光学显微镜自动聚焦的技术研究

在对普通光学显微镜进行一定改装的基础之上 ,设计了一套显微镜自动聚焦系统 ,并且给出了图像的灰度差分绝对值之和算子 ,解决了自动聚焦过程中是否正确聚焦的判断问题。最后还给出了一套基于启发式搜索算法的聚焦搜索策略 ,以加快焦平面的搜索。     

基于Internet的嵌入式数字图像自动对焦系统

提出了一种利用网络技术实现嵌入式数字图像自动对焦系统远程监控的方法,建立了基于DM642的数字显微镜自动调焦系统,设计了系统的网络传输应用程序。最后通过实验证明了该方法的可行性。     

Region sampling for robust and rapid autofocus in microscope

This paper proposes a region sampling based autofocus method for rapid and robust autofocus in microscope. Image content and region size are considered in region sampling criteria. An intelligent search algorithm which employs quartering hill climbing search and golden section search is developed, in which rule‐based evaluation of sampled focusing regions is applied. Experimental results demonstrate that the proposed method can significantly improve the performance of image‐based autofocus. Microsc. Res. Tech. 78:382–390, 2015. © 2015 Wiley Periodicals, Inc.     

Optical microscopy with flexible axial capabilities using a vari‐focus liquid lens

The axial imaging range of optical microscopy is restricted by its fixed working plane and limited depth of field. In this paper, the axial capabilities of an off‐the‐shelf microscope is improved by inserting a liquid lens, which can be controlled by a driving electrical voltage, into the optical path of the microscope. First, the numerical formulas of the working distance and the magnification with the variation of the focus of the liquid lens are inferred using a ray tracing method and conclusion is obtained that the best position for inserting a liquid lens with consistent magnification is the aperture plane and the rear focal plane of the objective lens. Second, with the liquid lens embedded in the microscope, the numerical relationship between the magnification and the working distance of the proposed flexible‐axial‐capability microscope and the liquid lens driving voltage is calibrated and fitted using the inferred numerical formulas. Third, techniques including autofocus, extending depth of field and three‐dimensional imaging are researched and applied, improving the designed microscope to not only flexibly control its working distance, but also to extend the depth of field near the variable working plane. Experiments show that the presented flexible‐axial‐capability microscope has a long working distance range of 8 mm, and by calibrating the magnification curve within the working distance range, samples can be observed and measured precisely. The depth of field can be extended to 400 μm from the variable working plane and is 20 times that of the off‐the‐shelf microscope.     

Precise observation of C. elegans dynamic behaviours under controlled thermal stimulus using a mobile phone–based microscope

We investigated the temperature‐dependent locomotion of Caenorhabditis elegans by using the mobile phone–based microscope. We developed the customized imaging system with mini incubator and smartphone to effectively control the thermal stimulation for precisely observing the temperature‐dependent locomotory behaviours of C. elegans. Using the mobile phone–based microscope, we successfully followed the long‐term progress of specimens of C. elegans in real time as they hatched and explored their temperature‐dependent locomotory behaviour. We are convinced that the mobile phone–based microscope is a useful device for real time and long‐term observations of biological samples during incubation, and can make it possible to carry out live observations via wireless communications regardless of location. In addition, this microscope has the potential for widespread use owing to its low cost and compact design.     

Bilateral prediction and intersection calculation autofocus method for automated microscopy

In this paper, a bilateral prediction and intersection calculation autofocus method for automated microscopy, which obtains the in‐focus position by calculating the intersection of the predicted left and right focus measure curves located respectively in the left and right sides of the peak position of the focus measure curve, is proposed and performed. According to the autofocus method, the area including the peak position of the focus measure curve and its left and right neighbourhoods should be determined firstly, and the left and right neighbourhoods are considered as the left and right sampling areas. The left and right focus measure curves are predicted by appropriate predicting models according to the two sample sequences, which comprise the focus values by evaluating the sampled images in the left sampling area and right sampling area, respectively and their corresponding sampling positions. The intersection of the predicted left and right focus measure curves is calculated and can be considered as the in‐focus position. The autofocus can be realized by moving the focus plane of the microscope to the intersection of the predicted left and right focus measure curves. The proposed bilateral prediction and intersection calculation autofocus method is experimentally verified in an automated light microscopy for implementing microassembly and micromanipulation. The theoretical analyses have shown that the proposed bilateral prediction and intersection calculation autofocus method can not only effectively avoid the principle error caused by assuming the symmetrical focus measure curve in the autofocus methods based on curve fitting, but also eliminate the possible waver search near the peak position in the modified fast climbing servo method. The experimental results have shown that the proposed bilateral prediction and intersection calculation autofocus method possesses the merits as follows: (1) the focusing accuracy is high and slightly affected by the sampling step size and (2) the focusing speed is higher than those of the 7‐point hill‐climbing search method and the quadratic curve fitting method with a determinate focusing accuracy.     

