Measurement-based evaluation of optical pathlength distributions reconstructed from simulated differential interference contrast images

Recently a method was presented for reconstructing optical pathlength distributions (OPDs) from images of weak phase objects obtained by a conventional differential interference contrast (DIC) microscope. A potential application of this technique is the determination of the mass of biological objects: by integrating the optical pathlength over the projected surface of the image of an object, a measure of the dry mass, i.e. the total mass of all solid constituents present in the object, is obtained. To assess the possibilities of DIC microscopy for this application, simulations were performed on computer-generated DIC images of objects of various sizes, shapes and orientation angles. After reconstructing the OPDs from these images, the integrated optical pathlength of each of the test objects was determined, and compared with the expected results. The parameter settings used in the reconstruction algorithm were found to be very important in obtaining a reliable measurement. Using optimal parameter settings, errors in the integrated OPD could be limited to a few per cent for circular objects within the investigated size range. For non-circular objects, however, the orientation angle of the object relative to the lateral shift was found to influence the measured values. Ellipses with their long axes perpendicular to the shift direction had a significantly higher integrated OPD than ellipses orientated parallel to the shift. By adjusting the reconstruction parameters the effect could be limited, but complete elimination of the artefact was not possible within the parameter range investigated.     

An aid to understanding differential interference contrast microscopy: computer simulation

Differential interference contrast (DIC) microscopy has been widely used for many years. However, many biologists employ DIC inefficiently and frequently misinterpret the resulting images. In an effort to improve the standard of use, a method of simulating DIC microscopy on the cathode-ray tube (CRT) and x–y plotter of a computer was therefore developed. This paper describes the program and gives examples of its application, emphasizing some common errors of operation of the DIC microscope and interpretation of the image. Simulations are made for objects of negligible thickness, and in one azimuth of the microscope at a time. The method shows clearly the profiles of the phases and amplitudes of the interfering waves obtained for a variety of objects and demonstrates unequivocally the kinds of images obtained. It is possible to show very quickly how the image changes as instrumental parameters, such as phase-bias, amplitude-ratio, extinction factor, and beam-splitter shear are altered. Simulation was found useful as a tool to learn DICM and as an aid to the interpretation of complicated images obtained with real objects. Examples of common errors in adjusting the microscope and interpreting images are given in this paper. The simulation program was developed for the computer of the Denver Universal Microspectroradiometer (David & Galbraith, 1975; Galbraith et al., 1975), but it can be used on a number of compatible computing systems. The program construction is explained to enable comparable programs to be written for different computers.     

Real-time image quality assessment with mixed Lagrange time delay estimation autoregressive (MLTDEAR) model

A proposal to assess the quality of scanning electron microscope images using mixed Lagrange time delay estimation technique is presented. With optimal scanning electron microscope scan rate information, online images can be quantified and improved. The online quality assessment technique is embedded onto a scanning electron microscope frame grabber card for real‐time image processing. Different images are captured using scanning electron microscope and a database is built to optimally choose filter parameters. An optimum choice of filter parameters is obtained. With the optimum choice of scan rate, noise can be removed from real‐time scanning electron microscope images without causing any sample contamination or increasing scanning time.     

A combined optical and atomic force microscope for live cell investigations

We present an easy-to-use combination of an atomic force microscope (AFM) and an epi-fluorescence microscope, which allows live cell imaging under physiological conditions. High-resolution AFM images were acquired while simultaneously monitoring either the fluorescence image of labeled membrane components, or a high-contrast optical image (DIC, differential interference contrast). By applying two complementary techniques at the same time, additional information and correlations between structure and function of living organisms were obtained. The synergy effects between fluorescence imaging and AFM were further demonstrated by probing fluorescence-labeled receptor clusters in the cell membrane via force spectroscopy using antibody-functionalized tips. The binding probability on receptor-containing areas identified with fluorescence microscopy ("receptor-positive sites") was significantly higher than that on sites lacking receptors.     

