Improvement in the resolution of three-dimensional data sets collected using optical serial sectioning

In this paper an approach for improving the quality of 3-D microscopic images obtained through optical serial sectioning is described and implemented. A serially sectioned image is composed of a sequence of 2-D images obtained by incrementing the focusing plane of the microscope through the specimen of interest; ideally, the image obtained at each focusing plane should be in focus, and should contain information lying only within that plane. In practice, however, the images obtained contain redundant information from neighbouring focusing planes and are blurred by a three-dimensional low-pass distortion. These degradations are a consequence of the limited aperture of any optical system; using principles of geometric optics and allowing for the passage of light through the specimen, we are able to demonstrate that the microscope distortion can be described as a linear system, if the absorption of the specimen is assumed to be linear and non-diffractive. The transfer function of the microscope is found to zero a biconic region of 3-D spatial frequencies orientated along the optical axis; a closed-form expression is derived for the low-pass transfer function of the microscope outside the region of missing frequencies. The planar resolution of the serial sections can be greatly improved by convolving the image obtained with the inverse of the low-pass distortion function, although the missing cone of frequencies is not recoverable. The reconstruction technique is demonstrated using both simulated images, to demonstrate more clearly the effects of the distortion and the accuracy of the subsequent reconstruction, and actual experiments with a pollen grain and a stained preparation of human cerebellum tissue.     

Why don't high-resolution simulations and images match?

Computer simulations have been used for many years to understand experimental high-resolution electron microscope images in a qualitative fashion, but the trend nowadays has been to attempt more quantitative image matching. This has led to the discovery that the contrast in experimental images is much less than in simulated images, typically by a factor of about three. There are many possible causes for this discrepancy, ranging from the mechanisms of scattering of electrons by the specimen through the calculations of the diffracted beam intensities and their focusing by the objective lens to the point spread function of the recording device. No single cause can explain all of the experimental contrast loss, although a combination of many factors could.     

A confocal laser microscope scanner for digital recording of optical serial sections

A confocal laser microscope scanner developed at our institute is described. Since an ordinary microscope is used, it is easy to view the specimen prior to scanning. Confocal imaging is obtained by laser spot illumination, and by focusing the reflected or fluorescent light from the specimen onto a pinhole aperture in front of the detector (a photomultiplier tube). Two rotating mirrors are used to scan the laser beam in a raster pattern. The scanner is controlled by a microprocessor which coordinates scanning, data display, and data transfer to a host computer equipped with an array processor. Digital images with up to 1024 × 1024 pixels and 256 grey levels can be recorded. The optical sectioning property of confocal scanning is used to record thin (～ 1 μm) sections of a specimen without the need for mechanical sectioning. By using computer-control to adjust the focus of the microscope, a stack of consecutive sections can be automatically recorded. A computer is then used to display the 3-D structure of the specimen. It is also possible to obtain quantitative information, both geometric and photometric. In addition to confocal laser scanning, it is easy to perform non-confocal laser scanning, or to use conventional microscopic illumination techniques for (non-confocal) scanning. The design has proved reliable and stable, requiring very few adjustments and realignments. Results obtained with this scanner are reported, and some limitations of the technique are discussed.     

Convolution and correlation: a case study of scanning imaging and analysis systems

The relationship between convolution/correlation operation and the data acquisition process of the scanning microscope and spectrometer families is analyzed. It is shown that a coordinate or event sensitive detector, and the intrinsic or extrinsic property of the specimen response, are two important factors in distinguishing the data acquisition mode of such systems. Four types of convolution- and correlation-based modes are extracted to illuminate the physical characteristics of scanning imaging and analysis systems by focusing on the probe, specimen, detector, and their relationships. Criteria for identifying these modes are explored. In addition, the physical meanings of general existing coefficients between the independent variables of convolution and correlation are investigated.     

