Shack-Hartmann wave front measurements in cortical tissue for deconvolution of large three-dimensional mosaic transmitted light brightfield micrographs

We present a novel approach for deconvolution of 3D image stacks of cortical tissue taken by mosaic/optical‐sectioning technology, using a transmitted light brightfield microscope. Mosaic/optical‐sectioning offers the possibility of imaging large volumes (e.g. from cortical sections) on a millimetre scale at sub‐micrometre resolution. However, a blurred contribution from out‐of‐focus light results in an image quality that usually prohibits 3D quantitative analysis. Such quantitative analysis is only possible after deblurring by deconvolution. The resulting image quality is strongly dependent on how accurate the point spread function used for deconvolution resembles the properties of the imaging system. Since direct measurement of the true point spread function is laborious and modelled point spread functions usually deviate from measured ones, we present a method of optimizing the microscope until it meets almost ideal imaging conditions. These conditions are validated by measuring the aberration function of the microscope and tissue using a Shack‐Hartmann sensor. The analysis shows that cortical tissue from rat brains embedded in Mowiol and imaged by an oil‐immersion objective can be regarded as having a homogeneous index of refraction. In addition, the amount of spherical aberration that is caused by the optics or the specimen is relatively low. Consequently the image formation is simplified to refraction between the embedding and immersion medium and to 3D diffraction at the finite entrance pupil of the objective. The resulting model point spread function is applied to the image stacks by linear or iterative deconvolution algorithms. For the presented dataset of large 3D images the linear approach proves to be superior. The linear deconvolution yields a significant improvement in signal‐to‐noise ratio and resolution. This novel approach allows a quantitative analysis of the cortical image stacks such as the reconstruction of biocytin‐stained neuronal dendrites and axons.     

High‐resolution wide‐field microscopy with adaptive optics for spherical aberration correction and motionless focusing

Live imaging in cell biology requires three‐dimensional data acquisition with the best resolution and signal‐to‐noise ratio possible. Depth aberrations are a major source of image degradation in three‐dimensional microscopy, causing a significant loss of resolution and intensity deep into the sample. These aberrations occur because of the mismatch between the sample refractive index and the immersion medium index. We have built a wide‐field fluorescence microscope that incorporates a large‐throw deformable mirror to simultaneously focus and correct for depth aberration in three‐dimensional imaging. Imaging fluorescent beads in water and glycerol with an oil immersion lens we demonstrate a corrected point spread function and a 2‐fold improvement in signal intensity. We apply this new microscope to imaging biological samples, and show sharper images and improved deconvolution.     

A method of PSF generation for 3D brightfield deconvolution

This paper addresses the problem of 3D deconvolution of through focus widefield microscope datasets (Z‐stacks). One of the most difficult stages in brightfield deconvolution is finding the point spread function. A theoretically calculated point spread function (called a ‘synthetic PSF’ in this paper) requires foreknowledge of many system parameters and still gives only approximate results. A point spread function measured from a sub‐resolution bead suffers from low signal‐to‐noise ratio, compounded in the brightfield setting (by contrast to fluorescence) by absorptive, refractive and dispersal effects. This paper describes a method of point spread function estimation based on measurements of a Z‐stack through a thin sample. This Z‐stack is deconvolved by an idealized point spread function derived from the same Z‐stack to yield a point spread function of high signal‐to‐noise ratio that is also inherently tailored to the imaging system. The theory is validated by a practical experiment comparing the non‐blind 3D deconvolution of the yeast Saccharomyces cerevisiae with the point spread function generated using the method presented in this paper (called the ‘extracted PSF’) to a synthetic point spread function. Restoration of both high‐ and low‐contrast brightfield structures is achieved with fewer artefacts using the extracted point spread function obtained with this method. Furthermore the deconvolution progresses further (more iterations are allowed before the error function reaches its nadir) with the extracted point spread function compared to the synthetic point spread function indicating that the extracted point spread function is a better fit to the brightfield deconvolution model than the synthetic point spread function.     

Adaptive optics in spinning disk microscopy: improved contrast and brightness by a simple and fast method

Multiconfocal microscopy gives a good compromise between fast imaging and reasonable resolution. However, the low intensity of live fluorescent emitters is a major limitation to this technique. Aberrations induced by the optical setup, especially the mismatch of the refractive index and the biological sample itself, distort the point spread function and further reduce the amount of detected photons. Altogether, this leads to impaired image quality, preventing accurate analysis of molecular processes in biological samples and imaging deep in the sample. The amount of detected fluorescence can be improved with adaptive optics. Here, we used a compact adaptive optics module (adaptive optics box for sectioning optical microscopy), which was specifically designed for spinning disk confocal microscopy. The module overcomes undesired anomalies by correcting for most of the aberrations in confocal imaging. Existing aberration detection methods require prior illumination, which bleaches the sample. To avoid multiple exposures of the sample, we established an experimental model describing the depth dependence of major aberrations. This model allows us to correct for those aberrations when performing a z‐stack, gradually increasing the amplitude of the correction with depth. It does not require illumination of the sample for aberration detection, thus minimizing photobleaching and phototoxicity. With this model, we improved both signal‐to‐background ratio and image contrast. Here, we present comparative studies on a variety of biological samples.     

