三维反卷积显微成像技术浅谈

三维宽场反卷积显微成像技术是应用光学切片方法获取三维标本的二维图像序列,然后通过反卷积图像处理方法进行图像恢复,进而进行三纺重建的一种以光学技术和图像处理技术为核心的业微成橡方法。本讲述了光学切片的基本原理,给出了反卷积处理中点扩展函数的理论模型和实验测试方法,然后对现存的反卷积算法做了对比。对这一领域的发展趋势做了预测。     

以三维荧光反卷积显微技术研究活体细胞中分泌囊泡的空间分布

获得活体细胞三维图像以观察细胞内分泌囊泡的空间分布有助于细胞分泌机制的研究。三维荧光反卷积显微技术可以为活体细胞观察提供低荧光漂白 ,低毒副作用的快速三维成像。研究了显微成像系统实验测定和理论计算点扩展函数之间的关系 ,并且实验验证了NA 1.6 5物镜条件下 ,理论计算点扩展函数可以较好地反映显微成像系统的特性。然后使用已知物理结构的三维样本对反卷积算法的有效性进行了研究。进而对使用吖啶橙(acridineorange)标记的大鼠胰腺 β细胞分泌囊泡进行观察。结果显示 ,反卷积算法可以有效地去除原始图像中因为焦外光影响产生的模糊 ,处理后图像清晰地显示了细胞内分泌囊泡的空间分布     

活体细胞三维图像科学可视化方法的研究

科学可视化是指运用计算机图形学和图像处理技术,将科学计算过程中或者是计算结果的数据转换为图形或图像,在屏幕上显示出来并进行交互式处理的理论技术或方法。介绍了用反卷积荧光显微成像技术获得活体大鼠胰腺B细胞三维图像及对其进行科学可视化的主要过程和两种常用可视化算法,并运用这两种方法对所得到的三维图像进行处理以分析和研究细胞内分泌囊泡的空间分布。结果显示,当仅观察细胞三维图像的二维切片时,三维图像中的某些重要信息会被忽略,而使用科学可视化方法则可以从三维角度直观观察活体细胞内分泌囊泡的空间分布,并且可以观察到分泌囊泡的释放趋势和整体分布,从而为细胞生物学研究提供重要的信息。     

用于动物分类学研究领域的显微成像新技术

UV-C光学共聚焦显微图像系统集成了光学和数字图像分析技术，特别适用于采集非平面样品（高度变化范围超过了显微镜影深范围）的图像，它彻底解决了传统光学显微镜成像时尚倍数与大景深不能共存的问题。     

用于动物分类学研究领域的显微成像新技术

UV-C光学共聚焦显微图像系统集成了光学和数字图像分析技术，特别适用于采集非平面样品（高度变化范围超过了显微镜景深范围）的图像，它彻底解决了传统光学显微镜成像时高倍数与大景深不能共存的问题，使样品的不同高低部位都能清晰成像，可以获得像电镜图像一样的巨大景深和精致的细节，     

共聚焦显微技术简介

共聚焦显微镜在生物学研究中得到广泛应用,共聚焦显微技术按照显微镜构造原理的不同分成激光扫描共聚焦和数字共聚焦显微技术两种,共聚焦技术具有成像清晰,获得三维图像,进行多标记观察,活细胞内动态生理反应的实时观察记录,定性定量分析等优势,与共聚焦显微技术相关的技术有荧光染料的选择,荧光指示剂装载以及图像数据处理等。     

用于海洋原位浮游生物探测的同轴数字全息显微技术研究

本文将数字全息与显微成像技术相结合,设计搭建了一套数字全息显微系统,用于对浮游生物进行光场获取。基于该系统获取的光场信息,通过在不同景深对图像进行再现,既可以获到单一浮游生物的清晰图像,也可以获得一定水体内浮游生物微粒在海水中的三维分布情况。通过实验测得系统分辨率可以达到7．8微米,景深可以达到10毫米,优于一般光学显微镜的技术指标。研究结果表明优势明显的数字全息显微系统是一种适合海洋原位探测的浮游生物研究的有效方法。基于数字同轴显微系统的水下仪器开发将是下一步工作的努力方向。     

