Optical microscopy with improved resolution using two-beam interference of low-coherence light

In recent years, high-resolution microscopy using structured illumination has been practically applied for fluorescent bio-imaging. However, there is a large amount of speckle noise in reflected- and scattered-light images, because structured illumination is typically generated by laser-beam interference. Hence, this high-resolution imaging technique cannot be effectively used in industrial applications. In this study, we attempted to generate structured illumination using two-beam interference of low-coherence light for high-resolution and low-speckle imaging. First, we constructed an optical system consisting of a Michelson interferometer configured in such a manner that it achieved zero optical path-length difference and allowed the interference fringes to be manipulated. Then, we confirmed that the generated structured illumination width corresponded to the coherence length of the light source. As a final result of the resolution improvement experiment, the narrow sample pitch of 0.4 μm was successfully resolved beyond the diffraction limit of 0.74 μm with relatively less speckle noise.     

Real-time phase microscopy using a phase-lock interferometer

A phase-lock interference microscope (PLIM) has been designed and constructed. The instrument measures optical phase in real time with analog output voltages proportional to phase and position. The PLIM has been used to measure gradient index profiles in optical fibers, wall thicknesses of microballoons for laser induced fusion, the phase of biological specimens, and the surface finish of optical surfaces. The peak-to-peak noise of the instrument is lambda/50.     

Atomic force microscopy: loading position dependence of cantilever spring constants and detector sensitivity

A simple and accurate experimental method is described for determining the effective cantilever spring constant and the detector sensitivity of atomic force microscopy cantilevers on which a colloidal particle is attached. By attaching large (approximately 85 microm diameter) latex particles at various positions along the V-shaped cantilevers, we demonstrate how the normal and lateral spring constants as well as the sensitivity vary with loading position. Comparison with an explicit point-load theoretical model has also been used to verify the accuracy of the method.     

Linear phase imaging using differential interference contrast microscopy

We propose an extension to Nomarski differential interference contrast microscopy that enables isotropic linear phase imaging. The method combines phase shifting, two directions of shear and Fourier‐space integration using a modified spiral phase transform. We simulated the method using a phantom object with spatially varying amplitude and phase. Simulated results show good agreement between the final phase image and the object phase, and demonstrate resistance to imaging noise.     

Phase behaviour and phase separation kinetics measurement using acoustic arrays

Speed of sound and acoustic wave attenuation are sensitive to fluid phase composition and to the presence of liquid-liquid interfaces. In this work, the use of an acoustic array comprising 64 elements as a non-intrusive sensor for liquid-liquid interface, phase separation kinetics measurement in bulk fluids, and local composition measurement in porous media is illustrated. Three benchmark examples: the phase behaviour of methanol + mixed hexanes and methanol + heptane mixtures at 25.0 °C and 1 bar, and Athabasca bitumen + heptane in a synthetic silica porous medium at 22.5 °C and 1 bar, illustrate the accuracy of liquid-liquid interface and potential research and industrial applications of the technique. Liquid-liquid interfaces can be detected independently using both speed of sound and acoustic wave attenuation measurements. The precision of the interface location measurement is 300 μm. As complete scans can be performed at a rate of 1 Hz, phase separation kinetics and diffusion of liquids within porous media are readily tracked. The technique is expected to find application where the fluids or porous media are opaque to visible light and where other imaging techniques are not readily applied, or are too costly. A current limitation is that the acoustic probes must be cooled to less than 315 K in order for them to operate.     

X-ray microscopy of plant cells by using LiF crystal as a detector

A lithium fluoride (LiF) crystal has been utilized as a new soft X-ray detector to image different biological samples at a high spatial resolution. This new type of image detector for X-ray microscopy has many interesting properties: high resolution (nanometer scale), permanent storage of images, the ability to clear the image and reuse the LiF crystal, and high contrast with greater dynamic range. Cells of the unicellular green algae Chlamydomonas dysosmos and Chlorella sorokiniana, and pollen grains of Olea europea have been used as biological materials for imaging. The biological samples were imaged on LiF crystals by using the soft X-ray contact microscopy and contact micro-radiography techniques. The laser plasma soft X-ray source was generated using a Nd:YAG/Glass laser focused on a solid target. The X-ray energy range for image acquisition was in the water-window spectral range for single shot contact microscopy of very thin biological samples (single cells) and around 1 keV for multishots microradiography. The main aim of this article is to highlight the possibility of using a LiF crystal as a detector for the biological imaging using soft X-ray radiation and to demonstrate its ability to visualize the microstructure within living cells.     

Contrast in confocal scanning microscopy with a finite detector

The optical properties of a general scanning microscope are determined within the framework of Fourier imaging theory. For a simple model optical system, with Gaussian lens and detector apertures, the contrast transfer function can be expressed in terms of elementary functions. The theory predicts that spatial resolution and depth discrimination vary continuously with detector aperture and that defocus phase contrast is present in transmission images obtained with a symmetric objective, collector lens confocal microscope.     

