Computer-aided microtomography with true 3-D display in electron microscopy

A novel research system has been designed to permit three-dimensional (3-D) viewing of high resolution image data from transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The system consists of front-end primary data acquisition devices, such as TEM and SEM machines, which are equipped with computer-controlled specimen tilt stages. The output from these machines is in analogue form, where a video camera attached to the TEM provides the sequential analogue image output while the SEM direct video output is utilized. A 10 MHz digitizer transforms the video image to a digital array of 512 X 512 pixel units of 8 bits deep-stored in a frame buffer. Digital images from multiple projections are reconstructed into 3-D image boxes in a dedicated computer. Attached to the computer is a powerful true 3-D display device which has hardware for graphic manipulations including tilt and rotate on any axis and for probing the image with a 3-D cursor. Data editing and automatic contouring functions are used to enhance areas of interest, and specialized software is available for measurement of numbers, distances, areas, and volumes. With proper archiving of reconstructed image sequences, a dynamic 3-D presentation is possible. The microtomography system is highly versatile and can process image data on-line or from remote sites from which data records would typically be transported on computer tape, video tape, or floppy disk.     

A Digital Image Analysis System for Comparing Groups of Small Nematodes

A digital imaging system was developed for measuring various physical characteristics of individual nematodes and for comparing groups of nematodes. The equipment consists of a microscope, a video camera, a video digitizer, interactive displays, and a computer. Various physical and mathematical methods were incorporated, algorithms devised, and computer software written for image acquisition, editing, and analysis. To test the system, four populations of an isolate of the pinewood nematode, Bursaphelenchus xylophilus, subjected to 100% relative humidity at 22 C for 0, 12, 24, or 48 hours were compared. The results showed that the system can be used to measure physical parameters of individual nematodes and to differentiate groups of nematodes.     

A microcomputer based system for three-dimensional reconstructions from tomographic or histologic sections

The reconstructions of three-dimensional (3-D) objects from serial two-dimensional (2-D) images can contribute to the understanding of many biologic structures, from organelles to organs and tissues. The 3-D reconstruction of sections can be divided into several major tasks: image acquisition, alignment of slices, internal object definition, object reconstruction and rotation of the completed image. A fast, versatile, interactive system was devised for the reconstruction of 3-D objects from serial 2-D images using a low-cost microcomputer, original programs and commercial software. The system allows reconstruction from any serial images, e.g., electron micrographs, histologic sections or computed tomograms. A photographic image or a microscopic field is acquired into the computer memory using a video digitizer. Slices are superimposed and aligned to each other using an operator-interactive program. A contour-(edge-) finding algorithm isolates an object of interest from the background image by "subtraction" of the image from an overlaid, slightly shifted identical image. Contours for each slice are input to a reconstruction procedure, which calculates the x, y and z coordinates of every point in a slice and the thickness and number of slices. It then calculates the illumination for every point using a given point source of light and an intensity-fading coefficient. Finally, the points are represented by cubes to provide dimension and reflective surfaces. A cube of appropriate shade and color represents in 2-D the equivalent of a 3-D object; this results in a very effective 3-D image. The reconstruction is rotated by recalculating the positions of every point defining the object and rebuilding the image.(ABSTRACT TRUNCATED AT 250 WORDS)     

A microcomputer system for video image analysis and diagnostic microdensitometry

A system for microdensitometry based on a microcomputer, video digitizer and solid-state camera has been developed. Image analysis and densitometry are achieved with convenient control over image editing and calibration. The linear photometric properties of the imaging device enable measurements of high accuracy. The system has proven to give rapid and repeatable performance for determining DNA content distribution from measurements of Feulgen-stained cell nuclei. The results show that a practical image analysis microdensitometer can be designed using a readily available microcomputer. The low cost and simple operation are of benefit for diagnostic applications in which flow cytometry is not possible, the time required for microscope photometry is too great or an automated image analyzer and support staff are not available.     

