A novel method for shape from focus in microscopy using Bezier surface approximation

In this article, we introduce a novel shape from focus method to compute 3D shape of microscopic objects, based on modified‐pixel intensities and Bezier surface approximations. A new and simple but effective focus measure is proposed. In our focus measure, the original intensities of a sequence of small neighborhood are modified by subtracting the maximum of the values of first and last frames. An initial depth map is calculated by finding the maximum of the pixel's focused energy and its corresponding frame number. Missing information between two consecutive frames, false depth detection, and enhancement of noise related intensities may provide inaccurate depth map. To overcome these problems and to produce an accurate depth map, we proposed Bezier surface approximation. The proposed method is tested using synthetic and real image sequences. The comparative analysis demonstrates the effectiveness of the proposed method. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Curvature measurement with reflected-light microscopy and its application to fly facet lenses

Two methods for microscopically measuring the curvature of strongly curved surfaces are compared: one using a Michelson interferometer-type microscope and one using a novel reflectometrical method implemented in an epi-illumination microscope. The curvature values obtained with the two methods were very similar, but the latter proved to be by far the simplest. Curvature measurements on the front surface of the facet lenses of various dipteran flies revealed that facet lens diameter and radius of curvature are linearly related over a wide range of facet lens sizes.     

Light exposure and cell viability in fluorescence microscopy

Test systems for measuring cell viability in optical microscopy (based on colony formation ability or lysosomal integrity) were established and applied to native cells as well as to cells incubated with fluorescence markers or transfected with genes encoding for fluorescent proteins. Human glioblastoma and Chinese hamster ovary cells were irradiated by various light doses, and maximum doses where at least 90% of the cells survived were determined. These tolerable light doses were in the range between 25 J cm^?2 and about 300 J cm^?2 for native cells (corresponding to about 250?3000 s of solar irradiance and depending on the wavelength as well as on the mode of illumination, e.g. epi‐ or total internal reflection illumination) and decreased to values between 50 J cm^?2 and less than 1 J cm^?2 upon application of fluorescent markers, fluorescent proteins or photosensitizers. In high‐resolution wide field or laser scanning microscopy of single cells, typically 10?20 individual cell layers needed for reconstruction of a 3D image could be recorded with tolerable dose values. Tolerable light doses were also maintained in fluorescence microscopy of larger 3D samples, e.g. cell spheroids exposed to structured illumination, but may be exceeded in super‐resolution microscopy based on single molecule detection.     

Mirrored pyramidal wells for simultaneous multiple vantage point microscopy

We report a novel method for obtaining simultaneous images from multiple vantage points of a microscopic specimen using size‐matched microscopic mirrors created from anisotropically etched silicon. The resulting pyramidal wells enable bright‐field and fluorescent side‐view images, and when combined with z‐sectioning, provide additional information for 3D reconstructions of the specimen. We have demonstrated the 3D localization and tracking over time of the centrosome of a live Dictyostelium discoideum. The simultaneous acquisition of images from multiple perspectives also provides a five‐fold increase in the theoretical collection efficiency of emitted photons, a property which may be useful for low‐light imaging modalities such as bioluminescence, or low abundance surface‐marker labelling.     

Robust incremental compensation of the light attenuation with depth in 3D fluorescence microscopy

Fluorescent signal intensities from confocal laser scanning microscopes (CLSM) suffer from several distortions inherent to the method. Namely, layers which lie deeper within the specimen are relatively dark due to absorption and scattering of both excitation and fluorescent light, photobleaching and/or other factors. Because of these effects, a quantitative analysis of images is not always possible without correction. Under certain assumptions, the decay of intensities can be estimated and used for a partial depth intensity correction. In this paper we propose an original robust incremental method for compensating the attenuation of intensity signals. Most previous correction methods are more or less empirical and based on fitting a decreasing parametric function to the section mean intensity curve computed by summing all pixel values in each section. The fitted curve is then used for the calculation of correction factors for each section and a new compensated sections series is computed. However, these methods do not perfectly correct the images. Hence, the algorithm we propose for the automatic correction of intensities relies on robust estimation, which automatically ignores pixels where measurements deviate from the decay model. It is based on techniques adopted from the computer vision literature for image motion estimation. The resulting algorithm is used to correct volumes acquired in CLSM. An implementation of such a restoration filter is discussed and examples of successful restorations are given.     