两种微体化石三维无损成像技术的对比

近年来,各种X射线三维无损成像技术在古生物学领域的应用越来越广泛。但是,不同的X射线三维无损成像技术针对不同保存类型和尺寸的化石标本在成像效果上各有利弊。本文以埃迪卡拉纪陡山沱组磷酸盐化的动物胚胎化石为研究对象,将目前应用最广的两种X射线三维无损成像方法,即基于实验室X光源的吸收衬度显微断层成像技术和基于同步辐射光源的相位衬度显微断层成像技术进行了对比分析。通过对两种技术的原理、效率、空间分辨率和图像衬度的对比,认为基于同步辐射光源的相位衬度显微断层成像技术是目前对于均一矿化的微体化石最佳的三维无损成像解决方案。     

用于动物分类学研究领域的显微成像新技术

UV-C光学共聚焦显微图像系统集成了光学和数字图象分析技术，特别适用于采集非平面样品（高度变化范围超过了显微镜景深范围）的图象，它彻底解决了传统光学显微镜成像时高倍数与大景深不能共存的问题，使样品的不同高低部位都能清晰成像，     

三维X射线显微技术与小型-微体化石高分辨率无损成像*

在古生物学研究中,以X射线断层成像(Computed Tomography)为代表的三维无损成像技术可以在不破坏化石标本的前提下,同时获得标本外观形态和内部结构的信息,相比传统的可见光成像手段有着明显优势。为推动化石三维无损成像技术在国内古生物学领域的发展,本文系统介绍一种新型显微CT技术——三维X射线显微术(Three-Dimensional X-ray Microscopy)。与基于几何放大和吸收衬度成像的传统显微CT技术相比,该技术有若干优势:(1)将同步辐射X射线显微断层成像的光学成像系统引入基于实验室X射线源的显微CT系统中,在几何放大的基础上增加了光学放大,优化了传统显微CT的系统架构,弥补了传统显微CT单纯依靠几何放大的不足,提高了空间分辨率;(2)采用可移动的X射线源和优化的光学成像系统,实现了低能X射线相位衬度成像,可以三维重构传统显微CT技术无法有效探测的、低吸收衬度的化石标本;(3)基于新的成像架构和成像算法,实现了厘米-分米级较大标本内部"感兴趣区域"(Region of Interest)精确导航和局部高分辨率(微米-亚微米空间分辨)成像;(4)可以实现小型扁平标本(宽厚比4,宽10cm)高效率、高分辨率成像和长条形微体标本长轴方向自动分段无缝拼接的微米至亚微米级高分辨率重建,弥补了传统工业显微CT针对小型扁平标本和长条形微体标本高分辨成像效果不佳的缺陷。这些优势使得基于实验室X射线源的显微CT成像技术可以获得接近同步辐射X射线源的成像质量,从而有效推动化石生物学研究。     

Confocal scanning optical microscopy and three-dimensional imaging

Summary— Confocal scanning optical microscopy has significant advantages over conventional fluorescence microscopy: it rejects the out-of-locus light and provides a greater resolution than the wide-field microscope. In laser scanning optical microscopy, the specimen is scanned by a diffraction-limited spot of laser light and the fluorescence emission (or the reflected light) is focused onto a photodetector. The imaged point is then digitized, stored into the memory of a computer and displayed at the appropriate spatial position on a graphic device as a part of a two-dimensional image. Thus, confocal scanning optical microscopy allows accurate non-invasive optical sectioning and further three-dimensional reconstruction of biological specimens. Here we review the recent technological aspects of the principles and uses of the confocal microscope, and we introduce the different methods of three-dimensional imaging.     