Blood velocity measurement in human conjunctival vessels

The bulbar conjunctiva is one of the few areas in which blood flow in the peripheral vasculature can be directly and noninvasively observed in the human. Although extensive literature exists describing morphological changes which correlate with a variety of systemic diseases in this vasculature, little quantitative data is available on hemodynamics in either normal or abnormal states. The hemodynamic data available are primarily subjective assessments of "low flow." Approaches to place the subjective assessment on more quantitative grounds have usually been based on photographic techniques that have intrinsic inadequacies. The objective of the work reported here was to develop a system capable of providing sequential blood velocity data potentially useful for providing quantitative information on blood flow and its change in the microvessels of the human conjunctiva. The method that has evolved uses a standard Zeiss slit-lamp to image a subject's conjunctival vessels by using a 1-inch Newvicon TV camera with an electronic magnification of 2x. The video image is simultaneously recorded on a video tape recorder (VTR) to an overall system magnification of approximately 4 microm/raster line. The data acquisition phase requires approximately 5 minutes of patient time, whereas the actual determination of blood velocity in individual vessels is done offline through a modification of the dual-slit videodensimetric method. Two independently controllable video cursors are placed axially over the vessel image with the VTR in the still-frame mode. For each consecutive video field, the position of two reference points on the vessel and the position of each cursor relative to these and to each other are encoded into a computer to track the moving image caused by normal eye movement. The computer then determines new cursor coordinates to ensure a constant position within the vessel. The electrical signals obtained for each cursor site and for each video field are cross-correlated to yield the average blood velocity over the sampled time interval. The system has been calibrated in vitro from 0.2 to 2.5 mm/sec, evaluated in experimental animals, and used to measure blood velocity (0.3 to 1.5 mm/sec) in human conjunctival venules with diameters ranging from 20 to 50 microm. At this writing, blood velocity has been recorded during a period of about 3 months in the same vessel of several postmyocardial infarction patients. Thus, the method appears suitable for determining sequential changes in small vessel blood flow in patients over extended periods of time.     

Mitochondrial fluorescence patterns in rhodamine 6G-stained myocardial cells in vitro

Cellular fluorescence in vitro has been studied employing a low light-level video system interfaced with a real-time image array-processing computer system. Changes in cytoplasmic (mitochondrial) fluorescence in myocytes employing the probe rhodamine 6G have been studied over real time with the aid of several computer-based programs. An oscillating pattern of fluorescence is observed that appears to reflect localized variations in mitochondrial activity. The low light level video computer system used in this study compares favorably with laser-stimulated microspot fluorescence photon-counting techniques for the detection of subcellular fluorescence.     

Mitochondrial fluorescence patterns in rhodamine 6G-stained myocardial cells in vitro. Analysis by real-time computer video microscopy and laser microspot excitation

Cellular fluorescence in vitro has been studied employing a low light-level video system interfaced with a real-time image array-processing computer system. Changes in cytoplasmic (mitochondrial) fluorescence in myocytes employing the probe rhodamine 6G have been studied over real time with the aid of several computer-based programs. An oscillating pattern of fluorescence is observed that appears to reflect localized variations in mitochondrial activity. The low light level video computer system used in this study compares favorably with laser-stimulated microspot fluorescence photon-counting techniques for the detection of subcellular fluorescence.     

High-performance confocal system for microscopic or endoscopic applications

We designed a high-performance confocal system that can be easily adapted to an existing light microscope or coupled with an endoscope for remote imaging. The system employs spatially and temporally patterned illumination produced by one of several mechanisms, including a micromirror array video projection device driven by a computer video source or a microlens array scanned by a piezo actuator in the microscope illumination path. A series of subsampled "component" video images are acquired from a solid-state video camera. Confocal images are digitally reconstructed using "virtual pinhole" synthetic aperture techniques applied to the collection of component images. Unlike conventional confocal techniques that raster scan a single detector and illumination point, our system samples multiple locations in parallel, with particular advantages for monitoring fast dynamic processes. We compared methods of patterned illumination and confocal image reconstruction by characterizing the point spread function, contrast, and intensity of imaged objects. Sample 3D reconstructions include a diatom and a Golgi-stained nerve cell collected in transmission.     