Using the Hilbert transform for 3D visualization of differential interference contrast microscope images

Differential interference contrast (DIC) is frequently used in conventional 2D biological microscopy. Our recent investigations into producing a 3D DIC microscope (in both conventional and confocal modes) have uncovered a fundamental difficulty: namely that the phase gradient images of DIC microscopy cannot be visualized using standard digital image processing and reconstruction techniques, as commonly used elsewhere in microscopy. We discuss two approaches to the problem of preparing gradient images for 3D visualization: integration and the Hilbert transform. After applying the Hilbert transform, the dataset can then be visualized in 3D using standard techniques. We find that the Hilbert transform provides a rapid qualitative pre-processing technique for 3D visualization for a wide range of biological specimens in DIC microscopy, including chromosomes, which we use in this study.     

数字化裂隙灯生物显微镜外眼图象分析系统

介绍了数字化裂隙灯生物显微镜外眼图象分析系统。系统通过改造传统裂隙灯生物显微镜的摄像光路,用数码相机取代普通光学相机,记录不同尺寸的放大图象,从而获得高清晰度数码图象,利用数字图象处理技术对裂隙灯生物显微镜图象进行处理,为临床提供客观和定量化的诊断依据,同时可以实现患者信息资料与图象的高交存储,管理与远程传输。     

A study on using scanning acoustic microscopy and neural network techniques to evaluate the quality of resistance spot welding

This paper shows that the quality of resistance spot welds can be evaluated using scanning acoustic microscopy (SAM). Two-layered coated spot-welded samples are investigated utilising a wide-field short-pulse scanning acoustic microscope with operation frequencies of 25, 50 and 100 MHz. Geometrical parameters, e.g. nugget area, maximum axis of nugget, and minimum axis of nugget, are acquired from C-scan images of weld nuggets using mathematical morphology techniques. These parameters serve as inputs for an artificial neural network (ANN) model to evaluate the quality of spot welds. The output of the model during the training process comprises the results of nugget peeling tests and expert opinions. The ANN can provide suggestions on weld quality with a higher than 95% correctness. A JAVA computer program is developed for image processing, ANN training, and ANN testing. With this model, the computer program can render the quality of spot welds that are close to those achieved using off-line destructive method.     

Nonmarski differential-interference contrast microscopy in transillumination: its use on unstained or stained sections, smears, and wet mounts, or on fluorochromed sections and cell-culture monolayers

Unstained, lightly stained and conventionally stained microtome tissue sections of two different thicknesses (ca. 4 μm and 1 μm) and also unstained or stained wet mounts of cells were photographed under the microscope using bright-field, positive phase contrast, and Nomarski differential-interference contrast (DIC) in transillumination. The photomicrographs were critically compared. It was found that the density of various stains did not adversely affect the better resolution of the DIC image (as compared to the bright-field image); however, Optical sectioning' of darkly stained objects is not possible. Unstained or stained smears of blood or of epithelial cells of buccal mucosa were examined with DIC in transillumination, then after certain preparatory techniques, the same preparation was examined in the scanning electron microscope, and finally the same areas of the slide were viewed with DIC in epiillumination. Particular attention was given to structures (nuclei and cytoplasm) which appeared in positive or negative relief in the photomicrographs taken by the various techniques. It was concluded that the optically more dense nucleus which always appeared in positive relief by the various methods of examination, was in fact geometrically raised from the surrounding cytoplasm. Acridine orange (AO) stained cell-culture monolayers and H and E stained sections were examined under a fluorescence microscope with DIC optics. By comparing photographs which had been taken with DIC, epi-fluorescence or fluorescence in transillumination, and DIC-fluorescence, it was concluded that the DIC image, which had been superimposed on the fluorescence image, contributed a definite gain in information. Some common errors in the interpretation of the DIC image are discussed; methods of avoiding improper use of equipment are given. The conclusion is drawn that the DIC system is superior to positive and negative phase contrast for the examination of a variety of unstained or stained preparations. Therefore, this method can be used to advantage not only for the examination of unstained preparations, but also on some specimens which have been routinely stained or fluorochromed.     