A study on observation and growth behavior of small surface cracks by remote measurement system

A new experimental system using remote measurement system (RMS) and image processing technique was applied for studying the growth behavior of small surface fatigue cracks in 1Cr ?1Mo?0.25V steel at room temperature. The system includes a long distance focusing microscope, a CCD camera, a light source, a 3-axis controller a monitor, a personal computer and a data translation card. The measurement error of the system appeared to be 0.8%. It is possible for this system to measure down to 30 μm of surface fatigue crack length. The length of surface fatigue crack could be successfully measured by the RMS during testing as well as after the test. The growth rate of small cracks on smooth specimens was represented in terms of stress intensity factor and J-integral. At equivalent elastic stress intensity factor levels, the growth rate of small surface cracks was not consistent and faster than that of long crack of CT specimen.     

In situ topography measurement in the SEM

An automatic technique for measuring heights in situ in the SEM based on a combination of stereo-metric and focusing methods has been developed. A pair of images of a surface element on the specimen is obtained by tilting the beam electrically in a manner such that the plane containing the tilt axis is coincident with the focal plane of the final lens. Cross-correlation is used to determine the parallax between the image pair which is then used to iteratively correct the height of the tilt axis by changing the lens focus. As a result, the lens focus tracks the specimen topography. With an appropriate specimen surface containing high resolution features for image correlation, the technique is capable of maintaining both its lateral and vertical resolutions over several decades of height displacement up to 100 μm. In an experimental system based on a commercial electron-optical column, spot, line profile, and three-dimensional measurements have been demonstrated.     

Automated electron tomography with scanning transmission electron microscopy

We report the successful implementation of a fully automated tomographic data collection system in scanning transmission electron microscopy (STEM) mode. Autotracking is carried out by combining mechanical and electronic corrections for specimen movement. Autofocusing is based on contrast difference of a focus series of a small sample area. The focus gradient that exists in normal images due to specimen tilt is effectively removed by using dynamic focusing. An advantage of STEM tomography with dynamic focusing over TEM tomography is its ability to reconstruct large objects with a potentially higher resolution.     

Automatic focus correction for spot-scan imaging of tilted specimens.

The variation in defocus within an image of a highly tilted specimen can be a serious source of artifact. Spot-scan imaging can be combined with dynamic focusing to greatly reduce this range of defocus. A protocol is described for determining the parameters required for the automatic focus compensation during the recording of a spot-scan image. Images of a gold test specimen demonstrate the efficacy of this procedure in extending the area of the image that contains high-quality data. In case the tilt angle or resolution is high enough that the height difference of the specimen within each small illuminated area is larger than the depth of field, the image must be treated to compensate for the focus variation. The same principle is used as was developed for compensation of conventional images of tilted specimens.     

Determination of the principal distance and the location of the perspective centre in low magnification SEM photogrammetry.

The principal distance, D, from the centre of perspective in the SEM optical projection to the tilt axis of the specimen stage must be accurately determined before photogrammetric evaluation of stereoscopic pairs of micrographs can proceed. A precise procedure for measuring D is described in which the specimen stage X micrometer is used to measure the width of the field scanned for a particular width of the CRT, when the specimen stage is moved along the electron beam axis by amounts measured with the stage Z micrometer. The Z micrometer is calibrated with an external dial gauge. A plot of field width against Z extrapolated to zero gives the location of the perspective centre. In SEM photogrammetry, it is usual to leave the lens currents unchanged whilst recording the stereo-pairs. The values of D measured with a constant final lens current show that the perspective centre is located close to the final aperture in its conventional position. Previous determinations of D for Stereoscans have used a changing lens current to keep the specimen in focus at varying Z, and found a virtual centre several millimetres above the final aperture. The value of D so obtained should only be used if the micrographs were recorded with dynamic or automatic focusing systems.     

Estimation of section thickness and quantification of iron standards with EELS

The possibilities of using electron energy-loss spectroscopy (EELS) for quantification of elemental concentrations in ultrathin sections are examined. Dynabeads, which are polystyrene beads with a known iron content, are proposed as internal iron standards. The quantity of an element present depends on the thickness of the specimen. A prerequisite for estimation of absolute section thicknesses with EELS is the knowledge of the mean free path λ of electrons in the specimen. This factor is determined for the embedding resins Epon and Nanoplast by comparing EELS data with directly observed thicknesses in re-embedded sections. Dynabeads were found to include iron in a homogeneous distribution and to be stable in the electron beam.