光学显微成像系统图像清晰度评价函数的对比

图像清晰度评价函数是评价各类成像系统成像质量的一个关键函数,为找到合适的图像清晰度评价算法,采用MATLAB软件对16种适用于光学显微成像系统的清晰度评价函数进行仿真,定量分析了不同算法的灵敏度、单峰性、无偏性以及运算速度。实验表明:Laplacian函数具有较高的单峰性、无偏性和灵敏度;存在高斯噪声时,Brenner函数、Tenengrad函数和基于Prewitt算子的函数以及中值滤波-离散余弦函数稳定性好;而存在椒盐噪声时,Roberts函数综合性能最优。     

Digital holographic microscopy and focusing methods based on image sharpness

Digital holographic microscope allows imaging of opaque and transparent specimens without staining. A digitally recorded hologram must be reconstructed numerically at the actual depth of the object to obtain a focused image. We have developed a high‐resolution digital holographic microscope for imaging amplitude and phase objects with autofocusing capability. If the actual depth of an object is not known a priori, it is estimated by comparing the sharpness of several reconstructions at different distances, which is very demanding in means of computational power when the recorded hologram is large. In this paper, we present 11 different sharpness metrics for estimating the actual focus depths of objects. The speed performance of focusing is discussed, and a scaling technique is introduced where the speed of autofocusing increases on the order of square of the scale ratio. We measured the performance of scaling on computer‐generated holograms and on recorded holograms of a biological sample. We show that simulations are in good agreement with the experimental results.     

Evaluating sharpness functions for automated scanning electron microscopy

Fast and reliable autofocus techniques are an important topic for automated scanning electron microscopy. In this paper, different autofocus techniques are discussed and applied to a variety of experimental through-focus series of scanning electron microscopy images with different geometries. The procedure of quality evaluation is described, and for a variety of scanning electron microscope samples it is demonstrated that techniques based on image derivatives and Fourier transforms are in general better than statistical, intensity and histogram-based techniques. Further, it is shown that varying of an extra parameter can dramatically increase quality of an autofocus technique.     

Image distortion correction for micromanipulation system based on SLM microscopic vision

Stereo light microscope (SLM) simulates stereo imaging principle of human eyes. Microscopic vision system based on SLM has become an important visual tool for micro measurement, micromanipulation, and microinjection. We develop a micromanipulation system based on SLM and present an image distortion correction method. We mainly correct two kinds of image distortions: lateral and vertical distortion. Distortion correction consists of two steps. First, a linear fitting algorithm for each row or column of target points is developed, and the fitting errors are calculated. If the fitting errors are smaller than a given threshold, the linear fitting results are kept and used. Otherwise polynomial fitting procedure will be used. Second, the parallelism of straight lines is corrected. The results show that a line in world coordinate frame (WCF) is not necessarily a straight line in image coordinate frame (ICF), or two parallel lines in WCF may be not parallel in ICF. Distortion correction can restore the parallel and linear relationship. For distorted left and right images, the magnitude of distortion exceeds 6 pixels and 4 pixels in the horizontal direction, and 1.2 pixels and 1.7 pixels in the vertical direction, respectively. After corrected, for left and right image, distortion can be reduced to 0.8 pixels and 0.7 pixels in the horizontal direction, and 0.96 pixels and 1.3 pixels in the vertical direction, respectively. The results show that distortion parameters obtained from the proposed method can effectively correct distorted images. Microsc. Res. Tech. 79:162–177, 2016. © 2016 Wiley Periodicals, Inc.     

