Single-shot, simultaneous incoherent and holographic microscopy

In this work, we demonstrate single-shot, simultaneous recording and subsequent retrieval of one incoherent and two holographic (intensity and phase) images from the same camera frame. Demultiplexing of incoherent and holographic signals in the spatial frequency domain is made possible by carrier frequency modulation and spatial oversampling intrinsic to the off-axis digital holographic configuration. In particular, we show applications to combined fluorescence and digital holographic microscopy, as well as combined bright field and holographic second harmonic generation microscopy.     

The inhomogeneity of the icosahedral phase in Al-Mn and Al-V

The inherent differences between the various current structural models for the determination of the scattering behaviour of icosahedral materials are reviewed with the conclusion that the nature of their apparently characteristic inhomogeneity needs further clarification. Here it is shown that the dark field reflection and deviation parameter dependence of transmission electron microscope (TEM) contrast variations can be used to provide useful qualitative evidence for the nature of quasicrystal inhomogeneities. Results obtained using the technique are discussed for the icosahedral phases i-(AlMn) and i-(AlV).     

The fractional Talbot effect in differential x‐ray phase‐contrast imaging for extended and polychromatic x‐ray sources

The influence of different physical parameters, such as the source size and the energy spectrum, on the functional capability of a grating interferometer applied for phase‐contrast imaging is discussed using numerical simulations based on Fresnel diffraction theory. The presented simulation results explain why the interferometer could be well combined with polychromatic laboratory x‐ray sources in recent experiments. Furthermore, it is shown that the distance between the two gratings of the interferometer is not in general limited by the width of the photon energy spectrum. This implies that interferometers that give a further improved image quality for phase measurements can be designed, because the primary measurement signal for phase measurements can be increased by enlargement of this distance. Finally, the mathematical background and practical instructions for the quantitative evaluation of measurement data acquired with a polychromatic x‐ray source are given.     

基于小波分析的显微序列图像合成系统

由于显微镜物镜焦深范围有限,故造成显微图像局部模糊,研究人员对图像的理解不完整等问题。根据聚焦评价函数分布,采用小波分析,将一序列局部聚焦图像合成全焦图像。改进的小波融合算法得到的融合图像极大地保留了系列图像聚焦部分的信息,合成全焦图像。由此得出的聚焦合成图像使研究人员可直接测量所需的显微结果。     

Autofocusing in computer microscopy: selecting the optimal focus algorithm

Autofocusing is a fundamental technology for automated biological and biomedical analyses and is indispensable for routine use of microscopes on a large scale. This article presents a comprehensive comparison study of 18 focus algorithms in which a total of 139,000 microscope images were analyzed. Six samples were used with three observation methods (brightfield, phase contrast, and differential interference contrast (DIC)) under two magnifications (100x and 400x). A ranking methodology is proposed, based on which the 18 focus algorithms are ranked. Image preprocessing was also conducted to extensively reveal the performance and robustness of the focus algorithms. The presented guidelines allow for the selection of the optimal focus algorithm for different microscopy applications.     

Wavefronts in the focus of a light microscope

We report the visualization of the wavefronts of light in the focal region of an objective lens. The focused wavefronts are measured for the highest available numerical aperture, i.e, 1·4 oil immersion. The measurement is accomplished by scanning a scattering probe through the focal region of a lens illuminated by plane waves. The scattered light is coherently added to an external reference beam and focused into a point-like detector. The registered signal allows the determination of the three-dimensional shape of the wavefronts in the focal region and their evolution in time.     

热透镜效应对激光光束聚焦的影响

利用高斯光束传播的ABCD定律,分析了热透镜效应对高斯光束聚焦的影响,对两种腔型做了数值计算和分析,并计算了不同焦距透镜对高斯光束聚焦后的腰斑半径和腰斑位置。通过计算分析表明对于同一高斯光束用短焦距透镜聚焦后得到的光斑比用长焦距透镜聚焦得到的光斑更稳定。     

Defocusing microscopy

Transparent objects (phase objects) are not visible in a standard brightfield optical microscope. In order to see such objects the most used technique is phase-contrast microscopy. In phase-contrast microscopy the contrast observed is proportional to the optical path difference introduced by the object. If the index of refraction is uniform, phase-contrast microscopy then yields a measure of the thickness profile of phase objects. We show that by slightly defocusing an optical microscope operating in brightfield, phase objects become visible. We modeled such an effect and show that the image contrast of a phase object is proportional to the amount of defocusing and proportional to the two-dimensional Laplacian of the optical path difference introduced by the object. For uniform index of refraction, defocusing microscopy then yields a measure of the curvature profile of phase objects. We extended our previous model for thin objects to thick objects. To check our theoretical model, we use as phase objects polystyrene spherical caps and compare their curvature radii obtained by defocusing microscopy (DM) to those obtained with atomic force microscopy (AFM). We also show that for thick curved phase objects one can reconstruct their thickness profiles from DM images. We illustrate the utility of defocusing microscopy in biological systems to study cell motility. In particular, we visualize and quantitatively measure real-time cytoskeleton curvature fluctuations of macrophages (a cell of the innate immune system). The study of such fluctuations might be important for a better understanding of the engulfment process of pathogens during phagocytosis.     

Improving focus measurements using logarithmic image processing

The logarithmic image processing (LIP) model is a mathematical framework which provides algebraic and functional operations for the processing of intensity images valued in a bounded range. The LIP model has been proved to be physically consistent, most notably with some image formation models and several laws and characteristics of human brightness perception. This paper addresses the image focus measurement problem using the LIP model. The three most classical image focus measurements: the sum-modified-Laplacian, the tenengrad and the variance, which aim at estimating the degree of focus of an acquired image by emphasizing and quantifying its sharpness information, are considered and reinterpreted using the LIP framework. These reinterpretations notably make attempts at evaluating degrees of focus in terms of human brightness (sensation) from physical light stimuli. Their potential is illustrated and validated on shape-from-focus issues on both simulated data and real acquisitions in digital optical microscopy. The concept of shape-from-focus involves recovering the shape of an observed thick sample by locally maximizing a focus measurement throughout a sequence of differently focused images. Finally, it is shown that the LIP-based focus measurements clearly outperform their respective classical ones.