoustic force microscopy

We present measurements of the reflection and mode conversion of surface acoustic waves (SAWs) by scanning acoustic force microscopy (SAFM). The SAFM offers a unique combination of high lateral resolution and high sensitivity towards acoustic modes of all polarizations. Since a SAW mixing experiment of two waves can be performed even if the amplitude difference between both waves is 40 dB, wavefields of extremely small amplitudes can be investigated. Using SAFM, the reflection of SAWs from a metallic wedge is investigated with submicron lateral resolution. We are able to identify two reflected wave modes, a Love and a non-coupling Rayleigh mode, by measuring their phase velocities. Received: 4 December 2000 / Accepted: 6 December 2000 / Published online: 9 February 2001     

Image contrast in X‐ray reflection interface microscopy: comparison of data with model calculations and simulations

The contrast mechanism for imaging molecular‐scale features on solid surfaces is described for X‐ray reflection interface microscopy (XRIM) through comparison of experimental images with model calculations and simulated measurements. Images of elementary steps show that image contrast is controlled by changes in the incident angle of the X‐ray beam with respect to the sample surface. Systematic changes in the magnitude and sign of image contrast are asymmetric for angular deviations of the sample from the specular reflection condition. No changes in image contrast are observed when defocusing the condenser or objective lenses. These data are explained with model structure‐factor calculations that reproduce all of the qualitative features observed in the experimental data. These results provide new insights into the image contrast mechanism, including contrast reversal as a function of incident angle, the sensitivity of image contrast to step direction (i.e. up versus down), and the ability to maximize image contrast at almost any scattering condition defined by the vertical momentum transfer, Q_z. The full surface topography can then, in principle, be recovered by a series of images as a function of incident angle at fixed momentum transfer. Inclusion of relevant experimental details shows that the image contrast magnitude is controlled by the intersection of the reciprocal‐space resolution function (i.e. controlled by numerical aperture of the condenser and objective lenses) and the spatially resolved interfacial structure factor of the object being imaged. Together these factors reduce the nominal contrast for a step near the specular reflection condition to a value similar to that observed experimentally. This formalism demonstrates that the XRIM images derive from limited aperture contrast, and explains how non‐zero image contrast can be obtained when imaging a pure phase object corresponding to the interfacial topography.     

Reflection-scanning near-field optical microscopy and spectroscopy of opaque samples

Opaque samples are imaged by Scanning Nearfield Optical Microscopy (SNOM) in reflection mode: A quartz glass fiber tip is used both to illuminate the sample and to collect light locally reflected from or emitted by the surface. The collected light is coupled out by a 2×2 fiber coupler and fed into a grating spectrometer for spectral analysis at each sampled point. The tip-sample distance is controlled by a shear-force feedback system. The simultaneous measurement of topography and optical signals allows an assessment of imaging artifacts, notably topography-induced intensity changes. It is demonstrated that an optical reflectance contrast not induced by topographic interference can be found on suitable samples. Local spectral analysis is shown in images of a photoluminescent layer.