Sample holder for axial rotation of specimens in 3D microscopy

In common light microscopy, observation of samples is only possible from one perspective. However, especially for larger three‐dimensional specimens observation from different views is desirable. Therefore, we are presenting a sample holder permitting rotation of the specimen around an axis perpendicular to the light path of the microscope. Thus, images can be put into a defined multidimensional context, enabling reliable three‐dimensional reconstructions. The device can be easily adapted to a great variety of common light microscopes and is suitable for various applications in science, education and industry, where the observation of three‐dimensional specimens is essential. Fluorescence z‐projection images of copepods and ixodidae ticks at different rotation angles obtained by confocal laser scanning microscopy and light sheet fluorescence microscopy are reported as representative results.     

Three-dimensional reconstruction of aqueous channels in human trabecular meshwork using light microscopy and confocal microscopy

Conventional two-dimensional imaging of the trabecular meshwork (TM) provides limited information about the size, shape, and interconnection of the aqueous channels within the meshwork. Understanding the three-dimensional (3-D) relationships of the channels within this tissue may give insight into its normal function and possible changes present in the eye disease glaucoma. The purpose of our study was to compare laser scanning confocal microscopy with standard 1 μm Araldite-embeddedhistologic sections for 3-D analysis of the trabecular meshwork. In addition, the study was done to determine whether computerized 3-D reconstruction could isolate the fluid spaces of the trabecular meshwork and determine the size of interconnections between the fluid spaces. Confocal microscopy appears comparable to 1 μm Araldite-embedded tissue sections and has the advantage of inherent registration of the serial tissue sections. Three-dimensional reconstruction allowed the isolation of the fluid spaces within the trabecular meshwork and revealed the presence of numerous interconnections between larger fluid spaces. The distribution of these interconnections was randomly arranged, with no predilection for specific regions within the trabecular meshwork. This distribution of constrictions and “expansion chambers” may provide a clue to the mechanism by which subtle histologic changes are associated with increased ocular pressure in glaucoma.     

基于单次成像的三维形貌拼接技术

针对大型物体的三维形貌测量,研究基于辅助靶标的三维形貌拼接技术,并在此基础上提出单摄像机单次成像求解转换矩阵的方法.该方法通过单摄像机单次成像,结合控制点的边长约束,求解出全局坐标系同局部测量坐标系的转换关系,进而将局部测量数据统一到全局坐标系下,实现拼接.此方法避免采用单摄像机多摄站测量时带来的繁琐操作以及产生的相关问题,大大提高拼接的速度,并且可以保证较高的精度.     

基于OpenGL的双目摄像机的实时3D显示

文章阐述了3D显示的基本原理以及几种主流的3D显示实现方法,并介绍了在Visual C++6.0环境下,如何应用开放图形库——OpenGL实现双目立体视觉摄像机的立体显示。     

Super-resolution microscopy using normal flow decoding and geometric constraints

Prior knowledge about the observed scene provides the key to restoration of frequencies beyond the bandpass of an imaging system (super-resolution). In conjunction with microscopy two super-resolution mechanisms have been mainly reported: analytic continuation of the frequency spectrum, and constrained image deconvolution. This paper describes an alternative approach to super-resolution. Prior knowledge is imposed through geometric and dynamic models of the scene. We illustrate our concept based on the stereo reconstruction of a micropipette moving in close proximity to a stationary target object. Information about the shape and the movement of the pipette is incorporated into the reconstruction algorithm. The algorithm was tested in a microrobot environment, where the pipette tip was tracked at sub-Rayleigh distances to the target. Based on the tracking results, a machine vision module controlled the manipulation of microscopic objects, e.g. latex beads or diamond mono-crystals. In the theoretical part of this paper we prove that knowledge of the form 'the pipette has moved between two consecutive frames of the movie' must result in a twofold increase in resolution. We used the normal flow of an image sequence to decode positional measures from motion evidence. In practice, super-resolution factors between 3 and 5 were obtained. The additional gain originates from the geometric constraints that were imposed upon the stereo reconstruction of the pipette axis.     

