Does light microscopy have a future?

The application of light microscopy in medicine and cell biology has been significantly influenced by both the availability of specific biological reagents such as monoclonal antibodies and nucleic acid probes, as well as by the enormous progress in microelectronics and computer technology. It is likely that specific reagents for a variety of cellular macromolecules will become available on a large scale in the coming years. Moreover, methods using both sensitive detection devices such as charge-coupled device (CCD) cameras and sophisticated image processing exist to quantify this information at the single molecule level in morphologically intact cells. This paper describes the impact of these two factors on the light microscope of the future, with special emphasis on fluorescence. It defines the improvements that still are required to solve some of the challenging problems such as the quantification of unique genes and their products in intact cells, the quantification of DNA adducts and the detection of rare mutant cells or circulating tumour cells.     

Problems of low-light detection in real-time at high resolution

Various kinds of low-light-level detectors and image intensifiers are described and specific charge-coupled device (CCD) developments are discussed from the perspective of the astronomical community, with possible application to optical microscopy. A number of research groups are reporting success at obtaining custom CCDs for application to high-quality cameras working at low-light levels. CCDs offer significant advantages in image quality and sensitivity compared with more traditional devices. Signal to noise (S/N) is derived for both CCDs and intensified devices. There is a tradeoff between the two and, because of the high read-out rate required by microscopists, intensified devices still offer a S/N advantage at the lowest light levels.     

Fluorescence microscopy: established and emerging methods, experimental strategies, and applications in immunology

Cutting-edge biophysical technologies including total internal reflection fluorescence microscopy, single molecule fluorescence, single channel opening events, fluorescence resonance energy transfer, high-speed exposures, two-photon imaging, fluorescence lifetime imaging, and other tools are becoming increasingly important in immunology as they link molecular events to cellular physiology, a key goal of modern immunology. The primary concern in all forms of microscopy is the generation of contrast; for fluorescence microscopy contrast can be thought of as the difference in intensity between the cell and background, the signal-to-noise ratio. High information-content images can be formed by enhancing the signal, suppressing the noise, or both. As improved tools, such as ICCD and EMCCD cameras, become available for fluorescence imaging in molecular and cellular immunology, it is important to optimize other aspects of the imaging system. Numerous practical strategies to enhance fluorescence microscopy experiments are reviewed. The use of instrumentation such as light traps, cameras, objectives, improved fluorescent labels, and image filtration routines applicable to low light level experiments are discussed. New methodologies providing resolution well beyond that given by the Rayleigh criterion are outlined. Ongoing and future developments in fluorescence microscopy instrumentation and technique are reviewed. This review is intended to address situations where the signal is weak, which is important for emerging techniques stressing super-resolution or live cell dynamics, but is less important for conventional applications such as indirect immunofluorescence. This review provides a broad integrative discussion of fluorescence microscopy with selected applications in immunology.     

Contrast matching in a sequence of images by histogram normalization

Variations in contrast between digital light microscopy images of a video sequence can be filtered out by matching the contrast of each individual image in the sequence to a reference image using the principle of histogram matching. This ensures the reliability of grey-scale threshold values in each frame within an image sequence. Here we describe examples of digital image-to-image contrast matching as applied to video light microscopy.     

Video microscopic image processing facilitates the evaluation of light microscopic autoradiography at high magnification

Light microscopic autoradiographs of H-thymidine labelled unstained semithin sections of Xenopus laevis embryonic nuclei were examined with conventional Nomarski differential interference contrast, phase-contrast and video microscopy. Whereas at low magnification it was possible to obtain a photograph of the nuclear structure and the silver grains in one focal plain, at high magnification, with small depths of focus, a satisfactory image was not attainable. Therefore, we stored the images of the two different focus levels with a digital image processing system and combined both images by an arithmetic operation. This video microscopic technique allows the use of high magnification light microscopy with oil immersion objectives and the application of additional electronic contrast enhancing methods for an adequate and rapid analysis of light microscopic autoradiographs.     

