Hard x-ray Zernike microscopy reaches 30 nm resolution

Since its invention in 1930, Zernike phase contrast has been a pillar in optical microscopy and more recently in x-ray microscopy, in particular for low-absorption-contrast biological specimens. We experimentally demonstrate that hard-x-ray Zernike microscopy now reaches a lateral resolution below 30 nm while strongly enhancing the contrast, thus opening many new research opportunities in biomedicine and materials science.     

高分辨率X射线显微成像及其进展

介绍了高分辨率X射线显微成像产生背景和发展过程,着重分析了基于光学元件波带片的放大成像的基本原理,并简述了高分辨率三维成像的有关理论。同时给出国内外高分辨率X射线显微成像研究的最新进展,展望了高分辨率X射线显微成像的应用前景。     

高分辨率X射线显微成像及其进展

介绍了高分辨率X射线显微成像产生背景和发展过程，着重分析了基于光学元件波带片的放大成像的基本原理，并简述了高分辨率三维成像的有关理论。同时给出国内外高分辨率X射线显微成像研究的最新进展，展望了高分辨率X射线显微成像的应用前景。     

高分辨率软X射线显微成像原理及其应用

软X射线显微成像具有分辨率高、辐射损伤小以及能够对含水的生物样品直接进行显微成像等独特的优势,目前已成为研究生命科学和生物学等学科中的超微观世界的特殊工具.本文简要介绍基于波带片的高分辨率软X射线显微成像的原理及其应用.     

An assessment of the resolution limitation due to radiation-damage in X-ray diffraction microscopy

X-ray diffraction microscopy (XDM) is a new form of X-ray imaging that is being practiced at several third-generation synchrotron-radiation X-ray facilities. Nine years have elapsed since the technique was first introduced and it has made rapid progress in demonstrating high-resolution three-dimensional imaging and promises few-nanometer resolution with much larger samples than can be imaged in the transmission electron microscope. Both life- and materials-science applications of XDM are intended, and it is expected that the principal limitation to resolution will be radiation damage for life science and the coherent power of available X-ray sources for material science. In this paper we address the question of the role of radiation damage. We use a statistical analysis based on the so-called “dose fractionation theorem” of Hegerl and Hoppe to calculate the dose needed to make an image of a single life-science sample by XDM with a given resolution. We find that the needed dose scales with the inverse fourth power of the resolution and present experimental evidence to support this finding. To determine the maximum tolerable dose we have assembled a number of data taken from the literature plus some measurements of our own which cover ranges of resolution that are not well covered otherwise. The conclusion of this study is that, based on the natural contrast between protein and water and “Rose-criterion” image quality, one should be able to image a frozen-hydrated biological sample using XDM at a resolution of about 10 nm.     

X-ray interferometry with microelectronvolt resolution

We demonstrate an interferometer for hard x rays with two back-reflecting sapphire crystal mirrors--a prototype x-ray Fabry-Pérot interferometer. A finesse of 15 and 0.76 mu eV broad Fabry-Pérot transmission resonances are measured by the time response of the interferometer. Interference patterns are observed directly in spectral dependences of reflectivity.     

X-ray microscopy

The wavelength limitation on resolution indicates that an x-ray microscope may surpass the optical microscope but will not compete with the electron microscope even if all the technical difficulties could be overcome. However, there are other advantages besides resolution that make x-rays an attractive medium for microscopy.

The penetration of x-rays allows the examination of the internal detail of a specimen on the micro scale without the need for sectioning and reconstruction. The large depth of focus permits the viewing of this internal detail with equal sharpness and stereographically if necessary. The specimen may be in air at atmospheric pressure, while the electron microscope demands high vacuum, and yet the resolution may be better than that of the optical microscope. Most important, the simple absorption and emission spectra of x-rays leads to the extraction of quantitative information from the image in terms of mass and elements present that in many cases cannot be obtained in any other way.

These and other reasons have led to the search for a usable x-ray microscope and the three more successful methods of reflection, contact and projection are discussed here. In addition, recent results on microanalysis show where the bulk of the future work will lie.     