Orientation independent retardation imaging using quantitative polarized phase microscopy

Simultaneous optical phase and retardation measurement of a birefringent specimen is demonstrated independently of a priori knowledge of the optic axis orientation. The two‐dimensional retardation distribution in both magnitude and angle of the fast axis orientation is uniquely determined from transverse phase images recorded with a bright field transmission microscope using light polarized at a minimum of three different polarization orientations. This approach opens a new possibility for stain‐free phase and orientation‐independent retardation characterization of samples using only one polarizer without needing other additional optical elements traditionally used in polarimetric measurements. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Scanning transmission X-ray microscopy with a fast framing pixel detector

Scanning transmission X-ray microscopy (STXM) is a powerful imaging technique, in which a small X-ray probe is raster scanned across a specimen. Complete knowledge of the complex-valued transmission function of the specimen can be gained using detection schemes whose every-day use, however, is often hindered by the need of specialized configured detectors or by slow or noisy readout of area detectors. We report on sub-50 nm-resolution STXM studies in the hard X-ray regime using the PILATUS, a fully pixelated fast framing detector operated in single-photon counting mode. We demonstrate a range of imaging modes, including phase contrast and dark-field imaging.     

Hard X-ray microscopy with Zernike phase contrast

Zernike phase contrast has been added to a full‐field X‐ray microscope with Fresnel zone plates that was in operation at 6.95 keV. The spatial resolution has also been improved by increasing the magnification of the microscope objective looking at the CsI(Tl) scintillation crystal. Cu no. 2000 meshes and a zone plate have been imaged to see the contrast as well as the spatial resolution. A Halo effect coming from the Zernike phase contrast was clearly visible on the images of meshes.     

Fast 3D imaging of giant unilamellar vesicles using reflected light-sheet microscopy with single molecule sensitivity

Observation of highly dynamic processes inside living cells at the single molecule level is key for a better understanding of biological systems. However, imaging of single molecules in living cells is usually limited by the spatial and temporal resolution, photobleaching and the signal-to-background ratio. To overcome these limitations, light-sheet microscopes with thin selective plane illumination, for example, in a reflected geometry with a high numerical aperture imaging objective, have been developed. Here, we developed a reflected light-sheet microscope with active optics for fast, high contrast, two-colour acquisition of -stacks. We demonstrate fast volume scanning by imaging a two-colour giant unilamellar vesicle (GUV) hemisphere. In addition, the high contrast enabled the imaging and tracking of single lipids in the GUV cap. The enhanced reflected scanning light-sheet microscope enables fast 3D scanning of artificial membrane systems and potentially live cells with single-molecule sensitivity and thereby could provide quantitative and molecular insight into the operation of cells.     

Design and analysis of multi-color confocal microscopy with a wavelength scanning detector

Spectral (or multi-color) microscopy has the ability to detect the fluorescent light of biological specimens with a broad range of wavelengths. Currently, the acousto-optic tunable filter (AOTF) is widely used in spectral microscopy as a substitute for a multiple-dichroic mirror to divide excitation and emission signals while maintaining sufficient light efficiency. In addition, systems which utilize an AOTF have a very fast switching speed and high resolution for wavelength selection. In this paper, confocal-spectral microscopy is proposed with a particular spectrometer design with a wavelength-scanning galvano-mirror. This enables the detection of broadband (480-700 nm) fluorescence signals by a single point detector (photomultiplier tube) instead of a CCD pixel array. For this purpose, a number of optical elements were applicably designed. A prism is used to amplify the dispersion angle, and the design of the relay optics matches the signals to the diameter of the wavelength-scanning galvano-mirror. Also, a birefringent material known as calcite is used to offset the displacement error at the image plane depending on the polarization states. The proposed multi-color confocal microscopy with the unique detection body has many advantages in comparison with commercial devices. In terms of the detection method, it can be easily applied to other imaging modalities.     

Transmission electron microscopy with Zernike phase plate.

The possibility of implementing a Zernike phase plate in a transmission electron microscope is investigated both theoretically and experimentally. The phase-retarding plate in the form of thin film with a hole in the center is positioned in the back-focal plane of the objective lens. The experiments show that the phase plate functions as predicted, producing a cosine-type phase contrast transfer function. Images of negatively stained horse spleen ferritin were highly improved in the contrast and the image-modulation, compared to those acquired without the phase plate. Charging and related difficulties were encountered during the phase plate experiments. In order to make the technique user-friendly a number of improvements have to be made, and are discussed in terms of the current level of technology and instrumentation.     