A new method in computer-assisted imaging in neuroanatomy

A procedure is described yielding computed images of postmortem brains with high topographic accuracy. Structures of the brain are traced and registered by means of a digitizer capable of measuring coordinates three-dimensionally. The information corresponding to one brain model is stored on a flexible disk with a capacity of 256 Kbytes. According to the output desired, the resulting brain images are either completely or partially displayed on the computer screen as stereo pairs. The brain models possess a local fidelity of about 1 mm. The images are useful in simultaneously studying superficial and central parts of the brain, spatial relationships of the various structures and the projection of deep structures onto the surface of the brain. A RAM of about 100 Kbytes is necessary for a program enabling the user to perform stereo projections, three-dimensional transformations and other image manipulations. The special features of anatomical computer imaging as compared to computed tomography (CT) and nuclear magnetic resonance imaging (NMR) are outlined. A combination of these different techniques seems to improve clinical diagnosis.     

A novel video-based method using projected light to measure trunk volumes during respiration

This paper proposes and evaluates an innovative video-based method for measuring the trunk volume during respiration, using projected light and surface reconstruction. The method consists of the following main steps: (a) to project a grid of circular light markers on the anterior and posterior human body trunk surface during breathing, (b) to register the subject's trunk surface using two pairs of pre-calibrated digital video cameras, (c) to segment the video stream and track the projected markers using pre-processing techniques, morphological operators and detection algorithms, (d) to label the corresponding markers in the video sequences registered by each pair of stereo cameras, (e) to reconstruct the 3-D coordinates of all markers, (f) to reconstruct the surfaces from the unordered cloud of points using the Power Crust method and (g) to calculate the trunk volume in function of time using the divergence theorem. The evaluation of the method was based on two experiments. (1) Comparison of the volume of a trunk model (mannequin) by immersion and using the proposed optical method. (2) Analysis of the applicability of the method for measuring a subject’s trunk volume during a vital capacity respiratory manoeuvre. The results showed that the method was able to automatically measure more than 2000 projected points per image and to provide a very detailed representation of the subject's trunk. The relative accuracy of the volume measurement was estimated to be better than 3%. The analysis of the experiments revealed that signals coherent with the respiratory cycles could be identified through this method. In conclusion, the method based on light projection and surface reconstruction provides an accurate, non-invasive and useful means to calculate human trunk volumes during breathing.     

Construction of a confocal microscope for real-time x-y and x-z imaging

We describe the construction of a simple 'real-time' laser-scanning confocal microscope, and illustrate its use for rapid imaging of elementary intracellular calcium signaling events. A resonant scanning galvanometer (8 kHz) allows x-y frame acquisition rates of 15 or 30 Hz, and the use of mirrors to scan the laser beam permits use of true, pin-hole confocal detection to provide diffraction-limited spatial resolution. Furthermore, use of a piezoelectric device to rapidly focus the objective lens allows axial (x-z) images to be obtained from thick specimens at similar frame rates. A computer with image acquisition and graphics cards converts the output from the microscope to a standard video signal, which can then be recorded on videotape and analyzed by regular image processing systems. The system is largely made from commercially available components and requires little custom construction of mechanical parts or electronic circuitry. It costs only a small fraction of that of comparable commercial instruments, yet offers greater versatility and similar or better performance.     

A novel video-based method using projected light to measure trunk volumes during respiration