Exploring nanomagnetism with soft X-ray microscopy

Magnetic soft X-ray microscopy images magnetism in nanoscale systems with a spatial resolution down to 15 nm provided by state-of-the-art Fresnel zone plate optics. X-ray magnetic circular dichroism (X-MCD) is used as the element-specific magnetic contrast mechanism similar to photoemission electron microscopy (PEEM), however, with volume sensitivity and the ability to record the images in varying applied magnetic fields which allows study of magnetization reversal processes at fundamental length scales. Utilizing a stroboscopic pump-probe scheme one can investigate fast spin dynamics with a time resolution down to 70 ps which gives access to precessional and relaxation phenomena as well as spin torque driven domain wall dynamics in nanoscale systems. Current developments in zone plate optics aim for a spatial resolution towards 10 nm and at next generation X-ray sources a time resolution in the fs regime can be envisioned.     

Imaging sclera with hard X-ray microscopy

In this study, the organization of collagen fibrils within the sclera of the eye was investigated using the 7 keV hard X-ray microscope of the Pohang light source and compared to images from electron and atomic force microscopy. From the captured X-ray images, individual collagen fibrils were observed clearly in a spatial resolution much better than 100 nm, both in longitudinal sections and in transverse sections. Some of the collagen fibrils showed evidence of axial periodicity. In some regions of the samples, we could see cross-bridge like structures between adjacent collagen fibrils. The X-ray microscope also allowed the observation of keratocytes and the lamella structure of the scleral stroma. The X-ray microscope has some unique advantages in the nano-scale imaging of bio-samples relative to other established imaging techniques.     

New directions in X-ray microscopy

The development of high brightness X-ray sources and high resolution X-ray optics has led to rapid advances in X-ray microscopy. Scanning microscopes and full-field instruments are in operation at synchrotron light sources worldwide, and provide spatial resolution routinely in the 25–50 nm range using zone plate focusing elements. X-ray microscopes can provide elemental maps and/or chemical sensitivity in samples that are too thick for electron microscopy. Lensless techniques, such as diffraction microscopy, holography and ptychography are also being developed. In high resolution imaging of radiation-sensitive material the effects of radiation damage needs to be carefully considered. This article is designed to provide an introduction to the current state and future prospects of X-ray microscopy for the non-expert.     

Color lensless digital holographic microscopy with micrometer resolution

Color digital lensless holographic microscopy with micrometer resolution is presented. Multiwavelength illumination of a biological sample and a posteriori color composition of the amplitude images individually reconstructed are used to obtain full-color representation of the microscopic specimen. To match the sizes of the reconstructed holograms for each wavelength, a reconstruction algorithm that allows for choosing the pixel size at the reconstruction plane independently of the wavelength and the reconstruction distance is used. The method is illustrated with experimental results.     

Saturated structured confocal microscopy with theoretically unlimited resolution

The resolution of fluorescence microscopes is limited by diffraction, which determines the extension of their point spread functions. We propose and study numerically a simple method, based on a combination of subtraction microscopy with regular and annular excitation beams, which permits to double the resolution compared to wide field microscopy. When combined with the fluorescence saturation phenomenon, this approach would be able to deliver a resolution of a few tens of nanometers.     

Photoselective laser photo-ion microscopy with 5 nm resolution

Spectral selectivity has been attained in the method of field-ion microscopy with a spatial resolution of about 5 nm and time-of-flight determination of the photo-ion masses. Light-absorbing CdS_xSe_1−x nanocrystals in a transparent glass matrix are detected by irradiating field tips made from red light filters with copper-vapor laser radiation. The nanocrystals appeared in the photo-ion images as bright spots on a dark background. Pis’ma Zh. éksp. Teor. Fiz. 67, No. 7, 450–454 (10 April 1998)     

Subwavelength depth resolution in near-field microscopy

A generalized Radon transform is presented that relates, for the case of an evanescent wave that is incident upon a weakly scattering medium, the homogeneous components of the scattered field to the three-dimensional Fourier transform of the dielectric susceptibility. This relationship is used within the context of total internal reflection microscopy to reconstruct the depth structure of the dielectric susceptibility from simulated scattered field data.     

Subdiffraction resolution in far-field fluorescence microscopy

We overcame the resolution limit of scanning far-field fluorescence microscopy by disabling the fluorescence from the outer part of the focal spot. Whereas a near-UV pulse generates a diffraction-limited distribution of excited molecules, a spatially offset pulse quenches the excited molecules from the outer part of the focus through stimulated emission. This results in a subdiffraction-sized effective point-spread function. For a 1.4 aperture and a 388-nm excitation wavelength spatial resolution is increased from 150 +/- 8 nm to 106 +/- 8 nm with a single offset beam. Superior lateral resolution is demonstrated by separation of adjacent Pyridine 2 nanocrystals that are otherwise indiscernible.     