Stereology meets electron tomography: towards quantitative 3D electron microscopy

Stereological tools are the gold standard for accurate (i.e., unbiased) and precise quantification of any microscopic sample. The past decades have provided a broad spectrum of tools to estimate a variety of parameters such as volumes, surfaces, lengths, and numbers. Some of them require pairs of parallel sections that can be produced by either physical or optical sectioning, with optical sectioning being much more efficient when applicable. Unfortunately, transmission electron microscopy could not fully profit from these riches, mainly because of the large depth of field. Hence, optical sectioning was a long-time desire for electron microscopists. This desire was fulfilled with the development of electron tomography that yield stacks of slices from electron microscopic sections. Now, parallel optical slices of a previously unimagined small thickness (2-5 nm axial resolution) can be produced. These optical slices minimize problems related to overprojection effects, and allow for direct stereological analysis, e.g., volume estimation with the Cavalieri principle and number estimation with the optical disector method. Here, we demonstrate that the symbiosis of stereology and electron tomography is an easy and efficient way for quantitative analysis at the electron microscopic level. We call this approach quantitative 3D electron microscopy.     

High-definition mapping of neural activity using voltage-sensitive dyes

The distribution of patterns of activity in different brain structures has been related to the encoding and processing of sensory information. Consequently, it is important to be able to image the distribution of these patterns to understand basic brain functions. The spatial resolution of voltage-sensitive dye (VSD) methods has recently been enhanced considerably by the use of video imaging techniques. The main factor that now hampers the resolution of VSD patterns is the inherent limitation of the optical systems. Unfortunately, the intrinsic characteristics of VSD images impose important limitations that restrict the use of general deconvolution techniques. To overcomes this problem, in this study an image restoration procedure has been implemented that takes into consideration the limiting characteristics of VSD signals. This technique is based on applying a set of imaging processing steps. First, the signal-to-noise (S/N) ratio of the images was improved to avoid an increase in the noise levels during the deconvolution procedures. For this purpose, a new filter technique was implemented that yielded better results than other methods currently used in optical imaging. Second, focal plane images were deconvolved using a modification of the well-known nearest-neighbor deconvolution algorithm. But to reduce the light exposure of the preparation and simplify image acquisition procedures, adjacent image planes were modeled according to the in-focus image planes and the empirical point spread function (PSF) profiles. Third, resulting focal plane responses were processed to reduce the contribution of optical responses that originate in distant image planes. This method was found to be satisfactory under simulated and real experimental conditions. By comparing the restored and unprocessed images, it was clearly demonstrated that this method can effectively remove the out-of-focus artifacts and produce focal plane images of better quality. Evaluations of the tissue optical properties allowed assessment of the maximum practical optical section thickness using this deconvolution technique in the optical system tested. Determination of the three-dimensional PSF permitted the correct application of deconvolution algorithms and the removal of the contaminating light arising from adjacent as well as distant optical planes. The implementation of this deconvolution approach in salamander olfactory bulb allowed the detailed study of the laminar distribution of voltage-sensitive changes across the bulb layer. It is concluded that (1) this deconvolution procedure is well suited to deconvolved low-contrast images and offers important advantages over other alternatives; (2) this method can be properly used only when the tissue optical properties are first determined; (3) high levels of light scattering in the tissue reduce the optical section capabilities of this technique as well as other deconvolution procedures; and (4) use of the highest numerical aperture in the objectives is advisable because this improves not only the light-collecting efficiency to detect poor-contrast images, but also the spatial frequency differences between adjacent image planes. Under this condition it is possible to overcome some of the limitations imposed by the light scattering/birefringence of the tissue.     

Reprint of “Stereology meets electron tomography: towards quantitative 3D electron microscopy” [J. Struct. Biol. 159 (2007) 443–450]

Stereological tools are the gold standard for accurate (i.e., unbiased) and precise quantification of any microscopic sample. The past decades have provided a broad spectrum of tools to estimate a variety of parameters such as volumes, surfaces, lengths, and numbers. Some of them require pairs of parallel sections that can be produced by either physical or optical sectioning, with optical sectioning being much more efficient when applicable. Unfortunately, transmission electron microscopy could not fully profit from these riches, mainly because of the large depth of field. Hence, optical sectioning was a long-time desire for electron microscopists.This desire was fulfilled with the development of electron tomography that yield stacks of slices from electron microscopic sections. Now, parallel optical slices of a previously unimagined small thickness (2–5 nm axial resolution) can be produced. These optical slices minimize problems related to overprojection effects, and allow for direct stereological analysis, e.g., volume estimation with the Cavalieri principle and number estimation with the optical disector method.Here, we demonstrate that the symbiosis of stereology and electron tomography is an easy and efficient way for quantitative analysis at the electron microscopic level. We call this approach quantitative 3D electron microscopy.     