基于短基线双目内窥镜成像系统的3D视频标定与校正

为了解决短基线双目内窥成像系统获得的视频图像在裸眼3D显示设备中观看到的视频纵深感和立体感较弱的问题,通过分析双目内窥镜的参数以及立体视频中图像对的视差,提出了基于短基线双目内窥成像系统的立体视频校正和视差调整方法。首先,对采用的双目结构内窥系统进行相机标定,获取各相机参数和相机间的位置参数;其次,利用获得的参数进行相机视频校正,再针对裸眼3D显示设备对视频源的参数要求进行图像对的视差调整,最终获得符合裸眼3D立体显示设备要求并适合人眼观看的双目内窥系统实时显示立体视频。通过实验验证了方法的可行性,实际搭建了一套基线距离为8 mm的短基线双目内窥成像系统,原始视差范围(0,64)像素,经视差调整后达到（-30, 30）像素,双路并行视频处理25 帧/s并实时显示。与实验室设计的裸眼3D立体显示系统匹配,可实现具有明显立体感的医用内窥镜实时裸眼3D成像。     

Digital image acquisition in in vivo confocal microscopy

A flexible system for the real-time acquisition of in vivo images has been developed. Images are generated using a tandem scanning confocal microscope interfaced to a low-light-level camera. The video signal from the camera is digitized and stored using a Gould image processing system with a real-time digital disk (RTDD). The RTDD can store up to 3200 512 times 512 pixel images at video rates (30 images s^?1). Images can be input directly from the camera during the study, or off-line from a Super VHS video recorder. Once a segment of experimental interest is digitized onto the RTDD, the user can interactively step through the images, average stable sequences, and identify candidates for further processing and analysis. Examples of how this system can be used to study the physiology of various organ systems in vivo are presented.     

Video-enhanced microscopy with a computer frame memory

Video-enhanced microscopy combined with the use of a computer frame memory extends considerably the useful range of our video enhanced contrast (AVEC) methods for polarizing, double-beam interference and differential interference contrast microscopy. Increased visual contrast is achieved by two stages of amplifications: the first optical, by using high bias retardation settings, and the second electronic. These steps are followed by a reduction of background brightness by means of a clamp voltage applied to a DC restoration circuit of the video camera. One of the limitations of the AVEC method alone is the inevitable appearance under high gain conditions of a pattern of mottle due to inaccessible dirt and defects in the lenses even of high quality. This limitation has been circumvented by storing the mottle pattern in the frame memory (frame store) and continuously subtracting it from each succeeding frame to clear the image. A major gain in image quality has resulted. In polarizing microscopy, the frame memory can be used also to subtract the image at one compensator setting from that at the equivalent setting of opposite sign, thus removing from the final image not only most of the mottle pattern but also the contrast due to the bright-field contrast. In the polarizing microscope, these manipulations of the raw video image make it possible to observe and measure the birefringence of various organelles and elements such as microtubules, intermediate filaments and bundles of as few as a half dozen actin filaments. Since scattered light is also removed from the image, features hidden from view in the unprocessed image become visible. In differential interference microscopy, the AVEC method makes visible (i.e. detectable) many linear elements and particles that are an order of magnitude smaller than the resolution limit and not visible in the optical image. Such features are inflated by diffraction, however, to Airy disk size.     

Analysis of heterogeneous fluorescence photobleaching by video kinetics imaging: The method of cumulants

The method of cumulants has been applied to digital video fluorescence microscopy. The method is used to reconstruct the distribution of fluorescent molecules before the initiation of fluorescence photobleaching, and to characterize heterogeneous photobleaching by imaging one or more of the cumulants of the bleaching decay rate. Using the pipelined pixel processor of the image analysis system for the bulk of the calculations, rather than the general-purpose host-computer CPU, the video kinetics imaging can be performed in near real-time. The method is applied to chick embryo myotubes labelled with fluorescein-conjugated α-bungarotoxin. The pre-bleach fluorescence distribution is derived, and the image of fluorescein fluorescence is separated from glutaraldehyde-induced autofluorescence on the basis of the spatially resolved average photobleaching decay rate.     

