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.     

Sialolith characterization by scanning electron microscopy and X-ray photoelectron spectroscopy

The objective of this study has been to characterize sialolith, a calcium phosphate deposit that develops in the human oral cavity, by high-resolution field emission scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The morphological and chemical data obtained helped in the determination of their formation mechanism in salivary glands. Sialoliths in the submandibular salivary glands may arise secondary to sialodenitis, but not via a luminal organic nidus. We believe this is the first study that characterizes a sialolith by XPS.     

Prospects in high resolution X-ray photoelectron microscopy

After a brief review of the present state of the art in X-ray photoelectron microscopy, the prospects to improve the nominal value of the spatial resolution are described. Progress in this method is also related to the development of dense X-ray sources, but possible radiation damage effects have to be considered.     

5.4 nm spatial resolution in biological photoemission electron microscopy

We report a spatial resolution of 5.4 nm in images of sarcoplasmic reticulum from rabbit muscle. The images were obtained in an aberration-corrected photoemission electron microscope with a hyperbolic mirror as the correcting element for spherical and chromatic aberration. In-situ measurements and numerical simulations confirm the low residual aberration in the instrument and indicate the ultimate resolution in this type of microscopy to be below 2 nm.     

Theoretical versus experimental resolution in optical microscopy

The aim of this article is to compare experimental resolution under different conditions with theoretical resolution predicted using electromagnetic diffraction theory. Imaging properties of fluorescent beads of three different diameters (0.1 microm, 0.2 microm, and 0.5 microm) as well as imaging properties of four different fluorescence-stained DNA targets (ABL gene, BCR gene, centromere 6, and centromere 17) are studied. It is shown how the dependence of the resolution on object size varies with wavelength (520 nm versus 580 nm), type of microscopy (wide-field, confocal using Nipkow disk, confocal laser scanning) and basic image processing steps (median and gaussian filters). Furthermore, specimen influence on the resolution was studied (the influence of embedding medium, coverglass thickness, and depth below the coverglass). Both lateral and axial resolutions are presented. The results clearly show that real objects are far from being points and that experimental resolution is often much worse than the theoretical one. Although the article concentrates on fluorescence imaging using high NA objectives, similar dependence can also be expected for other optical arrangements.     

Determination of oxygen in acid-treated high-purity niobium by X-ray photoelectron spectroscopy

High-purity niobium was immersed in nitric acid and hydrofluoric acid for two minutes and the resulting product was characterized. Scanning electron microscopy was employed to characterize the morphology of the acid treated product. Inert gas fusion was used for determination of oxygen in the high-purity niobium. X-ray photoelectron spectroscopy was employed to characterize the surface ratio of oxygen to niobium. The oxygen concentration was 30 ppm in the acid-treated material and 70 ppm in the untreated material. X-ray photoelectron spectroscopy was employed to characterize the reduction of oxygen at the surface. The ratio of oxygen to niobium decreased from 9.75 to 2.60 without treatment compared to acid treatment for two minutes. The concentrations of adsorbed water and niobium oxide decreased following etching. The acid-treated high-purity niobium was characterized by argon ion bombardment; adsorbed water molecules and niobium oxides were not present but non-lattice oxygen was observed.     

Increased resolution in neutral atom microscopy

The neutral atom microscope uses a beam of thermal noncharged atoms or molecules to probe an atomic surface with very low interaction energies (<70 meV). Continued optimization of the ‘pinhole’ neutral atom microscope has improved resolution to 0.35 μm. Recent images are presented demonstrating resolution and the contrast mechanisms identified so far. The future potential for sub‐100 nm resolution is discussed.     

Beam induced mass loss in high resolution biological microanalysis

Electron beam induced loss of mass from the organic matrix and from higher Z constituents of biological samples was measured by monitoring bremsstrahlung and peak changes in EDS spectra. When any effects of contamination, extraneous X-rays, beam current drift, specimen drift, and specimen shrinkage were monitored and corrected for, the three types of samples gave consistent and similar results at 296 K. Bremsstrahlung losses averaged 45%, 46% and 50% respectively for muscle homogenate, salivary gland sections and albumin. Sulphur losses average 74%, 72% and 86% for the same three sample types. No other elements suffered significant losses. D_l/e for bremsstrahlung averaged 0·14 C/cm². Bremsstrahlung loss at 93 K began approximately one order of magnitude higher in dose, and the extent of loss varied. Sulphur losses, however, were greatly reduced at low temperatures.     

