数码显微镜DIY及应用探讨

数码显微镜相对于光学显微镜而言,在观察效果、指示效果、图像记录、实验效率等方面均有很大的优点。作者利用现有的光学显微镜和一般的电脑摄像头,经过组装与调试,DIY成了完全可以实用的数码显微镜。并提供了作者在实际应用中总结的一些经验：如硬件和软件的选择与使用,图像的显示、指示、捕获、存储及动态摄像等。     

A reconfigurable real-time compressive-sampling camera for biological applications

Many applications in biology, such as long-term functional imaging of neural and cardiac systems, require continuous high-speed imaging. This is typically not possible, however, using commercially available systems. The frame rate and the recording time of high-speed cameras are limited by the digitization rate and the capacity of on-camera memory. Further restrictions are often imposed by the limited bandwidth of the data link to the host computer. Even if the system bandwidth is not a limiting factor, continuous high-speed acquisition results in very large volumes of data that are difficult to handle, particularly when real-time analysis is required. In response to this issue many cameras allow a predetermined, rectangular region of interest (ROI) to be sampled, however this approach lacks flexibility and is blind to the image region outside of the ROI. We have addressed this problem by building a camera system using a randomly-addressable CMOS sensor. The camera has a low bandwidth, but is able to capture continuous high-speed images of an arbitrarily defined ROI, using most of the available bandwidth, while simultaneously acquiring low-speed, full frame images using the remaining bandwidth. In addition, the camera is able to use the full-frame information to recalculate the positions of targets and update the high-speed ROIs without interrupting acquisition. In this way the camera is capable of imaging moving targets at high-speed while simultaneously imaging the whole frame at a lower speed. We have used this camera system to monitor the heartbeat and blood cell flow of a water flea (Daphnia) at frame rates in excess of 1500 fps.     

Comparison of different camera calibration approaches for underwater applications

The purpose of this study was to compare three camera calibration approaches applied to underwater applications: (1) static control points with nonlinear DLT; (2) moving wand with nonlinear camera model and bundle adjustment; (3) moving plate with nonlinear camera model. The DVideo kinematic analysis system was used for underwater data acquisition. The system consisted of two gen-locked Basler cameras working at 100 Hz, with wide angle lenses that were enclosed in housings. The accuracy of the methods was compared in a dynamic rigid bar test (acquisition volume-4.5×1×1.5 m(3)). The mean absolute errors were 6.19 mm for the nonlinear DLT, 1.16 mm for the wand calibration, 1.20 mm for the 2D plate calibration using 8 control points and 0.73 mm for the 2D plane calibration using 16 control points. The results of the wand and 2D plate camera calibration methods were less associated to the rigid body position in the working volume and provided better accuracy than the nonlinear DLT. Wand and 2D plate camera calibration methods presented similar and highly accurate results, being alternatives for underwater 3D motion analysis.     

A digital microscope for real time detection of moving microorganisms

A digital microscope which can store time sequences in order to analyse moving objects, without the aid of a videorecorder, is described. This digital microscope can also be used to highlight moving microorganisms, such as protozoa, by suppressing the images of any stationary object, such as particles of dirt, dead or immobile cells. This microscope which operates in real time, is equipped with such standard devices as a TV-camera and a digital frame grabber with hardware facilities (Zoom, Pan, Scroll).     

Comparison of digital focus criteria for a TV microscope system

Existing focusing methods, such as standard deviation of gray values, gradients, information content of the image and the lateral inhibition function, were compared, and a new combination criterion was developed. This combined method consists of using the standard deviation of the image for coarse focusing followed by a modified form of the lateral inhibition for the fine focus. The lateral inhibitor model of the eye is a recursive filter that can be simplified to a nonrecursive filtering algorithm, as postulated by Rechenberg. The reported focus method operates over a focusing range (z-axis) of +/- 300 micron and is accurate within +/- 0.1 micron in the transmission, dark-field, and phase contrast microscope.     

Remote monitoring of nests using digital camera technology

ABSTRACT.   Although cameras have been used for many years to collect data at birds' nests, recent advances in digital technology have led to increased storage capacity, faster and easier review of data, and reduced power consumption. The development of sophisticated triggering mechanisms, such as video motion detection, herald a new era of portable, energy-efficient systems that require less frequent maintenance. We used a digital infrared camera system to monitor predation events at the nests of ground-nesting Lapwings ( Vanellus vanellus ; N = 40) and tree-nesting Spotted Flycatchers ( Muscicapa striata ; N = 17). Eight predator species were recorded taking eggs or chicks at Lapwing ( N = 10) and Spotted Flycatcher ( N = 7) nests, including red fox ( Vulpes vulpes ), badger ( Meles meles ), sheep ( Ovis aries ), and Carrion Crow ( Corvus corone ) at Lapwing nests and Eurasian Jay ( Garrulus glandarius ), European Buzzard ( Buteo buteo ), Great-spotted Woodpecker ( Dendrocopus major ), and domestic cat ( Felis catus ) at flycatcher nests. We tested three system configurations in an attempt to minimize power requirements and maximize predation-event recording capability. We found that the use of a passive-infrared sensor to awaken the system from standby mode did not compromise reliability and reduced power consumption. With this system, a 38-A h battery operated the system for 120 h with no maintenance at a cost per unit of about $800 (US; or £400 UK Sterling). Further modifications would permit adaptation of the system for a wide range of scientific and nest surveillance operations.     

