Quantitative amplitude and phase contrast imaging in a scanning transmission X-ray microscope

Phase contrast in X-ray imaging provides lower radiation dose, and dramatically higher contrast at multi-keV photon energies when compared with absorption contrast. We describe here the use of a segmented detector in a scanning transmission X-ray microscope to collect partially coherent bright field images. We have adapted a Fourier filter reconstruction technique developed by McCallum, Landauer and Rodenburg to retrieve separate, quantitative maps of specimen phase shift and absorption. This is demonstrated in the imaging of a germanium test pattern using 525eV soft X-rays.     

Quantitative analysis of image contrast in phase contrast STEM for low dose imaging

This work quantitatively evaluates the contrast in phase contrast images of thin vermiculite crystals recorded by TEM and aberration-corrected bright-field STEM. Specimen movement induced by electron irradiation remains a major problem limiting the phase contrast in TEM images of radiation-sensitive specimens. While spot scanning improves the contrast, it does not eliminate the problem. One possibility is to utilise aberration-corrected scanning transmission electron microscopy (STEM) with an Ångstrom-sized probe to illuminate the sample, and thus further reduce irradiation-induced specimen movement. Vermiculite is relatively radiation insensitive in TEM to electron fluences below 100,000 e⁻/Ų and this is likely to be similar for STEM although different damage mechanisms could occur. We compare the performance of a TEM with a thermally assisted field emission electron gun (FEG) and charge coupled device (CCD) image capture to the performance of STEMs with spherical aberration correction, cold field emission electron sources and photomultiplier tube image capture at a range of electron fluences and similar illumination areas. We show that the absolute contrast of the phase contrast images obtained by aberration-corrected STEM is better than that obtained by TEM. Although the STEM contrast is higher, the efficiency of collection of electrons in bright field STEM is still much less than that in bright field TEM (where for thin samples virtually all the electrons contribute to the image), and the SNR of equivalent STEM images is three times lower. This is better than expected, probably due to the absence of a frequency dependent modulation transfer function in the STEM detection system. With optimisation of the STEM bright field collection angles, the efficiency may approach that of bright field TEM, and if reductions in beam-induced specimen movement are found, STEM could surpass the overall performance of TEM.     

Computer simulation and analysis of high-resolution electron microscope images and diffraction patterns with partial coherence,hollow cone illumination,and virtual apertures

A general method for computing high-resolution conventional transmission electron microscope images and diffraction patterns, when there are different types of partially coherent illumination conditions, is described. Examples of convergent beam, hollow cone, and virtual aperture illumination conditions are given in the context of interpreting image features. A comparison of real and computed diffraction patterns shows that, in practice, many innovative imaging modes are possible, which can be verified prior to real microscope experiments.     

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.     

Imaging of soft and hard materials using a Boersch phase plate in a transmission electron microscope

Using two levels of electron beam lithography, vapor phase deposition techniques, and FIB etching, we have fabricated an electrostatic Boersch phase plate for contrast enhancement of weak phase objects in a transmission electron microscope. The phase plate has suitable dimensions for the imaging of small biological samples without compromising the high-resolution capabilities of the microscope. A micro-structured electrode allows for phase tuning of the unscattered electron beam, which enables the recording of contrast enhanced in-focus images and in-line holograms. We have demonstrated experimentally that our phase plate improves the contrast of carbon nanotubes while maintaining high-resolution imaging performance, which is demonstrated for the case of an AlGaAs heterostructure. The development opens a new way to study interfaces between soft and hard materials.     

Differential phase contrast in scanning optical microscopy

High-quality high-resolution transmission and reflection images produced using a scanning optical microscope and the split-detector technique are presented. These images exhibit differential phase contrast, the method avoiding some drawbacks of the usual Nomarski DIC arrangement. Imaging is treated theoretically and compared with the Nomarski method.     

A study of the human hair structure with a Zernike phase contrast X-ray microscope

We have observed the internal structure of human hair shafts with a transmission Zernike phase contrast hard X‐ray microscope. Due to the high spatial resolution and the high contrast of the microscope, we could image scales, macrofibrils, medulla and melanin without staining. The structure of a black hair shaft is compared with that of a white one.     