Image compression and data integrity in confocal microscopy

To manage large volumes of image data generated routinely using real-time confocal microscopy, compressing image data using a lossy algorithm prior to sustained video rate transferring and/or storing is proposed. Test criteria for determining the acceptability of uncompressed data, both qualitatively and quantitatively, are described, and an empirical demonstration of the use of lossy compression in data management is provided. It is found that, if appropriately used, the lossy compression scheme could retain all the useful information in the data while providing better compression ratios (memory for the original/ memory for the compressed) when compared with a lossless scheme.     

Image comparisons of snow and ice crystals photographed by light (video) microscopy and low-temperature scanning electron microscopy

Light (video) microscopy and low-temperature scanning electron microscopy (SEM) were used to examine and record images of identical precipitated and metamorphosed snow crystals as well as glacial ice grains. Collection procedures enabled numerous samples from distant locations to be shipped to a laboratory for storage and/or observation. The frozen samples could be imaged with a video microscope in the laboratory at ambient temperatures or with the low-temperature SEM. Stereo images obtained by video microscopy or low-temperature SEM greatly increased the ease of structural interpretations. The preparation procedures that were used for low-temperature SEM did not result in sublimation or melting. However, this technique did provide far greater resolution and depth of focus over that of the video microscope. The advantage of resolution was especially evident when examining the small particles associated with rime and graupel (snow crystals encumbered with frozen water droplets), whereas the greater depth of focus provided clearer photographs of large crystals such as depth hoar, and ice. Because the SEM images contained only surface information while the video images were frequently confounded by surface and internal information, the SEM images also clarified the structural features of depth hoar crystals and ice grains. Low-temperature SEM appears to have considerable promise for future investigations of snow and ice.     

空间相机中的偏流角控制

像移补偿技术是高分辨力空间相机的移补偿技术是高分辨力空间相机的关键技术.由于偏流角的存在,使得像移速度在像面坐标系存在两个分量:前向像移速度和横向像移速度,偏流角控制本质上是消除横向像移速度,因此,偏流角控制是空间相机像移补偿的一部分.不同类型的空间相机有不同的像移补偿措施,也就有不同的偏流角控制方法,本文对像移补偿方法中的偏流角控制及作用进行分析,并且介绍和分析了采用TDICCD器件的图像传输型空间相机的偏流角控制的方法.     

A comparison between the use of a high-resolution CCD camera and 35 mm film for obtaining coloured micrographs

In light microscopy, colour CCD cameras are now capable of generating image data sets that contain more information than can be captured with slow 35 mm colour reversal film. The resolution of colour CCD cameras with a high density of sensor elements ( 3300 × 2200 per channel of colour) is equivalent to that of slow 35 mm colour film over typical fields of view for objectives with a wide range of magnifications and numerical apertures. The contrast that can be achieved in images derived from the data sets obtained with colour CCD cameras far exceeds that found with film and can exceed that of human vision. Finally, the data sets collected with high-resolution colour CCD cameras are capable of being displayed at a wide range (four-fold) of different magnifications easily and interchangeably. Consequently, the combination of a data set that describes a relatively large field of view with one or two data sets that describe specific details taken with an eight-fold increase in magnification are all that is necessary to describe the salient features of the vast majority of stained specimens examined with transmitted light microscopy.     

视频智能检测技术在视频矩阵系统中的应用

介绍一种在视频矩阵系统中可以广泛应用的视频检测技术,其原理是视频检测模块对视频信号的强度和同步信息进行实时检测,然后再将检测后的数据通过RS232通讯反馈到矩阵系统的主机,从而矩阵系统就能够实时获取所监控范围内各摄像机的视频质量信息,进而可以作出相关决策.     

Light sources and cameras for standard in vitro membrane potential and high‐speed ion imaging

Membrane potential and fast ion imaging are now standard optical techniques routinely used to record dynamic physiological signals in several preparations in vitro. Although detailed resolution of optical signals can be improved by confocal or two‐photon microscopy, high spatial and temporal resolution can be obtained using conventional microscopy and affordable light sources and cameras. Thus, standard wide‐field imaging methods are still the most common in research laboratories and can often produce measurements with a signal‐to‐noise ratio that is superior to other optical approaches. This paper seeks to review the most important instrumentation used in these experiments, with particular reference to recent technological advances. We analyse in detail the optical constraints dictating the type of signals that are obtained with voltage and ion imaging and we discuss how to use this information to choose the optimal apparatus. Then, we discuss the available light sources with specific attention to light emitting diodes and solid state lasers. We then address the current state‐of‐the‐art of available charge coupled device, electron multiplying charge coupled device and complementary metal oxide semiconductor cameras and we analyse the characteristics that need to be taken into account for the choice of optimal detector. Finally, we conclude by discussing prospective future developments that are likely to further improve the quality of the signals expanding the capability of the techniques and opening the gate to novel applications.     