Development of a digital infrared video camera system for recording and remote capturing

A digital infrared video camera system was developed for recording and remote capturing. The components are described in detail. Based on 2 years of experience within a wild boar (Sus scrofa) project, the system gave proof of its practicability, reliability and efficiency.     

The suitability of digital colposcopy for telematic applications]

Since carcinoma of the cervix is one of the most common cancers in women, screening of the cervix has acquired considerable importance. Colposcopy is a simple diagnostic method of detecting suspicious changes at an early stage. Shortcomings of this method are its low specificity and high inter- and intra-observer variability. A clinical pilot study was therefore carried out to investigate the advantages of a digital colposcopic system comprising a binocular colposcope coupled to a CCD camera and a computer. The aim of the study was to evaluate the reliability of diagnostic findings of the cervix obtained with digital colposcopy in comparison with standard binocular colposcopy, and to assess its suitability for telematic applications (teleconsultation, telediagnostics, treaching). A total of 315 patients were examined and statistically analysed. The patients were first submitted to a conventional colposcopic examination and a diagnosis was established. During the colposcopic examination camera images were stored on a computer, on the basis of which a second physician experienced in colposcopy reviewed the initial diagnosis. The primary and secondary findings of each patient were classified into 4 categories and compared following the Rome classification system. Agreement between the primary and secondary diagnosis was established in 69% of the cases (kappa = 0.60 +/- 0.03). No bias was observed in terms of under- or overrating. The percentage of non-assessable colposcopic examinations was 9.2%. Digital colposcopy is therefore suitable for reproducing diagnostic findings on the computer, given adequate digital image quality and a suitable classification model. The method has clear advantages with regard to follow-up, internal quality control of the diagnosis, and the training and further education of physicians and students. In the future, telecolposcopy may open up new opportunities in gynaecology.     

Biological applications of the scanning transmission electron microscope

The STEM has the capability to acquire spatial and chemical information from almost all scattered electrons. Dedicated STEMs do not have any post-specimen lenses but many different detectors instead.The STEM axial bright field mode is quite insensitive to multiple scattering occurring while imaging thick samples such as vitrified whole cells. The blur of small gold sphere has been simulated by Monte Carlo calculations.Direct electron detectors record the rich information of microdiffraction patterns from every irradiated spot while the STEM beam raster-scans over the sample.

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A custom confocal and two-photon digital laser scanning microscope

We describe a custom one-photon (confocal) and two-photon all-digital (photon counting) laser scanning microscope. The confocal component uses two avalanche photodiodes (APDs) as the fluorescence detector to achieve high sensitivity and to overcome the limited photon counting rate of a single APD ( approximately 5 MHz). The confocal component is approximately nine times more efficient than our commercial confocal microscope (fluorophore fluo 4). Switching from one-photon to two-photon excitation mode (Ti:sapphire laser) is accomplished by moving a single mirror beneath the objective lens. The pulse from the Ti:sapphire laser is 109 fs in duration at the specimen plane, and average power is approximately 5 mW. Two-photon excited fluorescence is detected by a fast photomultiplier tube. With a x63 1.4 NA oil-immersion objective, the resolution of the confocal system is 0.25 microm laterally and 0.52 microm axially. For the two-photon system, the corresponding values are 0.28 and 0.82 microm. The system is advantageous when excitation intensity must be limited, when fluorescence is low, or when thick, scattering specimens are being studied (with two-photon excitation).     

Uncertainties involved in leaf fall phenology detected by digital camera

We evaluated the uncertainty in the estimation of year-to-year variability in the timing of leaf fall detected by the analysis of red, green and blue (RGB) values extracted from daily phenological images in a deciduous broad-leaved forest in Japan. We examined (1) the spatial distribution of individual tree species within a 1-ha permanent plot and the spatio-temporal variability of leaf litter of various species for 8 years; and (2) the relationship between the year-to-year variability of leaf fall detected by leaf litter and that detected by phenological images of various species. Uncertainties were caused by (1) the heterogeneous distribution of each species within the whole forest community; (2) the year-to-year variability of the timing of leaf fall among species; and (3) differences in leaf colouring and leaf fall patterns among species. Our results indicate the importance of integrating RGB analysis of each species and of the whole canopy on the basis of spatial locations of individuals and proportions of tree species within a forest to reduce uncertainty.     

Early metallurgical applications of the electron microscope in Cambridge

A short account of a post-war (1948) survey of electron microscopes in Europe and of work done at Cambridge investigating metal replicas and dislocations in thin specimens.     