Fluorescent resolution target for super-resolution microscopy

We report methods of near‐field infrared microscopy with transient optically induced probes. The first technique – a transient aperture (TA) – uses photoinduced reflectivity in semiconductors to generate a relatively large transient mirror (TM) with a small aperture at its centre. We report the optical properties of the TM and TA and experiments performed on near‐field imaging with the TA. The second technique is based on solid immersion microscopy, in which high resolution is achieved when light is focused inside a solid with a high refractive index. By creating a transient Fresnel lens on the surface of a semiconductor wafer via photoinduction, we were able to form a solid immersion lens (SIL) for use as a near‐field probe. The use of transient probes eliminates the need for mechanical scanning of the lens or sample, and thus provides a much faster scanning rate and the possibility to work with soft and liquid objects.     

Emission microscopy and related techniques: resolution in photoelectron microscopy, low energy electron microscopy and mirror electron microscopy.

A unified treatment of the resolution of three closely related techniques is presented: emission electron microscopy (particularly photoelectron microscopy, PEM), low energy electron microscopy (LEEM), and mirror electron microscopy (MEM). The resolution calculation is based on the intensity distribution in the image plane for an object of finite size rather than for a point source. The calculations take into account the spherical and chromatic aberrations of the accelerating field and of the objective lens. Intensity distributions for a range of energies in the electron beam are obtained by adding the single-energy distributions weighted according to the energy distribution function. The diffraction error is taken into account separately. A working resolution is calculated that includes the practical requirement for a finite exposure time, and hence a finite non-zero current in the image. The expressions for the aberration coefficients are the same in PEM and LEEM. The calculated aberrations in MEM are somewhat smaller than for PEM and LEEM. The resolution of PEM is calculated to be about 50 A, assuming conventional UV excitation sources, which provide current densities at the specimen of 5 x 10(-5) A/cm2 and emission energies ranging up to 0.5 eV. A resolution of about 70 A has been demonstrated experimentally. The emission current density at the specimen is higher in LEEM and MEM because an electron gun is used in place of a UV source. For a current density of 5 x 10(-4) A/cm2 and the same electron optical parameters as for PEM, the resolution is calculated to be 27 A for LEEM and 21 A for MEM.     

Combining AFM and FRET for high resolution fluorescence microscopy

Here we demonstrate a new microscopic method that combines atomic force microscopy (AFM) with fluorescence resonance energy transfer (FRET). This method takes advantage of the strong distance dependence in Förster energy transfer between dyes with the appropriate donor/acceptor properties to couple an optical dimension with conventional AFM. This is achieved by attaching an acceptor dye to the end of an AFM tip and exciting a sample bound donor dye through far-field illumination. Energy transfer from the excited donor to the tip immobilized acceptor dye leads to emission in the red whenever there is sufficient overlap between the two dyes. Because of the highly exponential distance dependence in this process, only those dyes located at the apex of the AFM tip, nearest the sample, interact strongly. This limited and highly specific interaction provides a mechanism for obtaining fluorescence contrast with high spatial resolution. Initial results in which 400 nm resolution is obtained through this AFM/FRET imaging technique are reported. Future modifications in the probe design are discussed to further improve both the fluorescence resolution and imaging capabilities of this new technique.     

Axial resolution of confocal fluorescence microscopy

A simple analytic expression is given for the axial resolution of a confocal fluorescence microscope. The expression, which is based on the spatial frequency cut-off criterion of resolution, is valid for high aperture optics and arbitrary fluorescence wavelength.     