This paper proposes and evaluates an innovative video-based method for measuring the trunk volume during respiration, using projected light and surface reconstruction. The method consists of the following main steps: (a) to project a grid of circular light markers on the anterior and posterior human body trunk surface during breathing, (b) to register the subject's trunk surface using two pairs of pre-calibrated digital video cameras, (c) to segment the video stream and track the projected markers using pre-processing techniques, morphological operators and detection algorithms, (d) to label the corresponding markers in the video sequences registered by each pair of stereo cameras, (e) to reconstruct the 3-D coordinates of all markers, (f) to reconstruct the surfaces from the unordered cloud of points using the Power Crust method and (g) to calculate the trunk volume in function of time using the divergence theorem. The evaluation of the method was based on two experiments. (1) Comparison of the volume of a trunk model (mannequin) by immersion and using the proposed optical method. (2) Analysis of the applicability of the method for measuring a subject's trunk volume during a vital capacity respiratory manoeuvre. The results showed that the method was able to automatically measure more than 2000 projected points per image and to provide a very detailed representation of the subject's trunk. The relative accuracy of the volume measurement was estimated to be better than 3%. The analysis of the experiments revealed that signals coherent with the respiratory cycles could be identified through this method. In conclusion, the method based on light projection and surface reconstruction provides an accurate, non-invasive and useful means to calculate human trunk volumes during breathing.     

A quantitative method for the analysis of cell shape and locomotion

A rapid, semiautomated system to quantitate and analyze leukocyte shape and locomotion was developed. Video images of moving leukocytes were obtained using a Vidicon camera mounted on a Nikon phase microscope. The video signal was either inputted directly, or indirectly via a video cassette recorder, to a Datacube video analog-digital, digital-analog converter. A Digital Equipment Corporation LSI 11/23 computer using the RT-11/TSX-Plus operating system and computer programs written in FORTRAN and MARCO assembly language permitted image segmentation, image display, and calculation of position, speed, direction of movement and orientation of each leukocyte at 10 s intervals. These data were stored on a winchester disk for subsequent evaluation of the leukocyte orientation, speed and direction of movement using statistical and graphical methods. The reproducibility of measurements made with the video system was tested by comparison with manual measurements; a correlation coefficient of 0.998 was obtained for the two methods. Rates of chemokinesis were then determined for unstimulated and chemokinetically stimulated polymorphonuclear leukocytes (PMNs) and found to average 12.8 micron/min and 18.1 micron/min, respectively. The high speed, ease of data analysis, and potential for multiparameter evaluation makes this system useful for directly evaluating leukocyte locomotion.     

Three-dimensional video analysis of facial movements: a new method to assess the quantity and quality of the smile

The results of neuromuscular reconstructions of the paralyzed face are difficult to assess. Very sophisticated methods are necessary to measure the motor deficits of facial paralysis or the functional recovery in the face. The aim of this development was a relatively simple system for data acquisition, which is easy to handle and which makes it relatively cheap to delegate data acquisition to centers all over the world, which will not be able to derive a data analysis on their own, but will send their data to a center with specialized equipment. A complex mirror system was developed to get three different views of the face at the same time on the video screen. At each investigation, a digital video is taken from a calibration grid and from standardized facial movements of the patient. Secondary analysis of the digital videofilm is made possible at any time later on by the support of a computer program, which calculates distances and movements three-dimensionally from the frontal image and the right and left mirror images. Pathologies of the mimic movements can be identified as well as improvements after surgical procedures by this system. The significant advantage is the possibility to watch the same movement on the video which is under study and to apply any kind of study later on. Taking the video needs only a few minutes, and fatigue of the patient's mimic system is prevented. Measurements usually at the endpoints of the movements give excellent information on the quantity of the movement or the degree of the facial palsy, whereas the video itself is very informative regarding the quality of the smile. Specific computer software was developed for standardized three-dimensional analysis of the video-documented facial movements and for data presentation. There are options like two-dimensional graphs of single moving points in the face or three-dimensional graphs of the movements of all measured points at the same time during a standardized facial movement. By a comparison of the right- and left-sided alterations of specific distances between two points during the facial movements, the degree of normal symmetry or pathologic asymmetry is quantified. This system is more suitable for detailed scientific multicenter studies than any other system previously established. A very sensitive instrument for exact evaluation of mimic function is now available.     

Derivation of sonograph quality parameters by the use of point spread function analysis

A measuring system evaluating a Point Spread Function generated in an ultrasonographic image by scanning a spherical target was developed. The target is moved in measuring bath filled by water over scanned volume via 3D computer controlled positioning system. A video signal obtained is converted to digital form and analyzed by original software to derive various objective parameters of the imager as follows: Focal areas in both the azimuth and the elevation directions, Ultrasound scanning lines visualisation, Manufacturer preloaded TGC, Width of the scanning plane, Side lobe levels and Amplification uniformity in the azimuth direction. The method was verified by testing 18 different equipments in 282 measurements. Samples of particular measurement results in form of graphical outputs are included. Medical and physiological impacts of this approach are discussed.     

