Phase contrast enhancement in microscopy using spiral phase filtering

We demonstrate two methods based on Fourier plane filtering using (a) a fractional spiral phase plate (SPP) and (b) an off-axial SPP for phase contrast enhancement in optical microscopy. In comparison to previous works, a spatially incoherent LED is used in the Köhler illumination as the light source to illuminate the biological specimen. We demonstrate that both these methods can transform the phase specimen into a relief-like view even under such illumination. The degree and orientation of enhancement can be controlled by changing the phase structure of the filter. The SPP is displayed on a phase-only spatial light modulator, and can be integrated into the optical path of standard microscopes.     

Spatial and temporal coherence effects in interference microscopy and full‐field optical coherence tomography

Low‐coherence optical microscopy or optical coherence microscopy uses light with short coherence length. The well‐known case is: “white‐light interferometry”, which became recently more known as: “optical coherence tomography”. However, when lenses and microscope objectives are used to create interferometric images, in what is known classically as “interference microscopy” or today as “full‐field optical coherence tomography” the spatial coherence starts to play a critical role. In this article the coherence effects in low‐coherence optical microscopy are reviewed. As this technology is becoming increasingly publicized due to its importance in three‐dimensional imaging, particularly of scattering biological media and optical metrology, the understanding of the fundamental physics behind it is essential. The interplay between longitudinal spatial coherence and temporal coherence and the effects associated with them are discussed in detail particularly when high numerical apertures are used. An important conclusion of this study is that a high‐contrast, high‐resolution system for imaging of multilayered samples is the one that uses narrowband illumination and high‐NA objectives with an index‐matching fluid. Such a system, when combined with frequency‐domain operation, can reveal nearly real‐time three‐dimensional images, and is thus competitive with confocal microscopy.     

Synthetic holography in microscopy: opportunities arising from advanced wavefront shaping

The advent of computer-generated or synthetic holography has created a wealth of possibilities for wavefront shaping in optics. We discuss the impact this has had on optical microscopy. Synthetic Holographic Microscopy utilises wavefront shaping by a computer-generated ‘hologram’ (CGH) to modify light on the illumination or the detection side, or both. This enables modifications of the general sample appearance concerning contrast, resolution and other aspects. Multiplexing CGHs can perform several tasks at once, for instance splitting the image into sub-images corresponding to different depths in the sample, or displaying differently contrasted images of the sample, e.g. bright field, darkfield or (spiral) phase contrast, in different sub-images. We give an overview of the options and discuss the advantages and disadvantages of using programmable holographic elements inside an optical microscope.     

Shadow effects in spiral phase contrast microscopy

Recently it has been demonstrated that spatial filtering of images in microscopy with a spiral phase element in a Fourier plane of the optical path results in a strong edge enhancement of object structures. In principle the operation is isotropic, i.e., all phase edges of a sample object are highlighted simultaneously, independent of their local direction. However, here we demonstrate that the symmetry can be broken intentionally by controlling the phase of the central area of a spiral phase hologram, which is displayed at a computer controlled spatial light modulator. This produces an apparent shadow effect which can be rotated at video rate. The resulting relieflike impression of the sample topography with a longitudinal resolution in the subwavelength regime is demonstrated by imaging a standard low contrast test sample consisting of a human cheek cell.     

基于双螺旋点扩散函数工程的多焦点图像扫描显微

在传统共聚焦显微技术的基础上,图像扫描显微技术使用面阵探测器来代替单点探测器,结合虚拟数字针孔并利用像素重定位和解卷积图像重构算法将传统宽场显微镜的分辨率提高一倍,实现了高信噪比的超分辨共焦成像.但是,由于采用逐点扫描的方式,三维成像速度相对较慢,限制了其在活体样品成像中的应用.为了进一步提高图像扫描显微术的成像速度,本文提出了一种基于双螺旋点扩散函数工程的多焦点图像扫描显微成像方法和系统.在照明光路中,利用高速数字微镜器件产生周期分布的聚焦点阵对样品进行并行激发和快速二维扫描;在探测光路中,利用双螺旋相位片将激发点荧光信号的强度分布转换为双螺旋的形式;最终,利用后期数字重聚焦处理,从单次样品扫描数据中重构出多个样品层的超分辨宽场图像.在此基础上,利用搭建的系统分别对纤维状肌动蛋白和海拉细胞线粒体进行成像实验,证明了该方法的超分辨能力和快速三维成像能力.     