High resolution protein localization using soft X-ray microscopy

Soft X-ray microscopes can be used to examine whole, hydrated cells up to 10 µm thick and produce images approaching 30 nm resolution. Since cells are imaged in the X-ray transmissive 'water window', where organic material absorbs approximately an order of magnitude more strongly than water, chemical contrast enhancement agents are not required to view the distribution of cellular structures. Although living specimens cannot be examined, cells can be rapidly frozen at a precise moment in time and examined in a cryostage, revealing information that most closely approximates that in live cells. In this study, we used a transmission X-ray microscope at photon energies just below the oxygen edge (λ = 2.4 nm) to examine rapidly frozen mouse 3T3 cells and obtained excellent cellular morphology at better than 50 nm lateral resolution. These specimens are extremely stable, enabling multiple exposures with virtually no detectable damage to cell structures. We also show that silver-enhanced, immunogold labelling can be used to localize both cytoplasmic and nuclear proteins in whole, hydrated mammary epithelial cells at better than 50 nm resolution. The future use of X-ray tomography, along with improved zone plate lenses, will enable collection of better resolution (approaching 30 nm), three-dimensional information on the distribution of proteins in cells.     

Improved longitudinal resolution in tomographic diffractive microscopy with an ellipsoidal mirror

Tomographic diffractive microscopy is a technique, which is able to image transparent unstained samples with high resolution. The three‐dimensional distribution of the complex refractive index can be reconstructed quantitatively from the measured scattered fields under various illumination and detection angles, according to the diffraction tomography theorem. We propose a tomographic diffractive microscopy setup with an ellipsoidal mirror as the light collector. We demonstrate analytically and with numerical simulation that this approach permits to obtain images with drastically improved resolution.     

Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment

We compare the axial sectioning capability of multifocal confocal and multifocal multiphoton microscopy in theory and in experiment, with particular emphasis on the background arising from the cross‐talk between adjacent imaging channels. We demonstrate that a time‐multiplexed non‐linear excitation microscope exhibits significantly less background and therefore a superior axial resolution as compared to a multifocal single‐photon confocal system. The background becomes irrelevant for thin (< 15 µm) and sparse fluorescent samples, in which case the confocal parallelized system exhibits similar or slightly better sectioning behaviour due to its shorter excitation wavelength. Theoretical and experimental axial responses of practically implemented microscopes are given.     

A general model for resolution of digital holographic microscopy

For digital holographic microscopic imaging, the resolution in the reconstructed image is one of the most important parameters. To optimize the lateral resolution, a general model for the resolution of digital holographic microscopy (DHM) is proposed in this work, in which the effects of the sizes of each pixel, total area of the charge coupled device (CCD) and the microscopic objective lens are taken into account. Comparison between our model and others was carried out by calculating the point spread function (PSF) of DHM at different reconstruction distances and with different fill factors. It is shown that the effect of fill factors on the resolution of DHM becomes significant when the reconstruction distance is long. For high resolution DHM imaging the influence of fill factors must be taken into account when estimating the resolution of the reconstructed image. Furthermore, It is also demonstrated that the sidelobe of PSF can be cut effectively choosing appropriate values of the fill factors. Finally, the reconstructions of polyethylene microspheres have been implemented to demonstrate the theoretical analysis. These results obtained are helpful for estimation of the resolution and design of the DHM systems.     

The measurement and calculation of the X-ray spatial resolution obtained in the analytical electron microscope

The X-ray microanalytical spatial resolution is determined experimentally in various analytical electron microscopes by measuring the degradation of an atomically discrete composition profile across an interphase interface in a thin-foil of Ni-Cr-Fe. The experimental spatial resolutions are then compared with calculated values. The calculated spatial resolutions are obtained by the mathematical convolution of the electron probe size with an assumed beam-broadening distribution and the single-scattering model of beam broadening. The probe size is measured directly from an image of the probe in a TEM/STEM and indirectly from dark-field signal changes resulting from scanning the probe across the edge of an MgO crystal in a dedicated STEM. This study demonstrates the applicability of the convolution technique to the calculation of the microanalytical spatial resolution obtained in the analytical electron microscope. It is demonstrated that, contrary to popular opinion, the electron probe size has a major impact on the measured spatial resolution in foils < 150 nm thick.     

A numerical study of resolution and contrast in soft X-ray contact microscopy

We consider the case of soft X-ray contact microscopy using a laser-produced plasma. We model the effects of sample and resist absorption and diffraction as well as the process of isotropic development of the photoresist. Our results indicate that the micrograph resolution depends heavily on the exposure and the sample-to-resist distance. In addition, the contrast of small features depends crucially on the development procedure to the point where information on such features may be destroyed by excessive development. These issues must be kept in mind when interpreting contact microradiographs of high resolution, low contrast objects such as biological structures.