Confocal stereology and image analysis: methods for estimating geometrical characteristics of cells and tissues from three-dimensional confocal images.

A short review of confocal stereology and three-dimensional image analysis is presented, pointing out the achievements accomplished in this field by the Department of Biomathematics (Institute of Physiology, Prague). One of the methods of confocal stereology, the fakir method for surface area estimation, developed by this laboratory, is described. Methods for automatic measurement of geometrical characteristics of microscopical structures, based on 3-D image processing or surface triangulation, are discussed and compared with interactive stereological methods. Three-dimensional reconstruction programs and software implementation of stereological and digital methods as well as their practical applications are presented. The future trends are discussed.

STESYS2: extended STESYS software for MS Windows.

The STESYS2 software is a new version of the IBM PC software supporting interactive stereological measurements. In comparison with the previous STESYS, it is enhanced by a number of useful options, e.g. on-line image input via a TV camera coupled with a microscope operating under MS Windows OS. The main advantage, when compared with other such software packages, is the design of the STESYS2 as a module of the freeware image processing system Image Tool which provides a user-friendly environment including a number of image processing and preprocessing routines. Capabilities of the STESYS2 are illustrated by a practical example: estimation of the surface area of capillaries in the terminal villi of human placenta by the Sandau spatial grid method.

EEG patterns in theta and gamma frequency range and their probable relation to human voluntary movement organization.

In experiments with EEG accompanying continuous slow goal-directed voluntary movements we found abrupt short-term transients (STs) of the coefficients of EEG time-varying autoregressive (TVAR) model. The onset of STs indicated (i) a positive EEG wave related to an increase of 3-7 Hz oscillations in time period before the movement start, (ii) synchronization of 35-40 Hz prior to movement start and during the movement when the target is nearly reached. Both these phenomena are expressed predominantly over supplementary motor area, premotor and parietal cortices. These patterns were detected after averaging of EEG segments synchronized to the abrupt changes of the TVAR coefficients computed in the time course of EEG single records. The results are discussed regarding the cognitive aspect of organization of goal-directed movements.

An automatic 3D tracking system with a PC and a single TV camera.

We developed a novel system, for tracking a freely moving object (animal) in a limited 3D space, based on a closed loop TV and PC. Instead of using a pair of TV cameras to capture the 3D scene through the pair of its orthogonal projections, the system captures these projections by using a single TV camera. The direct view of the scene (e.g. its ground plan, i.e. projection onto the x-y plane) is captured together with its side projection, (i.e. projection onto the z-x plane) observed in a slanted mirror. The system uses a simple detection algorithm and hardware that were originally developed for 2D tracking of a single, contrast object on a stable and homogeneous background. All three coordinates of the tracked object are evaluated, displayed and stored in real-time, at 25 interlaced frames (samples) per second. The system was implemented on an IBM PC enhanced by an universal I/O board (Kaminsky, Yu, Krekule, I. Universal multifunctional IBM PC I/O board for clinical examinations and experimental research in neuroscience. Physiol Res 1994;43:193-199) and tested by tracking a LED marker, a small living fish in a water tank and a pair of LED markers which were activated one at the time in alternating TV frames thereby demonstrating the ability to simultaneously track two or more objects in 3D.

Alignment of adjacent picture frames captured by a CLSM.

Mosaicking a picture from its adjacent parts (called picture frames or tiles) is encountered in different fields of research and technology, e.g., photogrammetry, remote sensing, microscopy, etc. It is applied whenever the object of investigation is too large for the field of view of the sensor, e.g. a microscope. We describe mosaicking with a confocal laser-scanning microscope (CLSM) Bio-Rad, MRC 600 (U.K.). Aligning neighboring picture tiles was accomplished by registering the overlapped border areas of these tiles. Such registration procedures are constrained by: 1) the limited size of the registered samples (windows); 2) anisotropy of the form of the windows (usually narrow rectangles); and 3) the content of the windows, including changes of their intensity scale. Focusing on the latter problem, methods of registration were discussed and the robustness of the following three similarity based methods was studied with regard to the distortions of the intensity scales of the tiles to be registered: 1) the sum of absolute valued differences (SAVD); 2) normalized correlation coefficient (NCC); and 3) the mutual information function (MIF). Pilot experiments were extended to three-dimensional (3-D) stacks of pictures encountered in the framework of 3-D object rendering and visualization. MIF was found in most cases to be the most robust; however, it also demanded the most computational power. It is discussed how to choose a cost-effective method of the registration with regard to the content (texture, contrast, intensity scale distortion) of the tiles.

STESYS software for computer-assisted stereology.

A software system STESYS for interactive and flexible generation of stereological test systems is described. STESYS enables to implement many of the recent unbiased stereological methods applied to biomedical research and clinical diagnosis by using a simple personal computer. Advantages of the STESYS software are illustrated by several examples of stereological measurements for estimating the number, total and mean cross-sectional area, volume and surface area.

Spatial arrangement of fetal placental capillaries in terminal villi: a study using confocal microscopy.

The spatial arrangement of capillaries was studied in terminal villi of placentas at term by using confocal microscopy and methods for different types of 3-D reconstructions. Fixed specimens embedded in paraffin or glycol methacrylate resin were cut and stained with eosin. Digitized images of serial optical sections (approximately 0.5 microm) of individual terminal villi lying completely inside physical sections (100 microm) were captured by confocal laser scanning microscopy and analyzed. Topological reconstruction of the capillary bed and measurements of its Euler number, surface and volume rendering and wire-frame visualization were performed. Our findings showed that villous capillaries are arranged either in a single loop or in a more or less complicated anastomosing system. The results suggest that the combination of confocal microscope capture, methods for 3-D rendering and characterization of topological features reveals valuable information about spatial relationships of tissues in placental villi and the arrangement of the villous microcirculation, e.g. about the branching pattern of capillaries.