KBA型X射线显微系统

KBA型X射线显微镜的物与像不在同一水平面上,采用双对准方式解决了在靶室安装这套仪器的困难。与通常的光学系统不同,KBA显微镜的伪光轴与实轴夹角8.748 6,设计了观察系统,保证了掠入射角的要求。KBA显微镜的物方孔径角很小,用可见光装调焦深很大,无法满足要求,因此设计了辅助光学成像系统,满足了焦深为5 mm的要求。     

One micrometer resolution NMR microscopy

We obtained a magnetic resonance image of 1 microm resolution and 75 microm(3) voxel volume for a phantom filled with hydrocarbon oil within an hour at 14.1 T. For this work, a specially designed probe with a high sensitivity RF coil and gradient coils generating over 1000 G/cm was built. The optimal pulse sequence was analyzed in consideration of the bandwidth, diffusion coefficients, and T(1) and T(2) relaxations of the medium. The system was applied to the in vivo imaging of a geranium leaf stem to get the images of 2 microm resolution and 200 microm(3) voxel volume.     

Radiation damage in soft X-ray microscopy

The rates of chemical transformation by radiation damage of polystyrene (PS), poly(methyl methacrylate) (PMMA), and fibrinogen (Fg) in a X-ray photoemission electron microscope (X-PEEM) and in a scanning transmission X-ray microscope (STXM) have been measured quantitatively using synchrotron radiation. As part of the method of dose evaluation in X-PEEM, the characteristic (1/e) sampling depth of X-PEEM for polystyrene in the C 1s region was measured to be 4 ± 1 nm. Critical doses for chemical change as monitored by changes in the X-ray absorption spectra are 80 (12), 280 (40) and 1230 (180) MGy (1 MGy = 6.242*ρ eV/nm³, where ρ is the polymer density in g/cm³) at 300 eV photon energy for PMMA, Fg and PS, respectively. The critical dose for each material is comparable in X-PEEM and STXM and the values cited are thus the mean of the values determined by X-PEEM and STXM. C 1s, N 1s and O 1s spectroscopy of the damaged materials is used to gain insight into the chemical changes that soft X-rays induce in these materials.     

Digital resolution enhancement in surface plasmon microscopy

The use of photonic crystal and negative refractive index materials is known to improve the resolution of optical microscopy and lithography devices down to the 80 nm level. Here we demonstrate that utilization of well-known digital image recovery techniques allows us to further improve the resolution of optical microscopy down to the 30 nm level. Our microscope is based on a flat dielectric mirror deposited onto an array of nanoholes in thin gold film. This two-dimensional photonic crystal mirror may have either a positive or negative effective refractive index as perceived by surface plasmon polartions in the visible frequency range. The optical images formed by the mirror are enhanced using simple digital filters. PACS 73.20.Mf; 42.70.Qs; 07.60.Pb     

Transmission X-ray microscopy using X-ray magnetic circular dichroism

X-ray magnetic circular dichroism (X-MCD) was used as a large, element-specific and quantitative magnetic contrast mechanism in the soft X-ray microscopes at BESSY I (Berlin) and the ALS (Berkeley). The present state and potential of magnetic transmission X-ray microscopy (MTXM) is outlined. The possibility to record images in varying magnetic fields and the high spatial resolution down to 25 nm were used to image out-of-plane magnetized (4 ?Fe / 4 ?Gd)×75 systems. Magnetic domains could be studied in arrays of circular and square dots with lateral dimensions down to 180 nm. Hysteresis loops of individual dots were deduced using the direct proportionality of the X-MCD contrast to the sample magnetization. Images of a 3 nmCr / 50 nmFe / 6 nmCr film demonstrate for the first time that MTXM is also able to observe in-plane magnetized domains. In the future the possible applications of MTXM will be extended with regard to the strength of the external field, the available energy range and the sample conditions by building a dedicated transmission X-ray microscope for magnetic imaging at BESSY II. Received: 22 May 2001 / Accepted: 4 July 2001 / Published online: 5 October 2001