Super-resolution bright-field optical microscopy based on nanometer topographic contrast

By using an expectation-maximization maximum likelihood estimation algorithm to improve the lateral resolution of a recently developed non-interferometric wide-field optical profilometer, we obtain super-resolution bright-field optical images of nanometer features on a flat surface. The optical profilometer employs a 365-nm light source and an ordinary objective lens of a 0.95 numerical aperture. For objects of 100 nm thickness, lateral features about lambda/7 can be resolved in the restored images without fluorescence labeling. Current image acquisition rate is 0.1 frame/sec, which is limited by the brightness of the light source. With a brighter light source, the imaging speed can be fast enough for real-time observation of dynamic activities in the nanometer scale.     

Multimodal microscopy by digital image processing

A matching algorithm is proposed for aligning microscope images obtained using different modalities, making use of cross-correlations of outputs from Prewitt's edge filter. Brightfield, phase contrast and differential interference contrast microscope images of algal and bacterial cells from an experimental, high-rate algal pond are used for illustration. The information content of multimodal images is explored using principal components analysis and colour displays, and an image which represents optical thickness is constructed digitally.     

基于显微镜聚焦的微装配视觉伺服研究

利用显微镜聚焦理论,沿显微镜光轴方向移动物体,不断计算图像的灰度变化之和,可判断出物体沿光轴的坐标,将这一坐标集成在伺服控制方程中,可完成立体视觉跟踪。这样,采用单目视觉系统就可以获得物体的三维坐标,避免了双目立体视觉系统的复杂结构。为了提高系统图像处理速度,利用卡尔曼滤波器对跟踪的特征点进行预测,并用窗口处理技术减小图像处理区域。实验和仿真结果表明,上述方法可完成复杂微装配的视觉跟踪,系统有好的实时性。     

Specific features of detecting point objects in images formed by a detector array

The efficiency of detecting point objects in images obtained by a photodetector array is analyzed. It is shown that the detection efficiency depends both on the image processing method and on the relationship between the sizes of the array elements, the gaps between these elements, and the point spread function of the optical system forming the image. Results of a computational experiment are given, which confirm the existence of an optimal relationship between these parameters.     

A system to acquire and process scanning transmission electron microscope images

The numerical processing of electron microscope images is becoming important as more investigators need quantitative data on size and density of objects visualized by means of a STEM system [1–3]. We describe the hard- and software of a system for acquisition and processing of such images. Traditionally, the processed negatives of electromicrographs are scanned by a microdensitometer to provide a digital image [4,5]. We decided upon direct digitization of the transmission detector's output without intermediate optical or photochemical steps. The system's performance was tested on latex spheres.     

A simple method allowing DIC imaging in conjunction with confocal microscopy

Current optical methods to collect Nomarski differential interference contrast (DIC) or phase images with a transmitted light detector (TLD) in conjunction with confocal laser scanning microscopy (CLSM) can be technically challenging and inefficient. We describe for the first time a simple method that combines the use of the commercial product QPm (Iatia, Melbourne Australia) with brightfield images collected with the TLD of a CLSM, generating DIC, phase, Zernike phase, dark-field or Hoffman modulation contrast images. The brightfield images may be collected at the same time as the confocal images. This method also allows the calculation of contrast-enhanced images from archival data. The technique described here allows for the creation of contrast-enhanced images such as DIC or phase, without compromising the intensity or quality of confocal images collected simultaneously. Provided the confocal microscope is equipped with a motorized z-drive and a TLD, no hardware or optical modifications are required. The contrast-enhanced images are calculated with software using the quantitative phase-amplitude microscopy technique ( Barone-Nugent et al., 2002 ). This technique, being far simpler during image collection, allows the microscopist to concentrate on their confocal imaging and experimental procedures. Unlike conventional DIC, this technique may be used to calculate DIC images when cells are imaged through plastic, and without the use of expensive strain-free objective lenses.     

