基于贝叶斯自适应估计的光子计数集成成像

针对光子数极少环境下三维目标的重构问题,基于光子计数集成成像系统提出了一种贝叶斯自适应估计方法,来提高三维目标深度切片的重构质量。首先,通过光子计数集成成像系统获得一系列光子计数元素图像。接着,从光子计数过程的泊松分布出发,利用集成成像系统中对于同一个目标像素的多次采样特性,引入了局部自适应均值因子,从而建立起元素图像像素光子数估计的单参数后验概率模型。最后,通过后验概率模型的均值计算获得更新后的光子计数元素图像,并基于光束可逆原理重构出深度切片图像。实验结果表明:采用该方法在场景的两个深度处重构的切片图像相比传统贝叶斯重构图像的峰值信噪比提高了7.4dB和8.5dB,极大地提升了微弱光三维目标的重构质量。     

三维内部构造显微镜设计

通过连续切片获取生物体二维断层图像然后进行三维重建日益成为生物医学中研究生物体内部三维立体结构的主要途径.在对显微镜用薄片切片机结构及三维重建技术的研究的基础上,本文提出了三维内部构造显微镜的设计方案,解决了生物切片过程中的样品定位、样品切削、断层图像采集、系统控制诸多问题.以本系统获得的二维断层图像为原始数据,利用自行设计三维重建软件,得到了真彩的表面重建和任意切面重建结果.     

基于工业计算机断层成像技术的三维CAD模型重构方法

为解决复杂形状产品的三维计算机辅助设计模型的重构难题,提出了一种基于工业计算机断层成像技术的三维计算机辅助设计模型的重构方法.首先用工业计算机断层成像技术对产品进行扫描,得到产品切片图像,然后通过切片图像获取体数据;在采用高斯滤波对体数据进行预处理后,使用移动立方体算法重建三维表面,并用顶点删除法和二次误差测度算法简化三维表面;在采用Laplacian算法平滑三维表面后,将三维表面模型保存为STL格式的文件;最后,将STL格式的文件导入到UG中,重构出产品的三维计算机辅助设计模型.实际应用验证了该方法的有效性和正确性.     

一种新颖的微机电系统微结构显微图像三维自动拼接方法

提出一种基于白光垂直扫描显微镜(White-light vertical scanning interference microscopy,WVSIM)的微机电系统(Micro electro mechanical systems,MEMS)微结构三维自动拼接方法,来获得包含完整MEMS信息的大视场、高分辨率的MEMS微结构显微图像,以满足MEMS微结构功能特征分析、评定的需要。通过分析白光垂直扫描显微成像原理,将MEMS微结构显微图像的三维拼接分解为x-y向拼接与z向高度校正。将二维图像配准的尺度不变特征变换特征匹配算法应用到MEMS微结构显微图像的三维拼接中,实现MEMS微结构显微图像的智能、鲁棒三维自动拼接。试验表明,该方法不需要超高精度的硬件,解决了任意形状MEMS微结构显微图像的高精度三维自动拼接问题。横向拼接精度可达0.8μm,纵向拼接精度小于1 nm。     

扫描多光谱显微图像的重构及处理

本文介绍了一种新的显微成像一扫描多光谱显微成像的基本原理,着重描述了显微成像中多光谱图像的特点以及图像的获取和重构方法,最后介绍了系统软件的编程。     

邻域法在三维显微图像复原中的应用研究

三维显微图像复原方法通常分为两类邻域法和三维去卷积法.本文详细介绍了邻域法的基本原理;对邻域法公式中参数c,α的不同选择和相应的复原结果进行了评价,通过仿真实验得出了考虑不同切片数时c,α的最佳选择以及不同显微成像系统所需考虑切片数M的最佳选择.与三维去卷积法相比,邻域法具有简单、计算量小,容易实现等优点,适合于观察活的生物和进行实时连续观察.     