基于IOS平台的移动终端实时监控系统

以移动终端视频监控系统为研究背景,设计和实现了基于IOS平台的移动终端实时监控系统。系统基于移动终端实现了实时视频数据的传输、解码和优化。使用摄像头采集的视频流,通过服务器将监控视频进行分流发送到iPhone移动终端,用户可以随时随地在终端查看监控视频。     

Omnifocus video camera

The omnifocus video camera takes videos, in which objects at different distances are all in focus in a single video display. The omnifocus video camera consists of an array of color video cameras combined with a unique distance mapping camera called the Divcam. The color video cameras are all aimed at the same scene, but each is focused at a different distance. The Divcam provides real-time distance information for every pixel in the scene. A pixel selection utility uses the distance information to select individual pixels from the multiple video outputs focused at different distances, in order to generate the final single video display that is everywhere in focus. This paper presents principle of operation, design consideration, detailed construction, and over all performance of the omnifocus video camera. The major emphasis of the paper is the proof of concept, but the prototype has been developed enough to demonstrate the superiority of this video camera over a conventional video camera. The resolution of the prototype is high, capturing even fine details such as fingerprints in the image. Just as the movie camera was a significant advance over the still camera, the omnifocus video camera represents a significant advance over all-focus cameras for still images.     

Natural color scanning electron microscopy based on the frequency characteristics of the human visual system

The principles of image formation in natural color scanning electron microscopy (NC-SEM) are discussed in detail. This method is based on the frequency characteristic of the human visual system. It is shown that the Mach effect and masking effect are important in the characteristics. The former, which can enhance structural details, is visually similar to the edge effect in secondary electron (SE) images, and the latter is required for proper representation of very degraded color information obtained from a light microscope. When using these effects suitably, an NC-SEM image with the resolution equivalent to that of an SEM image can be acquired, though it is composed of an SEM image and a special video microscopy (VM) image with a resolution much lower than the SEM image of the identical view. The NC-SEM is more effective than the SEM in observation. interpretation, and analysis of various samples with important color information.     

Laser scanning phase modulation microscope

We describe the concept and first implementation of an innovative new instrument for quantitative light microscopy. Currently, it provides selective imaging of optical path differences due to birefringence; with further development, it is also possible to selectively image several optical properties, including refractive path differences, optical rotation, and linear and circular dichroism, all with diffraction-limited resolution. An image consists of a 512×512 element array, with each pixel displaying one of 256 grey levels, linearly proportional to the specific optical property being observed. Additionally, conventional brightfield and polarized light microscopy are available, with the accompanying advantages of laser scanning and digital image processing. The microscope consists of three subsystems, representing three distinct technologies. The laser scanning subsystem moves a focused, microspot across the specimen; the output of a photodetector is an electric signal corresponding to a scanned image. The image display subsystem digitizes this signal and displays it as an image on a video monitor. When used in conjunction with a phase modulation feedback loop, the image formed is of the specimen's birefringent retardation or other selected optical property. The digitized images are also available for computer enhancement.     

智能视频分析在输电线舞动监测中的应用

为了获得有效的测量信息,建立架空输电线舞动自动监测系统,采用图像与视觉分析方法,提出了一种智能视频分析在舞动监测中的应用的系统设计。该方法采用固定于杆塔上的摄像头拍摄杆塔和电缆图像,在电力线上加装参照物的方法增强识别效果,采用智能视频分析算法获得舞动轨迹、幅度与角度。该系统通过ZigBee技术构建无线传感器网络进行可靠通信,采用风光互补供电方式保证野外供电的持续可靠。为输电线舞动的动态监测与预警提供了一种新的方法和思路。