Correlation of two‐photon in vivo imaging and FIB/SEM microscopy

Advances in the understanding of brain functions are closely linked to the technical developments in microscopy. In this study, we describe a correlative microscopy technique that offers a possibility of combining two‐photon in vivo imaging with focus ion beam/scanning electron microscope (FIB/SEM) techniques. Long‐term two‐photon in vivo imaging allows the visualization of functional interactions within the brain of a living organism over the time, and therefore, is emerging as a new tool for studying the dynamics of neurodegenerative diseases, such as Alzheimer's disease. However, light microscopy has important limitations in revealing alterations occurring at the synaptic level and when this is required, electron microscopy is mandatory. FIB/SEM microscopy is a novel tool for three‐dimensional high‐resolution reconstructions, since it acquires automated serial images at ultrastructural level. Using FIB/SEM imaging, we observed, at 10 nm isotropic resolution, the same dendrites that were imaged in vivo over 9 days. Thus, we analyzed their ultrastructure and monitored the dynamics of the neuropil around them. We found that stable spines (present during the 9 days of imaging) formed typical asymmetric contacts with axons, whereas transient spines (present only during one day of imaging) did not form a synaptic contact. Our data suggest that the morphological classification that was assigned to a dendritic spine according to the in vivo images did not fit with its ultrastructural morphology. The correlative technique described herein is likely to open opportunities for unravelling the earlier unrecognized complexity of the nervous system.     

Near real time in vivo fibre optic confocal microscopy: sub-cellular structure resolved

We have built a fibre optic confocal reflectance microscope capable of imaging biological tissue in near real time. The measured lateral resolution is 3 µm and axial resolution is 6 µm. Images of epithelial cells, excised tissue biopsies, and the human lip in vivo have been obtained at 15 frames s^?1. Both cell morphology and tissue architecture can be appreciated from images obtained with this microscope. This device has the potential to enable reflected light confocal imaging of internal organs for in situ detection of pathology.     

Multichannel wide‐field microscopic FRET imaging based on simultaneous spectral unmixing of excitation and emission spectra

Simultaneous spectral unmixing of excitation and emission spectra (ExEm unmixing) has inherent ability resolving spectral crosstalks, two key issues of quantitative fluorescence resonance energy transfer (FRET) measurement, of both the excitation and emission spectra between donor and acceptor without additional corrections. We here set up a filter‐based multichannel wide‐field microscope for ExEm unmixing‐based FRET imaging (m‐ExEm‐spFRET) containing a constant system correction factor (f_sc) for a stable system. We performed m‐ExEm‐spFRET with four‐ and two‐wavelength excitation respectively on our system to quantitatively image single living cells expressing FRET tandem constructs, and obtained accurate FRET efficiency (E) and concentration ratio of acceptor to donor (R_C). We also performed m‐ExEm‐spFRET imaging for single living cells coexpressing CFP‐Bax and YFP‐Bax, and found that the E values were about 0 for control cells and about 28% for staurosporin‐treated cells when R_C were larger than 1, indicating that staurosporin induced significant oligomerisation.     

A novel autofocusing method using the angle of hilbert space for microscopy

Autofocusing technology is indispensable for routine use of microscopes on a large scale in biological field. The autofocusing method using the angle of Hilbert space is brought forward to measure whether the image is focused or not. The angle of Hillbert space can be used to evaluate accurately the similarity degree of two images. The experiment results show that the autofocusing method can decrease the computational cost and get accuracy for real‐time biological and biomedical images with noise robustness. The focus curves are smooth and possess the unimodality, the monotonicity and the symmetry. Compared with other classic and optimum focus method, the Hilbert method demonstrates its robustness to noise and can improve the focus speed. The experiments showed that the proposed method can increase the overall performance of an autofocus system and has strong applicability in various autofocusing algorithms. Microsc. Res. Tech. 77:289–295, 2014. © 2014 Wiley Periodicals, Inc.     

A wavefront generator for complex pupil function synthesis and point spread function engineering

We describe a simple method to produce an arbitrary complex optical field using a ferroelectric liquid crystal spatial light modulator. The system is configured so as to act as a pupil plane filter in a confocal microscope. The ability to tune the complex pupil function permits the system to be used both to modify the imaging performance by effectively engineering the point spread function as well as to remove optical aberrations present in the optical system.     

Note: Automated maskless micro-multidomain photoalignment

We present a fully automated maskless exposure system for the fabrication of microscopic orientational surface alignment patterns. The maskless system allows us to fabricate arbitrary surface patterns over a 2 mm × 2 mm area with a resolution of 2.2 μm. A confocal autofocus system insures accurate and repeatable focus. Microscopic orientational surface patterns have been demonstrated to exhibit a variety of novel functionalities, such as surface alignment multi-stability.     