New data‐driven method from 3D confocal microscopy for calculating phytoplankton cell biovolume

Confocal laser scanner microscopy coupled with an image analysis system was used to directly determine the shape and calculate the biovolume of phytoplankton organisms by constructing 3D models of cells. The study was performed on Biceratium furca (Ehrenberg) Vanhoeffen, which is one of the most complex‐shaped phytoplankton. Traditionally, biovolume is obtained from a standardized set of geometric models based on linear dimensions measured by light microscopy. However, especially in the case of complex‐shaped cells, biovolume is affected by very large errors associated with the numerous manual measurements that this entails. We evaluate the accuracy of these traditional methods by comparing the results obtained using geometric models with direct biovolume measurement by image analysis. Our results show cell biovolume measurement based on decomposition into simple geometrical shapes can be highly inaccurate. Although we assume that the most accurate cell shape is obtained by 3D direct biovolume measurement, which is based on voxel counting, the intrinsic uncertainty of this method is explored and assessed. Finally, we implement a data‐driven formula‐based approach to the calculation of biovolume of this complex‐shaped organism. On one hand, the model is obtained from 3D direct calculation. On the other hand, it is based on just two linear dimensions which can easily be measured by hand. This approach has already been used for investigating the complexities of morphology and for determining the 3D structure of cells. It could also represent a novel way to generalize scaling laws for biovolume calculation.     

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.     

Quantitative polarized light microscopy

We describe a simple modification to a confocal microscope, which analyses the state of polarization of light emerging from the specimen so as to permit quantitative polarized light microscopy to be performed. The system uses a novel form of rotating analyser which, together with lock‐in detection, permits images to be obtained where the image contrast corresponds to both specimen retardance and orientation (e.g. in the case of a birefringent specimen). Images are presented from a wide range of specimens and the origin of the contrast observed from simple point scatterers is investigated both theoretically and experimentally.     

Blind deconvolution of 3D fluorescence microscopy using depth‐variant asymmetric PSF

The 3D wide‐field fluorescence microscopy suffers from depth‐variant asymmetric blur. The depth‐variance and axial asymmetry are due to refractive index mismatch between the immersion and the specimen layer. The radial asymmetry is due to lens imperfections and local refractive index inhomogeneities in the specimen. To obtain the PSF that has these characteristics, there were PSF premeasurement trials. However, they are useless since imaging conditions such as camera position and refractive index of the specimen are changed between the premeasurement and actual imaging. In this article, we focus on removing unknown depth‐variant asymmetric blur in such an optical system under the assumption of refractive index homogeneities in the specimen. We propose finding few parameters in the mathematical PSF model from observed images in which the PSF model has a depth‐variant asymmetric shape. After generating an initial PSF from the analysis of intensities in the observed image, the parameters are estimated based on a maximum likelihood estimator. Using the estimated PSF, we implement an accelerated GEM algorithm for image deconvolution. Deconvolution result shows the superiority of our algorithm in terms of accuracy, which quantitatively evaluated by FWHM, relative contrast, standard deviation values of intensity peaks and FWHM. Microsc. Res. Tech. 79:480–494, 2016. © 2016 Wiley Periodicals, Inc.     