基于半盲解卷积复原的高分辨率视网膜成像系统

为获得高分辨率视网膜图像,建立了基于自适应光学的视网膜成像系统,并以成像时获得的残余像差作为图像复原的估计参数,通过半盲解卷积进行图像复原以获得高质量图像.通过Hartmann-Shark波前传感器和微机械薄膜变形镜组成的自适应光学系统对活体人眼像差进行测量与校正,并在成像时记录系统残余像差,据此重建光学传递函数作为图像复原模型初始参数估计,对获得的视网膜图像进行条件约束迭代半盲解卷积复原,消除像差对成像质量的影响,从而得到高分辨率视网膜图像.实验表明,系统获得的图像经该方法处理后可获得较满意视网膜图像,图像质量提高近一倍,成像成功率由38％提高至78％,成像时间缩短为原来的1/7.该方法在满足使用要求的前提下有效缩短了校正时间,提高了成像的成功率,提升了系统的适用范围.     

The resolution of photoelectron microscopes with UV, X-ray, and synchrotron excitation sources

The resolution of emission electron microscopes is calculated by determining the intensity distribution in the image. The object is a small disc of uniform brightness centered on the axis. A finite object, as distinct form a point source, provides a non-zero current in the image without the requirement of infinite object brightness and the consequent infinities in the geometrical intensity distribution. The minimum object size, which in turn affects the resolution of the microscope, depends on the minimum current or contrast required in the image. In photoelectron microscopes with UV illumination just above the threshold for emission the predominant aberrations are the chromatic and spherical aberrations of the accelerating field and the spherical aberration of the objective lens. For higher energies, e.g. in the soft X-ray range, the chromatic aberration of the objective lens must also be taken into account, as the aberration coefficients of the accelerating field are greatly reduced. The intensity distributions in the image are calculated first for single energies. The intensity distribution for a beam with a range of energies is obtained by adding a series of single-energy distribution curves weighted according to the energy distribution function. In the presence of spherical aberration the position of the image formed by the electrons depends on the angle of emission. In image planes between the paraxial and marginal planes the combination of spherical aberration and defocus causes the the image spot to have a retrograde type of behavior as the angle of emission increases. The image spot initially moves away from the axis in the azimuth of emission and then returns to the axis and moves away in the opposite azimuth. As a result the intensity in the central portion of the image plane is enhanced. The single-energy intensity distribution curves calculated as a function of depth in the image reveal the existence of a compact, high-intensity image peak in an image plane located between the paraxial and marginal planes. This peak occurs in the plane in which the image spot has a maximum retrograde displacement equal to its radius. The present analysis shows that the resolution in the high-intensity plane is better than in the plane of least confusion, and the effects of aberrations in these two planes are quite different.(ABSTRACT TRUNCATED AT 400 WORDS)     

On the importance of fifth-order spherical aberration for a fully corrected electron microscope

Next generation aberration correctors will not only eliminate the third-order spherical aberration, but also improve the information limit by correction of chromatic aberration. As a result of these improvements, higher order aberrations, which have largely been neglected in image analysis, will become important. In this paper, we concern ourselves with situations where sub-A resolution can be achieved, and where the third-order spherical aberration is corrected and the fifth-order spherical aberration is measurable. We derive formulae to explore the maximum value of the fifth-order spherical aberration for directly interpretable imaging and discuss the optimum imaging conditions and their applicable range.     

High-resolution imaging with an aberration-corrected transmission electron microscope

Recently an electromagnetic hexapole system for the correction of the spherical aberration of the objective lens of a 200 kV transmission electron microscope has been constructed by Haider and coworkers. By appropriately exciting the hexapole elements it is possible to adjust specific values of the spherical aberration coefficient ranging from the value of the original uncorrected instrument over zero even to negative values. In the first part of the paper the consequences of the tunable spherical aberration are investigated. New imaging modes are available: By adjustment of an optimum value for the spherical-aberration coefficient, the point resolution of phase-contrast imaging can be extended to the information limit. Phase-contrast imaging can be improved by a reduced level of contrast delocalisation. For zero aberration contrast delocalisation does not occur. In this case high-resolution investigations are carried out under amplitude-contrast conditions, where the local image intensity of crystalline objects is controlled by electron diffraction channelling. The defocus and spherical aberration values related to the new imaging modes are given. In the second part novel applications of the instrument to semiconductor heterostructures and ceramic grain boundaries are examined.     

Subject index to volume 10

The scanning transmission electron microscope with a field emission electron source operated at 100 kV allows X-ray microanalysis using electron probes as small as 1 to 2 nm. Measurements of the probe in a Vacuum Generators HB-501 STEM show that spherical aberration in the objective lens controls the probe size and shape at beam convergence half-angles of 10 mrad and greater typically used for X-ray microanalysis. A virtual objective aperture eliminates X-ray contributions from the probe-forming system, but must be aligned exactly to avoid asymmetrical broadening of the probe by spherical aberration. It is estimated that 5 nm X-ray spatial resolution can be achieved in low to medium atomic number materials. Even at this resolution however, probe broadening in the specimen controls the resolution; the main limitation is one of specimen preparation and a knowledge of the final specimen thickness. Determination of composition profiles near voids, dislocations and other individual defects in thin foils also requires a knowledge of the defect depth position and deconvolution of the probe and composition profiles.