Full‐field X‐ray reflection microscopy of epitaxial thin‐films

Novel X‐ray imaging of structural domains in a ferroelectric epitaxial thin film using diffraction contrast is presented. The full‐field hard X‐ray microscope uses the surface scattering signal, in a reflectivity or diffraction experiment, to spatially resolve the local structure with 70 nm lateral spatial resolution and sub‐nanometer height sensitivity. Sub‐second X‐ray exposures can be used to acquire a 14 µm × 14 µm image with an effective pixel size of 20 nm on the sample. The optical configuration and various engineering considerations that are necessary to achieve optimal imaging resolution and contrast in this type of microscopy are discussed.     

Towards tender X‐rays with Zernike phase‐contrast imaging of biological samples at 50 nm resolution

X‐ray microscopy is a commonly used method especially in material science application, where the large penetration depth of X‐rays is necessary for three‐dimensional structural studies of thick specimens with high‐Z elements. In this paper it is shown that full‐field X‐ray microscopy at 6.2 keV can be utilized for imaging of biological specimens with high resolution. A full‐field Zernike phase‐contrast microscope based on diffractive optics is used to study lipid droplet formation in hepatoma cells. It is shown that the contrast of the images is comparable with that of electron microscopy, and even better contrast at tender X‐ray energies between 2.5 keV and 4 keV is expected.     

基于螺旋相位调制的非相干全息点扩散函数研究

分析了菲涅耳非相干相关全息(Fresnel incoherent correlation holography,FINCH)系统中纯相位空间光调制器(spatial light modulator,SLM)加载螺旋相位掩模时的点扩散函数.以氙灯为照明光源搭建了FINCH系统,电荷耦合器记录的点源全息图与点扩散函数模拟结果一致.采用该系统分别在SLM上加载双透镜掩模和螺旋相位调制双透镜掩模两种情况下对分辨率板和非染色洋葱细胞成像,给出了成像对比结果.结果表明:采用螺旋相位调制的FINCH系统可以在几乎不牺牲分辨率的情况下提高图像的边缘对比度;同样,对相位物体也可以实现图像的边缘提取和识别.该方法在实时监测活细胞的分裂、形变等方面具有重要应用前景.     

纳米分辨相干反斯托克斯拉曼散射显微成像

采用附加探测光声子耗尽法来实现超衍射极限相干反斯托克斯拉曼散射显微成像. 此方法引入一束环形分布的附加探测光来消耗点扩展函数周边的相干声子, 实现点扩展函数的改造, 从而达到超越衍射极限的空间分辨率. 为了获得更高的空间分辨率和更佳的相位匹配条件, 通常需采用高数值孔径物镜对抽运光、斯托克斯光和探测光进行聚焦, 此时标量衍射理论不再成立. 基于矢量衍射理论, 分析了线偏振光、圆偏振光先后经过螺旋相位片和高数值孔径物镜后的光强分布, 结果表明: 圆偏振光在高数值孔径物镜后焦平面的光强分布呈中心对称状, 较线偏振环形光更适合作为附加探测光. 此外, 采用全量子理论分析了附加探测光声子耗尽法. 结果表明: 当附加探测光与探测光强度比为80时, 成像系统的横向空间分辨率可以达到45 nm; 继续提高附加探测光强度, 空间分辨将进一步提高.     