Nanostructural analysis by atomic force microscopy followed by light microscopy on the same archival slide

Integrated information on ultrastructural surface texture and chemistry increasingly plays a role in the biomedical sciences. Light microscopy provides access to biochemical data by the application of dyes. Ultrastructural representation of the surface structure of tissues, cells, or macromolecules can be obtained by scanning electron microscopy (SEM). However, SEM often requires gold or coal coating of biological samples, which makes a combined examination by light microscopy and SEM difficult. Conventional histochemical staining methods are not easily applicable to biological material subsequent to such treatment. Atomic force microscopy (AFM) gives access to surface textures down to ultrastructural dimensions without previous coating of the sample. A combination of AFM with conventional histochemical staining protocols for light microscopy on a single slide is therefore presented. Unstained cores were examined using AFM (tapping mode) and subsequently stained histochemically. The images obtained by AFM were compared with the results of histochemistry. AFM technology did not interfere with any of the histochemical staining protocols. Ultrastructurally analyzed regions could be identified in light microscopy and histochemical properties of ultrastructurally determined regions could be seen. AFM-generated ultrastructural information with subsequent staining gives way to novel findings in the biomedical sciences. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

A comparison study of detecting gold nanorods in living cells with confocal reflectance microscopy and two‐photon fluorescence microscopy

Two‐photon fluorescence microscopy and confocal reflectance microscopy were compared to detect intracellular gold nanorods in rat basophilic leukaemia cells. The two‐photon photoluminescence images of gold nanorods were acquired by an 800 nm fs laser with the power of milliwatts. The advantages of the obtained two‐photon photoluminescence images are high spatial resolution and reduced background. However, a remarkable photothermal effect on cells was seen after 30 times continuous scanning of the femto‐second laser, potentially affecting the subcellular localization pattern of the nanorods. In the case of confocal reflectance microscopy the images of gold nanorods can be obtained with the power of light source as low as microwatts, thus avoiding the photothermal effect, but the resolution of such images is reduced. We have noted that confocal reflectance images of cellular gold nanorods achieved with 50 μW 800 nm fs have a relatively poor resolution, whereas the 50 μW 488 nm CW laser can acquire reasonably satisfactory 3D reflectance images with improved resolution because of its shorter wavelength. Therefore, confocal reflectance microscopy may also be a suitable means to image intracellular gold nanorods with the advantage of reduced photothermal effect.     

Virtual electron microscopy in cell biology

Virtual microscopy of histological glass slides can emulate conventional light microscopy. Up till now, such a digital simulation does not exist for ultrathin electron microscopic slides. Because of the relative inaccessibility of electron microscopy, evaluation of subcellular structures by (bio)medical students is performed with the aid of photographic prints. In this article, the generation and evaluation of virtual electron microscopic slides is discussed. A T‐lymphoblastic cell was used as an example. Electron microscopic pictures were taken at two magnifications (25,000 and 50,000), processed in an analogue or digital way and stitched to reconstruct the image of the total cell. This image is viewed with a webviewer equipped with pan and zoom functions. The possibility of distinguishing the trilaminar structure of cellular membranes was the requisite. Virtual images obtained at an original magnification of 25,000, scanned at a resolution of 800 ppi could compete with pictures developed directly from negatives obtained by electron microscopy. It is possible to navigate and zoom into details in a way emulating electron microscopy. Virtual electron microscopy is innovative and offers new perspectives to interpret cytological pictures and to teach cell biology in an interactive and unique way. Microsc. Res. Tech., 2010. © 2010 Wiley‐Liss, Inc.     

数字全息三维显示关键技术与系统综述

三维全息显示能够表现出与真实物体一样的深度和视差,是一种理想的三维显示方法.但是,三维物体计算全息图计算复杂且计算量巨大,因此,如何快速生成三维物体计算全息图是数字三维全息动态显示中的关键问题之一.本文首先论述了数字全息三维显示的关键技术,包括物点散射法、体视全息法、层析法等三种三维物体计算全息图实现方法,一种RGB分离的真彩色全息显示实现方法和若干提高全息再现像质的方法;然后对几种最新典型的数字三维全息显示系统进行了技术分析;最后总结了数字全息三维显示领域的发展动态,指出三维全息显示技术会朝着实时、动态、更大尺寸、更高分辨率方向发展.