扫描探针显微镜及其在生物医学研究中的应用

本文介绍了一类可以从原子水平到微米尺寸观察物质结构的三维成像工具——扫描探针显微镜(SPM),重点介绍了扫描隧道显微镜(STM)和原子力显微镜(AFM)的基本原理,以及SPM在细胞生物学、核酸和小分子成像等生物医学研究领域的一些应用。SPM不久将可能成为大多数生命科学实验室的一项重要技术。     

Software for quantification of labeled bacteria from digital microscope images by automated image analysis

Automated image analysis software, CellC, was developed and validated for quantification of bacterial cells from digital microscope images. CellC enables automated enumeration of bacterial cells, comparison of total count and specific count images [e.g., 4',6-diamino-2-phenylindole (DAPI) and fluorescence in situ hybridization (FISH) images], and provides quantitative estimates of cell morphology. The software includes an intuitive graphical user interface that enables easy usage as well as sequential analysis of multiple images without user intervention. Validation of enumeration reveals correlation to be better than 0.98 when total bacterial counts by CellC are compared with manual enumeration, with all validated image types. The software is freely available and modifiable: the executable files and MATLAB source codes can be obtained at www. cs. tut.fi/sgn/csb/cellc.     

Single beam optical trapping integrated in a confocal microscope for biological applications.

Confocal microscopy is very useful in biology because of its three dimensional imaging capacities and has proven to be an excellent tool to study the 3D organization of, for instance, cell structures. This property of confocal microscopy makes it also very suitable for observation during guidance of the three dimensional manipulation of single cells or cell elements. Therefore we decided to integrate a confocal microscope and a single beam optical manipulator into a single instrument. The advantage of optical manipulation over mechanical techniques is that it is non-invasive and therefore may be applied on living (micro-) organisms and cells. The creation of an effective single beam optical trap requires the use of a high numerical aperture (N.A.) objective to focus the laser beam. In this paper we briefly discuss the vertical or axial force exerted on a sphere in a single beam trap. The axial force on a sphere placed on the optical axis, caused by reflection and refraction, is calculated applying a electromagnetic vector diffraction theory to determine the field distribution in the focal region. One of the results is that the particle also experiences a vertical trapping force towards the focusing lens when it is in the strongly convergent part of the field in addition to the known negative signed trapping force in the divergent part of the field. Further we describe an instrumental approach to realize optical trapping in which the optical trap position is controlled by moving the focusing objective only.(ABSTRACT TRUNCATED AT 250 WORDS)     

应用数码相机拍摄人体解剖学标本的技巧

在运用数码相机拍摄解剖标本时,注意处理好以下几个方面的技巧因素:①相机的选择;②实物的制作与准备;③讲究用光角度④选择背景;⑤正确使用三脚架;⑥调整白平衡,掌握曝光时间;⑦图片的后期处理.     

Estimating the spatial position of marine mammals based on digital camera recordings

Estimating the spatial position of organisms is essential to quantify interactions between the organism and the characteristics of its surroundings, for example, predator–prey interactions, habitat selection, and social associations. Because marine mammals spend most of their time under water and may appear at the surface only briefly, determining their exact geographic location can be challenging. Here, we developed a photogrammetric method to accurately estimate the spatial position of marine mammals or birds at the sea surface. Digital recordings containing landscape features with known geographic coordinates can be used to estimate the distance and bearing of each sighting relative to the observation point. The method can correct for frame rotation, estimates pixel size based on the reference points, and can be applied to scenarios with and without a visible horizon. A set of R functions was written to process the images and obtain accurate geographic coordinates for each sighting. The method is applied to estimate the spatiotemporal fine‐scale distribution of harbour porpoises in a tidal inlet. Video recordings of harbour porpoises were made from land, using a standard digital single‐lens reflex (DSLR) camera, positioned at a height of 9.59 m above mean sea level. Porpoises were detected up to a distance of ~3136 m (mean 596 m), with a mean location error of 12 m. The method presented here allows for multiple detections of different individuals within a single video frame and for tracking movements of individuals based on repeated sightings. In comparison with traditional methods, this method only requires a digital camera to provide accurate location estimates. It especially has great potential in regions with ample data on local (a)biotic conditions, to help resolve functional mechanisms underlying habitat selection and other behaviors in marine mammals in coastal areas.     

Acquisition of chemiluminescent signals from immunoblots with a digital single-lens reflex camera

We found that certain mid-range consumer-level digital single-lens reflex (SLR) cameras using full-frame complementary metal oxide semiconductor (CMOS) sensors outperform X-ray film in acquiring signals from immunoblots that use enhanced chemiluminescence for detection. These cameras exhibit a sensitivity comparable to X-ray film, yet they provide a 3-fold increase in linear dynamic range and substantial cost savings over time, are more convenient to use, and eliminate the chemical waste associated with processing film.