ApoTome光学成像系统

荧光显微镜已被广泛地应用于生物样本的荧光成像。然而在观察较厚样本时,来自非焦平面的荧光信息会使荧光图像变得模糊、对比度不够、背景较亮,甚至有的时候一些很重要的结构因此被掩盖而无法被观察到。目前有很多种光学切片方法可以用来去除这种非焦平面荧光的影响,例如激光共聚焦扫描显微技术或3D去卷积技术等。本文介绍了一种可以在普通荧光显微镜上实现光学切片成像的方法,并着重介绍了ApoTome光学成像系统的构造和原理。     

光子计数三维成像激光雷达反转误差的校正

实施光子飞行时间测量法时,光子飞行时间测量值受激光回波信号能量的影响会出现测量反转误差,从而影响系统三维成像的精度.本文描述了一种光子计数三维成像激光雷达系统反转误差的校正方法及其实验.提出的反转误差校正方法包含先验模型标定和反转误差校正两个步骤.首先,通过标定法得到系统反转误差相对于激光脉冲响应率的函数关系,建立系统的反转误差预测函数.然后,由系统反转误差函数预测出原始三维图像的反转误差图像并实现原始三维图像的反转误差校正.实验搭建了光子计数三维成像激光雷达系统,采用盖格-雪崩光电二极管(Gm-APD)作为光子探测器,由高速扫描振镜二维扫描获取三维图像.通过时间相关记录仪获取光子到达时间分布,分别得到原始三维图像和激光脉冲响应率.在反转误差校正的测试实验中,系统的测量均方差由校正前的33.2 mm提高至8.1 mm.实验结果表明,该反转误差校正方法可以有效降低光子计数三维成像激光雷达的反转误差.     

三维显微图像带约束的迭代解卷积复原算法

阐述显微镜透明厚样本成像原理和三维显微图像带约束的迭代解卷积复原算法。根据显微镜透明厚样本成像原理,对已知三维清晰图像进行退化处理,并且使用带约束的迭代解卷积算法去除退化图像中的散焦信息。试验结果表明,图像散焦信息的干扰得到有效的去除,清晰度和信噪比得到明显的改善,并且该算法可以恢复成像过程中丢失的部分频率成分,实现超分辨率复原。当迭代次数较大时,复原效果优于邻域法和线性方法。     

数字共焦显微仪序列光学切片自动采集方法研究

提出一种基于图像处理的数字共焦显微仪序列光学切片自动采集方法。方法分两个步骤:第一步是对细胞进行自动聚焦;第二步是细胞的起始点自动调焦定位及进行光学切片。方法采用清晰度评价函数作为判别标准,将灰度差分算法与拉普拉斯函数法相结合,通过计算机实现两个步骤的自适应调焦控制,进而实现光学切片的自动采集。实验结果表明,方法有较高的自动聚焦和细胞起始点定位的速度和准确性,对三维球形细胞序列光学切片图像采集,简单易行,效果良好。     

Three‐dimensional structural imaging of starch granules by second‐harmonic generation circular dichroism

Chirality is one of the most fundamental and essential structural properties of biological molecules. Many important biological molecules including amino acids and polysaccharides are intrinsically chiral. Conventionally, chiral species can be distinguished by interaction with circularly polarized light, and circular dichroism is one of the best‐known approaches for chirality detection. As a linear optical process, circular dichroism suffers from very low signal contrast and lack of spatial resolution in the axial direction. It has been demonstrated that by incorporating nonlinear interaction with circularly polarized excitation, second‐harmonic generation circular dichroism can provide much higher signal contrast. However, previous circular dichroism and second‐harmonic generation circular dichroism studies are mostly limited to probe chiralities at surfaces and interfaces. It is known that second‐harmonic generation, as a second‐order nonlinear optical effect, provides excellent optical sectioning capability when combined with a laser‐scanning microscope. In this work, we combine the axial resolving power of second‐harmonic generation and chiral sensitivity of second‐harmonic generation circular dichroism to realize three‐dimensional chiral detection in biological tissues. Within the point spread function of a tight focus, second‐harmonic generation circular dichroism could arise from the macroscopic supramolecular packing as well as the microscopic intramolecular chirality, so our aim is to clarify the origins of second‐harmonic generation circular dichroism response in complicated three‐dimensional biological systems. The sample we use is starch granules whose second‐harmonic generation‐active molecules are amylopectin with both microscopic chirality due to its helical structure and macroscopic chirality due to its crystallized packing. We found that in a starch granule, the second‐harmonic generation for right‐handed circularly polarized excitation is significantly different from second‐harmonic generation for left‐handed one, offering excellent second‐harmonic generation circular dichroism contrast that approaches 100%. In addition, three‐dimensional visualization of second‐harmonic generation circular dichroism distribution with sub‐micrometer spatial resolution is realized. We observed second‐harmonic generation circular dichroism sign change across the starch granules, and the result suggests that in thick biological tissue, second‐harmonic generation circular dichroism arises from macroscopic molecular packing. Our result provides a new method to visualize the organization of three‐dimensional structures of starch granules. The second‐harmonic generation circular dichroism imaging method expands the horizon of nonlinear chiroptical studies from simplified surface/solution environments to complicated biological tissues.     