Automating fruit fly Drosophila embryo injection for high throughput transgenic studies

To decipher and manipulate the 14 000 identified Drosophila genes, there is a need to inject a large number of embryos with transgenes. We have developed an automated instrument for high throughput injection of Drosophila embryos. It was built on an inverted microscope, equipped with a motorized xy stage, autofocus, a charge coupled device camera, and an injection needle mounted on a high speed vertical stage. A novel, micromachined embryo alignment device was developed to facilitate the arrangement of a large number of eggs. The control system included intelligent and dynamic imaging and analysis software and an embryo injection algorithm imitating a human operator. Once the injection needle and embryo slide are loaded, the software automatically images and characterizes each embryo and subsequently injects DNA into all suitable embryos. The ability to program needle flushing and monitor needle status after each injection ensures reliable delivery of biomaterials. Using this instrument, we performed a set of transformation injection experiments. The robot achieved injection speeds and transformation efficiencies comparable to those of a skilled human injector. Because it can be programed to allow injection at various locations in the embryo, such as the anterior pole or along the dorsal or ventral axes, this system is also suitable for injection of general biochemicals, including drugs and RNAi.     

Compact non‐contact total emission detection for in vivo multiphoton excitation microscopy

We describe a compact, non‐contact design for a total emission detection (c‐TED) system for intra‐vital multiphoton imaging. To conform to a standard upright two‐photon microscope design, this system uses a parabolic mirror surrounding a standard microscope objective in concert with an optical path that does not interfere with normal microscope operation. The non‐contact design of this device allows for maximal light collection without disrupting the physiology of the specimen being examined. Tests were conducted on exposed tissues in live animals to examine the emission collection enhancement of the c‐TED device compared to heavily optimized objective‐based emission collection. The best light collection enhancement was seen from murine fat (5×–2× gains as a function of depth), whereas murine skeletal muscle and rat kidney showed gains of over two and just under twofold near the surface, respectively. Gains decreased with imaging depth (particularly in the kidney). Zebrafish imaging on a reflective substrate showed close to a twofold gain throughout the entire volume of an intact embryo (approximately 150 μm deep). Direct measurement of bleaching rates confirmed that the lower laser powers, enabled by greater light collection efficiency, yielded reduced photobleaching in vivo. The potential benefits of increased light collection in terms of speed of imaging and reduced photo‐damage, as well as the applicability of this device to other multiphoton imaging methods is discussed.     

A universal fluid cell for the imaging of biological specimens in the atomic force microscope

Recently, atomic force microscope (AFM) manufacturers have begun producing instruments specifically designed to image biological specimens. In most instances, they are integrated with an inverted optical microscope, which permits concurrent optical and AFM imaging. An important component of the set‐up is the imaging chamber, whose design determines the nature of the experiments that can be conducted. Many different imaging chamber designs are available, usually designed to optimize a single parameter, such as the dimensions of the substrate or the volume of fluid that can be used throughout the experiment. In this report, we present a universal fluid cell, which simultaneously optimizes all of the parameters that are important for the imaging of biological specimens in the AFM. This novel imaging chamber has been successfully tested using mammalian, plant, and microbial cells. Microsc. Res. Tech. 76:357–363, 2013. © 2013 Wiley Periodicals, Inc.     

Combined AFM and confocal fluorescence microscope for applications in bio-nanotechnology

We present a custom-designed atomic force fluorescence microscope (AFFM), which can perform simultaneous optical and topographic measurements with single molecule sensitivity throughout the whole visible to near-infrared spectral region. Integration of atomic force microscopy (AFM) and confocal fluorescence microscopy combines the high-resolution topographical imaging of AFM with the reliable (bio)-chemical identification capability of optical methods. The AFFM is equipped with a spectrograph enabling combined topographic and fluorescence spectral imaging, which significantly enhances discrimination of spectroscopically distinct objects. The modular design allows easy switching between different modes of operation such as tip-scanning, sample-scanning or mechanical manipulation, all of which are combined with synchronous optical detection. We demonstrate that coupling the AFM with the fluorescence microscope does not compromise its ability to image with a high spatial resolution. Examples of several modes of operation of the AFFM are shown using two-dimensional crystals and membranes containing light-harvesting complexes from the photosynthetic bacterium Rhodobacter sphaeroides.