A combined light sheet fluorescence and differential interference contrast microscope for live imaging of multicellular specimens

We describe a microscope capable of both light sheet fluorescence microscopy and differential interference contrast microscopy (DICM). The two imaging modes, which to the best of our knowledge have not previously been combined, are complementary: light sheet fluorescence microscopy provides three‐dimensional imaging of fluorescently labelled components of multicellular systems with high speed, large fields of view, and low phototoxicity, whereas differential interference contrast microscopy reveals the unlabelled neighbourhood of tissues, organs, and other structures with high contrast and inherent optical sectioning. Use of a single Nomarski prism for differential interference contrast microscopy and a shared detection path for both imaging modes enables simple integration of the two techniques in one custom microscope. We provide several examples of the utility of the resulting instrument, focusing especially on the digestive tract of the larval zebrafish, revealing in this complex and heterogeneous environment anatomical features, the behaviour of commensal microbes, immune cell motions, and more.     

Some aspects of optimizing contrasts for the investigation of joint materials in the environmental scanning electron microscope

In the environmental scanning electron microscope, material joints of different atomic mass and different electrical conducting properties can easily be observed simultaneously without coating the specimen. For such heterogeneous materials, the quality of the image can be optimized with respect to contrast and resolution if the contrast types as well as their significance to the composition of the image are known.     

Automated noninvasive epithelial cell counting in phase contrast microscopy images with automated parameter selection

Cell counting is commonly used to determine proliferation rates in cell cultures and for adherent cells it is often a ‘destructive’ process requiring disruption of the cell monolayer resulting in the inability to follow cell growth longitudinally. This process is time consuming and utilises significant resource. In this study a relatively inexpensive, rapid and widely applicable phase contrast microscopy‐based technique has been developed that emulates the contrast changes taking place when bright field microscope images of epithelial cell cultures are defocused. Processing of the resulting images produces an image that can be segmented using a global threshold; the number of cells is then deduced from the number of segmented regions and these cell counts can be used to generate growth curves. The parameters of this method were tuned using the discrete mereotopological relations between ground truth and processed images. Cell count accuracy was improved using linear discriminant analysis to identify spurious noise regions for removal. The proposed cell counting technique was validated by comparing the results with a manual count of cells in images, and subsequently applied to generate growth curves for oral keratinocyte cultures supplemented with a range of concentrations of foetal calf serum. The approach developed has broad applicability and utility for researchers with standard laboratory imaging equipment.     

Variable multimodal light microscopy with interference contrast and phase contrast; dark or bright field

Using the optical methods described, specimens can be observed with modified multimodal light microscopes based on interference contrast combined with phase contrast, dark‐ or bright‐field illumination. Thus, the particular visual information associated with interference and phase contrast, dark‐ and bright‐field illumination is joined in real‐time composite images appearing in enhanced clarity and purified from typical artefacts, which are apparent in standard phase contrast and dark‐field illumination. In particular, haloing and shade‐off are absent or significantly reduced as well as marginal blooming and scattering. The background brightness and thus the range of contrast can be continuously modulated and variable transitions can be achieved between interference contrast and complementary illumination techniques. The methods reported should be of general interest for all disciplines using phase and interference contrast microscopy, especially in biology and medicine, and also in material sciences when implemented in vertical illuminators.     

A hybrid scanning force and light microscope for surface imaging and three-dimensional optical sectioning in differential interference contrast

The design of a scanned-cantilever-type force microscope is presented which is fully integrated into an inverted high-resolution video-enhanced light microscope. This set-up allows us to acquire thin optical sections in differential interference contrast (DIC) or polarization while the force microscope is in place. Such a hybrid microscope provides a unique platform to study how cell surface properties determine, or are affected by, the three-dimensional dynamic organization inside the living cell. The hybrid microscope presented in this paper has proven reliable and versatile for biological applications. It is the only instrument that can image a specimen by force microscopy and high-power DIC without having either to translate the specimen or to remove the force microscope. Adaptation of the design features could greatly enhance the suitability of other force microscopes for biological work.     