In vivo three-dimensional imaging of plants with optical coherence microscopy

Achieving the ability to non‐destructively, non‐invasively examine subsurface features of living multicellular organisms at a microscopic level is currently a challenge for biologists. Optical coherence microscopy (OCM) is a new photonics‐based technology that can be used to address this challenge. OCM takes advantage of refractive properties of biological molecules to generate three‐dimensional images that can be viewed with a computer. We describe new data processing techniques and a different visualization algorithm that substantially improve OCM images. We have applied OCM imaging, in conjunction with these improvements, to a variety of structures of plants, including leaves, flowers, ovules and germinating seeds, and describe the visualization of cellular and subcellular structures within intact plants. We present evidence, based on detailed examination of our OCM images, comparisons to classical plant anatomy studies, and current knowledge of light scattering by cells and their components, that we can distinguish nuclei, organelles and vacuoles. Detailed examination of vascular tissue, which contains cells with elaborate wall structure, shows that cell walls produce no significant OCM signal. These improvements to the visualization process, together with the powerful non‐invasive, non‐destructive aspects of the technology, will broaden the application of OCM to questions in studies of plants as well as animals.     

Quantitative imaging of Candida utilis and its organelles by soft X‐ray Nano‐CT

Soft X‐ray microscopy has excellent characteristics for imaging cells and subcellular structures. In this paper, the yeast strain, Candida utilis, was imaged by soft X‐ray microscopy and three‐dimensional volumes were reconstructed with the SART‐TV method. We performed segmentation on the reconstruction in three dimensions and identified several types of subcellular architecture within the specimen cells based on their linear absorption coefficient (LAC) values. Organelles can be identified by the correlation between the soft X‐ray LAC values and the subcellular architectures. Quantitative analyses of the volume ratio of organelles to whole cell in different phases were also carried out according to the three‐dimensional datasets. With such excellent features, soft X‐ray imaging has a great influence in the field of biological cellular and subcellular research.     

Automated high-resolution three-dimensional fluorescence imaging of large biological specimens

We describe a novel automated technique for visualizing the three‐dimensional distribution of fluorochrome‐labelled components, in which image resolution is uncoupled from specimen size. This method is based on computer numerically controlled milling technology and combines an arrayed imaging technique with fluorescence capabilities. Fluorescent signals are segmented by emission spectra such that multiple fluorochromes present within a single specimen may be reconstructed and visualized individually or as a group. The automated nature of the system minimizes the workload and time involved in image capture and volume reconstruction. As an application, the system was used to image zones of fluorochrome‐labelled microdamage within an 8‐mm diameter cylinder of trabecular bone at a voxel size of 3 × 3 × 8 μm³. Our reconstruction of this specimen provides a visual map and quantitative measures of the volume of damage present throughout the cylinder, clearly demonstrating the interpretive power afforded by three‐dimensional visualization. The three‐dimensional nature of this highly automated and adaptable system has the potential to facilitate new diagnostic tools and techniques with application to a wide range of biological and medical research fields.     