Fast Keyhole MR Imaging Using Optimized k-Space Data Acquisition

In some dynamic magnetic resonance imaging (MRI) applications, the sample is still, and only the signal intensity changes with time. For such cases, the keyhole imaging principle can be used. In standard keyhole imaging, a low-frequency image signal is acquired, using a limited number of phase-encoding steps, which correspond to the rectangular sampling region in the k-space center. However, such a region practically never coincides with the position of the k-space points, which carry the most relevant low-frequency image information. In this paper we propose an improved keyhole method, which allows dynamic acquisition of a low-frequency image signal from selected most relevant k-space points via fast imaging mechanisms. Dynamic data acquisition is executed in the presence of time-varying magnetic-field (MF) gradients after single sample excitation. Special care has been taken in the design of the gradient sequence to minimize gradient load. This improved keyhole imaging method has been considered theoretically and verified experimentally on a model system.     

A quantitative method for the analysis of cell shape and locomotion

Summary A rapid, semiautomated system to quantitate and analyze leukocyte shape and locomotion was developed. Video images of moving leukocytes were obtained using a Vidicon camera mounted on a Nikon phase microscope. The video signal was either inputted directly, or indirectly via a video cassette recorder, to a Datacube video analog-digital, digital-analog converter. A Digital Equipment Corporation LSI 11/23 computer using the RT-11/TSX-Plus operating system and computer programs written in FORTRAN and MARCO assembly language permitted image segmentation, image display, and calculation of position, speed, direction of movement and orientation of each leukocyte at 10 s intervals. These data were stored on a winchester disk for subsequent evaluation of the leukocyte orientation, speed and direction of movement using statistical and graphical methods. The reproducibility of measurements made with the video system was tested by comparison with manual measurements; a correlation coefficient of 0.998 was obtained for the two methods. Rates of chemokinesis were then determined for unstimulated and chemokinetically stimulated polymorphonuclear leukocytes (PMNs) and found to average 12.8 m/min and 18.1 m/min, respectively. The high speed, ease of data analysis, and potential for multiparameter evaluation makes this system useful for directly evaluating leukocyte locomotion.In honour of Prof. P. van Duijn     

Low-cost image processing system for analyzing molecular RNA fingerprints.

A complete image digitizing and processing system is described for capturing, enhancing and analyzing molecular fingerprints. The low-cost, high-resolution system features a Motorola 68000 processor, multi-tasking, a separate video coprocessor, and color or gray scale processing. Thousands of manipulations are possible using functions which include histographic equalization, edge detection, filtering, overlays, false coloring, zoom, pan and print. All operations are initiated and controlled with a mouse. Techniques for enhancing, scaling and comparing molecular fingerprints are described. The techniques all involve using a graphical interface to select and manipulate the various processes. The system has been used successfully for about 1.5 years, and it has been ideal for our application which requires human judgment at many steps between processing and which probably would not lend itself to a completely automated analysis. Similar techniques could probably be used with this system on many other applications.     

A low-cost microcomputer-based data acquisition and analysis system for an electron spin resonance spectrometer: data handling of dilute spin labeled nucleic acids

This article describes the construction of an inexpensive and reliable data acquisition system for a Varian E-line Century Series ESR spectrometer utilizing an Apple II Plus microcomputer. All necessary hardware is readily available and used without modification. A BASIC program for routine collection, display, plotting and disk storage of experimental data has been written and subsequently compiled into machine code for high speed operation. The interface offers distinct advantages in spectral resolution as well as instrument control. An example of signal enhancement via computer controlled time averaging is presented for a spin labeled DNA experiment. The technique has recently been applied to studies of relative binding affinities of gene-32 protein for various spin-labeled polynucleotides.