A light efficient optically sectioning microscope

We describe a simple method by which optically sectioned images may be obtained. The system geometry is similar to that of a tandem scanning microscope but a one-dimensional grid pattern is used rather than an array of pinholes. This produces a composite image consisting of an optically sectioned image superimposed on a conventional image. A blank sector on the disc is used to provide a wide-field image. Image subtraction yields the optically sectioned image in real time.     

Image restoration for TV‐scan moving images acquired through a semiconductor backscattered electron detector

A semiconductor backscattered electron (BSE) detector has become popular in scanning electron microscopy session. However, detectors of semiconductor type have a serious disadvantage on the frequency characteristics. As a result, fast scan (e.g. TV‐scan) BSE image should be blurred remarkably. It is the purpose of this study to restore this degradation by using digital image processing technology. In order to improve it practically, we have to settle several problems, such as noise, undesirable processing artifacts, and ease of use. Image processing techniques in an impromptu manner like a conventional mask processing are unhelpful for this study, because a complicated degradation of output signal affects severely the phase response as well as the amplitude response of our SEM system. Hence, based on the characteristics of an SEM signal obtained from the semiconductor BSE detector, a proper inverse filter in Fourier domain is designed successfully. Finally, the inverse filter is converted to a special convolution mask, which is skillfully designed, and applied for TV‐scan moving BSE images. The improved BSE image is very effective in the work for finding important objects. SCANNING 31: 229–235, 2009. © 2010 Wiley Periodicals, Inc.     

Sequential processing of quantitative phase images for the study of cell behaviour in real‐time digital holographic microscopy

Transmitted light holographic microscopy is particularly used for quantitative phase imaging of transparent microscopic objects such as living cells. The study of the cell is based on extraction of the dynamic data on cell behaviour from the time‐lapse sequence of the phase images. However, the phase images are affected by the phase aberrations that make the analysis particularly difficult. This is because the phase deformation is prone to change during long‐term experiments. Here, we present a novel algorithm for sequential processing of living cells phase images in a time‐lapse sequence. The algorithm compensates for the deformation of a phase image using weighted least‐squares surface fitting. Moreover, it identifies and segments the individual cells in the phase image. All these procedures are performed automatically and applied immediately after obtaining every single phase image. This property of the algorithm is important for real‐time cell quantitative phase imaging and instantaneous control of the course of the experiment by playback of the recorded sequence up to actual time. Such operator's intervention is a forerunner of process automation derived from image analysis. The efficiency of the propounded algorithm is demonstrated on images of rat fibrosarcoma cells using an off‐axis holographic microscope.     

Efficiency of Bilateral Filter Application in Problems of Optical Flow Calculation

It is proposed to use weight coefficients of the bilateral filter to calculate the measure of similarity of image regions in optical flow calculation algorithms. The efficiency of using this measure is studied by an example of a three-dimensional recursive search algorithm. Weight coefficients are used for calculating the optical flow with subpixel accuracy by the Lucas–Kanade algorithm. A possibility of reducing the optical flow calculation error by means of using the proposed approaches is demonstrated by an example of processing of various types of images. A method of choosing the parameters of the weight functions of the bilateral filter is described and analyzed.     

DIC image reconstruction on large cell scans

The width of the emission spectrum of a common fluorophore allows only for a limited number of spectral distinct fluorescent markers in the visible spectrum, which is also the regime where CCD-cameras are used in microscopy. For imaging of cells or tissues, it is required to obtain an image from which the morphology of the whole cell can be extracted. This is usually achieved by differential interference contrast (DIC) microscopy. These images have a pseudo-3D appearance, easily interpreted by the human brain. In the age of high throughput and high content screening, manual image processing is not an option. Conventional algorithms for image processing often use threshold-based criteria to identify objects of interest. These algorithms fail for DIC images as they have a range from dim to bright with an intermediate intensity equal to the background, so as to produce no clear object boundary. In this article we compare different reconstruction methods for up to 100 MB-large DIC images and implement a new iterative reconstruction method based on the Hilbert Transform that enables identification of cell boundaries with standard threshold algorithms.