A comparative study of the quantitative accuracy of three-dimensional reconstructions of spinal cord from serial histological sections

We evaluated the accuracy of estimating the volume of biological soft tissues from their three‐dimensional (3D) computer wireframe models, reconstructed from histological data sets obtained from guinea‐pig spinal cords. We compared quantification from two methods of three‐dimensional surface reconstruction to standard quantitative techniques, Cavalieri method employing planimetry and point counting and Geometric Best‐Fitting. This involved measuring a group of spinal cord segments and test objects to evaluate the accuracy of our novel quantification approaches. Once a quantitative methodology was standardized there was no statistical difference in volume measurement of spinal segments between quantification methods. We found that our 3D surface reconstructions’ ability to model precisely actual soft tissues provided an accurate volume quantification of complex anatomical structures as standard approaches of Cavalieri estimation and Geometric Best‐Fitting. Additionally, 3D reconstruction quantitatively interrogates and three‐dimensionally images spinal cord segments and obscured internal pathological features with approximately the same effort required for standard quantification alone.     

X-ray high-resolution vascular network imaging

This paper presents the first application of high‐resolution X‐ray synchrotron tomography to the imaging of large microvascular networks in biological tissue samples. This technique offers the opportunity of analysing the full three‐dimensional vascular network from the micrometre to the millimetre scale. This paper presents the specific sample preparation method and the X‐ray imaging procedure. Either barium or iron was injected as contrast agent in the vascular network. The impact of the composition and concentration of the injected solution on the X‐ray synchrotron tomography images has been studied. Two imaging modes, attenuation and phase contrast, are compared. Synchrotron high‐resolution computed tomography offers new prospects in the three‐dimensional imaging of in situ biological vascular networks.     

Three-dimensional microscopic biopsy of in vivo human skin: a new technique based on a flexible confocal microscope

A new noninvasive microscopic technique of three-dimensional optical biopsy from in vivo human skin based on real-time confocal microscopy and computer reconstruction is demonstrated. A tandem scanning confocal microscope is a prototype of a mobile, flexible design for the in-depth microscopic exploration of the skin on the human body. The various skin layers were observed in real-time, at the subcellular level down to a depth of 200 μm with a vertical resolution of 2 μm. Rapid video recording of the Z-series through the ventral aspect of the forearm avoided shifts caused by subject movement and blood flow pulsations. Two video frames were averaged, and the average was digitized, providing a stack of 64 optical sections in 1-μm vertical steps. Three-dimensional reconstructions of in vivo human skin were obtained with sets of orthogonal slices, and slices at arbitrary planes through a volume containing the stack of slices. This method clearly shows the spatial relationships between the different cell layers. The use of orthogonal cutting planes is preferred because of its analogy with classical vertical sections of histopathology. Linear structures (surface lines) within the stratum corneum are described and their global orientations were determined by the use of Fourier transform analysis. En face optical sections constitute unusual views of this tissue, since typical pathohistological studies are based on sagittal (vertical) slices. The noninvasive optical microscopic technique provides a three-dimensional optical biopsy of in vivo human skin.     

Evaluation of X‐ray tomography contrast agents: A review of production,protocols, and biological applications

X‐ray computed tomography is a strong tool that finds many applications both in medical applications and in the investigation of biological and nonbiological samples. In the clinics, X‐ray tomography is widely used for diagnostic purposes whose three‐dimensional imaging in high resolution helps physicians to obtain detailed image of investigated regions. Researchers in biological sciences and engineering use X‐ray tomography because it is a nondestructive method to assess the structure of their samples. In both medical and biological applications, visualization of soft tissues and structures requires special treatment, in which special contrast agents are used. In this detailed report, molecule‐based and nanoparticle‐based contrast agents used in biological applications to enhance the image quality were compiled and reported. Special contrast agent applications and protocols to enhance the contrast for the biological applications and works to develop nanoparticle contrast agents to enhance the contrast for targeted drug delivery and general imaging applications were also assessed and listed.     

Focused ion beam scanning electron microscopy in biology

Since the end of the last millennium, the focused ion beam scanning electron microscopy (FIB‐SEM) has progressively found use in biological research. This instrument is a scanning electron microscope (SEM) with an attached gallium ion column and the 2 beams, electrons and ions (FIB) are focused on one coincident point. The main application is the acquisition of three‐dimensional data, FIB‐SEM tomography. With the ion beam, some nanometres of the surface are removed and the remaining block‐face is imaged with the electron beam in a repetitive manner. The instrument can also be used to cut open biological structures to get access to internal structures or to prepare thin lamella for imaging by (cryo‐) transmission electron microscopy. Here, we will present an overview of the development of FIB‐SEM and discuss a few points about sample preparation and imaging.