Using the Hilbert transform for 3D visualization of differential interference contrast microscope images

Differential interference contrast (DIC) is frequently used in conventional 2D biological microscopy. Our recent investigations into producing a 3D DIC microscope (in both conventional and confocal modes) have uncovered a fundamental difficulty: namely that the phase gradient images of DIC microscopy cannot be visualized using standard digital image processing and reconstruction techniques, as commonly used elsewhere in microscopy. We discuss two approaches to the problem of preparing gradient images for 3D visualization: integration and the Hilbert transform. After applying the Hilbert transform, the dataset can then be visualized in 3D using standard techniques. We find that the Hilbert transform provides a rapid qualitative pre-processing technique for 3D visualization for a wide range of biological specimens in DIC microscopy, including chromosomes, which we use in this study.     

硬X射线相衬成像及其生物应用

X射线相衬技术大大拓宽X射线的应用领域。本文综述X射线相衬成像的物理学原理、X射线相衬成像装置的一般结构和应用方法,并对各种不同相衬成像的方法、成像算法和生物应用逐一阐述。     

Characterizing voltage contrast in photoelectron emission microscopy

A non‐destructive technique for obtaining voltage contrast information with photoelectron emission microscopy is described. Samples consisting of electrically isolated metal lines were used to quantify voltage contrast in photoelectron emission microscopy. The voltage contrast behaviour is characterized by comparing measured voltage contrast with calculated voltage contrast from two electrostatic models. Measured voltage contrast was found to agree closely with the calculated voltage contrast, demonstrating that voltage contrast in photoelectron emission microscopy can be used to probe local voltage information in microelectronic devices in a non‐intrusive fashion.     

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.     

Laser scanning phase modulation microscope

We describe the concept and first implementation of an innovative new instrument for quantitative light microscopy. Currently, it provides selective imaging of optical path differences due to birefringence; with further development, it is also possible to selectively image several optical properties, including refractive path differences, optical rotation, and linear and circular dichroism, all with diffraction-limited resolution. An image consists of a 512×512 element array, with each pixel displaying one of 256 grey levels, linearly proportional to the specific optical property being observed. Additionally, conventional brightfield and polarized light microscopy are available, with the accompanying advantages of laser scanning and digital image processing. The microscope consists of three subsystems, representing three distinct technologies. The laser scanning subsystem moves a focused, microspot across the specimen; the output of a photodetector is an electric signal corresponding to a scanned image. The image display subsystem digitizes this signal and displays it as an image on a video monitor. When used in conjunction with a phase modulation feedback loop, the image formed is of the specimen's birefringent retardation or other selected optical property. The digitized images are also available for computer enhancement.     

Contrast enhancement and depth perception in three-dimensional representations of differential interference contrast and confocal scanning laser microscope images

A commonly used three-dimensional reconstruction method in confocal scanning laser microscopy (CSLM) involves the generation of stereo views by stacking optical sections. Under certain conditions the resulting stereo pairs exhibit inferior quality with respect to contrast and depth perception. A method based on digital image processing is described in which the individual images are enhanced prior to reconstruction, thereby increasing the number of usable optical sections by a factor of up to five. Furthermore, we introduce a new contrast enhancement transformation based upon local statistics and a grey-level probability density function that provides improved depth perception. Digital image processing methods map a discrete grey scale onto a continuous, unbounded interval. Inasmuch as a closed, discrete grey scale is required for computer display purposes, we present an appropriate mapping function derived from an entropy criterion.     

The role of induced contrast in images obtained using the environmental scanning electron microscope

Generation of contrast in images obtained using the environmental scanning electron microscope (ESEM) is explained by interpretation of images acquired using the gaseous secondary electron detector (GSED), ion current, and the Everhart-Thornley detector. We present a previously unreported contrast component in GSED and ion current images attributed to signal induction by changes in the concentration of positive ions in the ESEM chamber during image acquisition. Changes in positive ion concentration are caused by changes in electron emission from the sample during image acquisition and by a discrepancy between the drift velocities of negative and positive charge carriers in the imaging gas. The proposed signal generation mechanism is used to explain contrast reversal in images produced using the GSED and ion current signals and accounts for discrepancies in contrast observed, under some conditions, in these types of images. Combined with existing models of signal generation in the ESEM, the proposed model provides a basis for correct interpretation of ESEM images.