Contact X‐ray microscopy of living cells by using LiF crystal as imaging detector

In this paper, the use of lithium fluoride (LiF) as imaging radiation detector to analyse living cells by single‐shot soft X‐ray contact microscopy is presented. High resolved X‐ray images on LiF of cyanobacterium Leptolyngbya VRUC135, two unicellular microalgae of the genus Chlamydomonas and mouse macrophage cells (line RAW 264.7) have been obtained utilizing X‐ray radiation in the water window energy range from a laser plasma source. The used method is based on loading of the samples, the cell suspension, in a special holder where they are in close contact with a LiF crystal solid‐state X‐ray imaging detector. After exposure and sample removal, the images stored in LiF by the soft X‐ray contact microscopy technique are read by an optical microscope in fluorescence mode. The clear image of the mucilaginous sheath the structure of the filamentous Leptolyngbya and the visible nucleolus in the macrophage cells image, are noteworthiness results. The peculiarities of the used X‐ray radiation and of the LiF imaging detector allow obtaining images in absorption contrast revealing the internal structures of the investigated samples at high spatial resolution. Moreover, the wide dynamic range of the LiF imaging detector contributes to obtain high‐quality images. In particular, we demonstrate that this peculiar characteristic of LiF detector allows enhancing the contrast and reveal details even when they were obscured by a nonuniform stray light.     

Hard x-ray phase contrast imaging using single absorption grating and hybrid semiconductor pixel detector

A method for x-ray phase contrast imaging is introduced in which only one absorption grating and a microfocus x-ray source in a tabletop setup are used. The method is based on precise subpixel position determination of the x-ray pattern projected by the grating directly from the pattern image. For retrieval of the phase gradient and absorption image (both images obtained from one exposure), it is necessary to measure only one projection of the investigated object. Thus, our method is greatly simplified compared with the phase-stepping method and our method can significantly reduce the time-consuming scanning and possibly the unnecessary dose. Furthermore, the technique works with a fully polychromatic spectrum and gives ample variability in object magnification. Consequently, the approach can open the way to further widespread application of phase contrast imaging, e.g., into clinical practice. The experimental results on a simple testing object as well as on complex biological samples are presented.     

显微数字全息图相位的滤波法提取

显微数字全息的关键技术之一是其相位信息的提取。通过分析显微数字全息图的空间频谱特点,提出了一种新的相位提取方法。将数字全息图频谱进行搬移,使物光频谱中心移到频域坐标原点;设计有限脉冲响应(FIR)数字滤波器滤出物光频谱;求解滤波后信号的辐角得到全息图相位。对设计的实验系统进行了验证,实验结果表明采用提出的方法提取显微数字全息相位,完全可行,效果良好。     

Quality improvement of environmental secondary electron detector signal using helium gas in variable pressure scanning electron microscopy

The quality of the image signal obtained from the environmental secondary electron detector (ESED) employed in a variable pressure (VP) SEM can be dramatically improved by using helium gas. The signal-to-noise ratio (SNR) increases gradually in the range of the pressures that can be used in our modified SEM. This method is especially useful in low-voltage VP SEM as well as in a variety of SEM operating conditions, because helium gas can more or less maintain the amount of unscattered primary electrons. In order to measure the SNR precisely, a digital scan generator system for obtaining two images with identical views is employed as a precondition.     

Electron phase microscopy at 1 MeV with graphite crystal supports

Thin phase objects can be seen with good contrast with 1 MeV electrons if they are supported on a graphite crystal approximately half an extinction distance thick and viewed near 101 or 112 bend contours. The shape of unstained tobacco mosaic virus is easily seen with crystal phase contrast although it is almost invisible in a normal bright field micrograph taken with 1 MeV electrons. Phase contrast images of both TMV and 109 nm diameter latex spheres are observed to be bright against a grey background on one side of a 101 or 112 bend contour and dark against a grey background on the other side of the bend contour. For strong contrast the crystal diffraction vector g must have a component parallel to the beam. Contours such as 100 and 110 do not give strong phase contrast. The phase contrast images for biological material gradually increase in contrast as the specimens are irradiated by the electron beam. An electron exposure of about 10⁴ electrons nm^?2 is required to obtain maximum contrast.     

Recording of single-particle hysteresis loops with differential phase contrast microscopy

The magnetic behaviour of laterally patterned structures on a micrometer or nanometer scale in external magnetic fields can be described by their hysteresis loops. However, it is very difficult to record the hysteresis loop of a single (sub-) micron-sized particle. Furthermore, extensive calculations are necessary in order to interpret the shape of the loop and to conclude the micromagnetic domain configuration in the sample from the hysteresis loop only. We have developed a technique which yields both the hysteresis loop and the magnetic domain structure simultaneously. This method uses differential phase contrast microscopy and a microscope with remote control capability for the automatic recording of a single-particle hysteresis loop.