Quantitative phase retrieval in X‐ray Zernike phase contrast microscopy

In recent years, increasing attention has been devoted to X‐ray phase contrast imaging, since it can provide high‐contrast images by using phase variations. Among the different existing techniques, Zernike phase contrast microscopy is one of the most popular phase‐sensitive techniques for investigating the fine structure of the sample at high spatial resolution. In X‐ray Zernike phase contrast microscopy, the image contrast is indeed a mixture of absorption and phase contrast. Therefore, this technique just provides qualitative information on the object, which makes the interpretation of the image difficult. In this contribution, an approach is proposed for quantitative phase retrieval in X‐ray Zernike phase contrast microscopy. By shifting the phase of the direct light by π/2 and 3π/2, two images of the same object are measured successively. The phase information of the object can then be quantitatively retrieved by a proper combination of the measured images. Numerical experiments were carried out and the results confirmed the feasibility of the proposed method. It is expected that the proposed method will find widespread applications in biology, materials science and so on.     

Hybrid Rayleigh,Raman and two‐photon excited fluorescence spectral confocal microscopy of living cells

A hybrid fluorescence–Raman confocal microscopy platform is presented, which integrates low‐wavenumber‐resolution Raman imaging, Rayleigh scatter imaging and two‐photon fluorescence (TPE) spectral imaging, fast ‘amplitude‐only’ TPE‐fluorescence imaging and high‐spectral‐resolution Raman imaging. This multi‐dimensional fluorescence–Raman microscopy platform enables rapid imaging along the fluorescence emission and/or Rayleigh scatter dimensions. It is shown that optical contrast in these images can be used to select an area of interest prior to subsequent investigation with high spatially and spectrally resolved Raman imaging. This new microscopy platform combines the strengths of Raman ‘chemical’ imaging with light scattering microscopy and fluorescence microscopy and provides new modes of correlative light microscopy. Simultaneous acquisition of TPE hyperspectral fluorescence imaging and Raman imaging illustrates spatial relationships of fluorophores, water, lipid and protein in cells. The fluorescence–Raman microscope is demonstrated in an application to living human bone marrow stromal stem cells. Copyright © 2009 John Wiley & Sons, Ltd.     

激光直写制作高阶螺旋相位板及其性能

将拓扑荷为4的螺旋相位光束与平面光干涉的计算全息图输入到空间光调制器中,得到含有多个衍射级次的高阶涡旋光束。为提高衍射效率,利用激光直写技术制作拓扑荷为4的高阶螺旋相位板,经测定,相位板深度理论数值为1.073 m,测量数值为1.082 m,相位板制作误差在0.83%以内。平行光束通过此相位板时,在夫琅和费衍射场获得一个高质量的高阶光学涡旋,光强分布与理论数值基本吻合,衍射效率达到86%。     

Photoacoustic microscopy

Photoacoustic microscopy (PAM) is a hybrid in vivo imaging technique that acoustically detects optical contrast via the photoacoustic effect. Unlike pure optical microscopic techniques, PAM takes advantage of the weak acoustic scattering in tissue and thus breaks through the optical diffusion limit (∼1 mm in soft tissue). With its excellent scalability, PAM can provide high‐resolution images at desired maximum imaging depths up to a few millimeters. Compared with backscattering‐based confocal microscopy and optical coherence tomography, PAM provides absorption contrast instead of scattering contrast. Furthermore, PAM can image more molecules, endogenous or exogenous, at their absorbing wavelengths than fluorescence‐based methods, such as wide‐field, confocal, and multi‐photon microscopy. Most importantly, PAM can simultaneously image anatomical, functional, molecular, flow dynamic and metabolic contrasts in vivo. Focusing on state‐of‐the‐art developments in PAM, this Review discusses the key features of PAM implementations and their applications in biomedical studies.     

Image edge-enhancement in optical microscopy with a phase mismatched spiral phase plate

We present a spiral phase filtering system with a large tolerance for edge enhancement of both phase and amplitude objects in optical microscopy.The method is based on a Fourier 4-f spatial filtering system.A phase mismatched spiral phase plate (SPP) fabricated by electron beam lithography is employed as the radial Hilbert transform for image edge enhancement.Compared with holography,SPP is simple,economical,reliable,and easy to integrate.     