Structure brings clarity: Structured illumination microscopy in cell biology

Biological samples are three dimensional and, therefore, optical sectioning is mandatory for microscopic images to precisely show the localization or function of structures within biological samples. Today, researchers can choose from a variety of methods to obtain optical sections. This article focuses on structured illumination microscopy, which is a group of techniques utilizing a combination of optics and mathematics to obtain optical sections: A structure is imaged onto the sample by optical means and the additional information thereby encoded in the image is used to calculate an optical section from several acquired images. Different methods of structured illumination microscopy (mainly grid projection and aperture correlation) are discussed from a practical point of view, concentrating on advantages, limitations and future prospects of these techniques and their use in cell biology. Structured illumination can also be used to obtain super-resolution information if structures of higher frequency are projected onto the sample. This promising approach to super-resolution microscopy is also briefly discussed from a user's perspective.     

Widefield deconvolution epifluorescence microscopy combined with fluorescence in situ hybridization reveals the spatial arrangement of bacteria in sponge tissue

Widefield deconvolution epifluorescence microscopy (WDEM) combined with fluorescence in situ hybridization (FISH) was performed to identify and characterize single bacterial cells within sections of the mediterranean sponge Chondrosia reniformis. Sponges were embedded in paraffin wax or plastic prior to the preparation of thin sections, in situ hybridization and microscopy. Serial digital images generated by widefield epifluorescence microscopy were visualized using an exhaustive photon reassignment deconvolution algorithm and three-dimensional rendering software. Computer processing of series of images taken at different focal planes with the deconvolution technique provided deblurred three-dimensional images with high optical resolution on a submicron scale. Results from the deconvolution enhanced widefield microscopy were compared with conventional epifluorescent microscopical images. By the application of the deconvolution algorithm on digital image data obtained with widefield epifluorescence microscopy after FISH, the occurrence and spatial arrangement of Desulfovibrionaceae closely associated with micropores of Chondrosia reniformis could be visualized.     

Low‐photobleaching line‐scanning confocal microscopy using dual inclined beams

Confocal microscopy is an indispensable tool for biological imaging due to its high resolution and optical sectioning capability. However, its slow imaging speed and severe photobleaching have largely prevented further applications. Here, we present dual inclined beam line‐scanning (LS) confocal microscopy. The reduced excitation intensity of our imaging method enabled a 2‐fold longer observation time of fluorescence compared to traditional LS microscopy while maintaining a good sectioning capability and single‐molecule sensitivity. We characterized the performance of our method and applied it to subcellular imaging and three‐dimensional single‐molecule RNA imaging in mammalian cells.      

Cell imaging and manipulation by nonlinear optical microscopy

Advances in the technologies for labeling and imaging biological samples drive a constant progress in our capability of studying structures and their dynamics within cells and tissues. In the last decade, the development of numerous nonlinear optical microscopies has led to a new prospective both in basic research and in the potential development of very powerful noninvasive diagnostic tools. These techniques offer large advantages over conventional linear microscopy with regard to penetration depth, spatial resolution, three-dimensional optical sectioning, and lower photobleaching. Additionally, some of these techniques offer the opportunity for optically probing biological functions directly in living cells, as highlighted, for example, by the application of second-harmonic generation to the optical measurement of electrical potential and activity in excitable cells. In parallel with imaging techniques, nonlinear microscopy has been developed into a new area for the selective disruption and manipulation of intracellular structures, providing an extremely useful tool of investigation in cell biology. In this review we present some basic features of nonlinear microscopy with regard both to imaging and manipulation, and show some examples to illustrate the advantages offered by these novel methodologies.