Optical Fourier techniques for medical image processing and phase contrast imaging

This paper briefly reviews the basics of optical Fourier techniques (OFT) and applications for medical image processing as well as phase contrast imaging of live biological specimens. Enhancement of microcalcifications in a mammogram for early diagnosis of breast cancer is the main focus. Various spatial filtering techniques such as conventional 4f filtering using a spatial mask, photoinduced polarization rotation in photosensitive materials, Fourier holography, and nonlinear transmission characteristics of optical materials are discussed for processing mammograms. We also reviewed how the intensity dependent refractive index can be exploited as a phase filter for phase contrast imaging with a coherent source. This novel approach represents a significant advance in phase contrast microscopy.     

Dark-field optical coherence microscopy

Dark-field illumination is known to enhance scattering contrast in optical microscopy. We combined this concept with Fourier domain optical coherence microscopy (OCM). The detection and illumination paths are decoupled, and only the scattered light originating from the sample generates the tomogram signal, whereas any specular reflection is highly suppressed. We analyze and discuss this dark-field OCM concept and present its superior imaging quality on live cell samples.     

When optical microscopy meets all-optical analog computing: A brief review

As a revolutionary observation tool in life science, biomedical, and material science, optical microscopy allows imaging of samples with high spatial resolution and a wide field of view. However, conventional microscopy methods are limited to single imaging and cannot accomplish real-time image processing. The edge detection, image enhancement and phase visualization schemes have attracted great interest with the rapid development of optical analog computing. The two main physical mechanisms that enable optical analog computing originate from two geometric phases: the spin-redirection Rytov-Vlasimirskii-Berry (RVB) phase and the Pancharatnam-Berry (PB) phase. Here, we review the basic principles and recent research progress of the RVB phase and PB phase based optical differentiators. Then we focus on the innovative and emerging applications of optical analog computing in microscopic imaging. Optical analog computing is accelerating the transformation of information processing from classical imaging to quantum techniques. Its intersection with optical microscopy opens opportunities for the development of versatile and compact optical microscopy systems.     

Non-conventional joint-transform correlations for pattern recognition by use of grating filters and heterodyne scanning

The spatial light modulators (SLMs) at the Fourier planes of a conventional joint-transform correlation (JTC) are eliminated using simple grating filters such as rectangular and triangular ones. To achieve this goal, two non-conventional real-time JTC architectures are proposed: (a) the grating filter is used along with a heterodyning and one-dimensional optical scanning technique to capture the cross-correlation functions of the input images without major processing and (b) the one-dimensional optical scanning is eliminated to achieve a faster (but a little bit more complicated) processing. The proposed techniques significantly reduce the time processing needed for real-time applications by eliminating the drawbacks of the non-ideal characteristics of the SLMs.     

表面等离激元结构光照明显微成像技术研究进展

结构光照明显微成像技术(SIM)因其高分辨、宽场、快速成像的优势,在生物医学成像领域发挥了不可估量的作用.结构光照明显微成像技术与动态可控的亚波长表面等离激元条纹相结合,可以在不借助非线性效应的情况下,将传统SIM的分辨率从2倍于衍射极限频率提升到3 4倍,此外还有抑制背景噪声、提升信噪比的能力,在近表面的生物医学成像应用中有重要价值.本文介绍了表面等离激元结构光照明显微成像技术的原理,并总结了近几年国内外的相关研究进展.     

Transient absorption microscopy: advances in chemical imaging of photoinduced dynamics

This article reviews the state of the art of ultrafast transient absorption microscopy, discusses current experimental concepts and highlights future challenges. The advantages of transient absorption microscopy over other micro‐spectroscopic techniques are its high optical resolution combined with high temporal resolution as well as its ability to study non‐fluorescent and weakly fluorescent molecular species and to probe excited‐state processes. In conventional transient absorption spectroscopy the spectroscopic information usually presents a spatial average over the focal spot of the typically weakly focused probe beam. Transient absorption microscopy, however, enables investigations of the excited state dynamics in individual microscopic areas of a sample. Hence, the technique does not only yield detailed morphological information based on a label‐free molecular contrast, but also gives insight into the ultrafast morphology‐dependent photoinduced processes in heterogeneous samples. Different variations of transient absorption microscopy have found a number of applications ranging from material sciences to biology, which are discussed in this review together with different setup modifications and approaches towards transient absorption spectroscopy with spatial resolution below the diffraction limit.