Application of computer-controlled, real-time TV single-object tracking in behavioural biology and rehabilitation medicine.

A PC-based system with TV input for automatic tracking of a single and contrast object in 2D in a homogeneous and stationary environment has been developed and applied to Morris water maze experiments. Further development of the system aimed at broader support of experiments, reduction of requirements on the stationarity and homogeneity of the scene background and on multiple-object tracking is discussed. The computer control of active light markers of the tracked object applicable to multiple-objects tracking in a time-sharing regime is also mentioned in the conclusion. The latter extension of the system can be applied to kinematic studies in biomechanics, sport and rehabilitation medicine.

Computer graphics editor GSE and some of its applications in morphology.

A simple, vector-based graphics editor, designed for experimental data presentation, schematics, large structured chemical formulae etc. in a modest IBM PC, MS DOS environment is described. Its applications in semi-automatic image analysis, especially in the implementation of design-based stereological methods for estimating global geometrical characteristics of studied samples and in 3D volume reconstruction from serial sections are discussed.

Universal multifunctional IBM PC I/O board for clinical examinations and experimental research in neuroscience.

The PC is and will remain a basic instrument in the laboratory arsenal in the next few years. The key role of the IBM PC and its clones prompted us to develop a universal multifunctional I/O board (UNIMUL) for this computer. The board will make it possible to use the IBM PC for a wide range of tasks from a simple interface for laboratory processing of data to complex IBM PC-based instruments, e.g. a stimulator, signal analyzer, chart recorder. The present article summarizes the experience gathered during the design and application of the described I/O board in more than 10 different IBM PC-based laboratory and clinical systems listed in the Appendix. An example of the application of the I/O board is presented in the conclusion of this report together with the discussion of the future role of new Application-Specific Integration Circuits (ASICs) and single chip processors in this domain.

Application of digital image processing to evaluation of eye fundus pictures. Preliminary report.

An application of computerized digital analysis to photographically recorded pictures of the fundus of the eye is described. This approach has been found as a reproducible and objective method for detection and description of different changes in ocular tissues (exudative diabetic maculopathy especially) for the diagnostic or prognostic purposes (progress of the illness, results of the treatment). Possibilities offered by this method in other relevant retrospective studies are also discussed.

IBM PC based visual evoked potential stimulator and averager.

Custom made hard and software enhancement of an IBM PC is described which makes it possible to: i) provide patterned visual stimulation by using the EGA or higher level display, ii) average recorded potentials and iii) monitor the experiment simultaneously. The description is illustrated by examples from a study of retinal evoked potentials in the rat.

Immunofluorescence detection of the vimentin epitope in chromatin structures of cell nuclei and chromosomes.

We describe immunofluorescence detection of the vimentin epitope, recognized by monoclonal antibody VI-01, in chromatin structures of eukaryotic cell nuclei and chromosomes. The approach used is based on increased sensitivity of 5-bromodeoxyuridine (BrdU)-substituted DNA to UV irradiation-induced crosslinking of DNA with proteins in vivo, by which the proteins interacting with chromosomal DNA can be immunovisualized in situ.

[Imaging and computer processing of the ocular fundus picture]. / K moznostem snímání a pocítacového zpracování obrazu ocního pozadí.

Computer analysis of a picture in ophthalmology makes objective evaluation of the clinical finding possible. After analogue-digital transfer the pictures of the fundus on a black and white negative and coloured positive 24 x 36 mm were analyzed on a Pericolor 2000 E computer. We measured the area of the glaucoma excavation of the papilla of the optic nerve, calculated the C/D ratio, assessed changes in the picture of the fundus in exudative diabetic maculopathy on paired slides obtained after a one-year interval. We evaluated changes of fluorescein penetration during fluoroangiography in different diseases.

Image analysis of transparent particle systems by mathematical morphology methods.

The particle images are often distorted by apparent holes due to undeflected transmitted light. These apparent holes must be disregarded for a consequent image analysis. The reconstruction of the real particles by methods of mathematical morphology was used. The problems associated with touching particles were also dealt with. The method is illustrated on an ion-exchanger system.

Does nondirectional signalization of target distance contribute to navigation in the Morris water maze?

The possibility that rats can navigate in the Morris water maze by reducing the difference between the memorized platform scene and the current sensory input was tested in nine blind rats. A computerized videosystem monitored the rats' movements in the pool and converted the rat-target distance into tones the frequency of which increased in 64 equal steps from 120 Hz at 128 cm to 7680 Hz at 0 cm. During 15 days of training to find a fixed platform position from different starting points (12 trials per day) average escape latencies decreased from 39.0 to 25.4 s. The performance significantly deteriorated when the acoustic distance signalization was omitted and/or when the target position was changed form trial to trial. It is concluded that blind rats solved the task by simultaneously employing search strategy based on position responses, mapping using acoustic background beacons, and distance reduction navigation. It is argued that the various strategies are additive and that their relative significance depends of the conditions of the experiment.

On-demand platform improves accuracy of the Morris water maze procedure.

In order to prevent chance finding of the hidden target in the Morris water tank task, the rigid underwater platform is replaced with a collapsible platform, resting at the bottom of the pool. A computerized videosystem tracks the rat's movement across the pool and raises the platform when the animal has stayed in the target area for a predetermined time. Acquisition of the task with the collapsible platform proceeds at a similar rate as with the rigid platform when the criterion conditions are easy (target distance 15 cm, target time 2.5 s), but gradually deteriorates when the target time increases to 10.0 s. Successful solution of the modified task requires accurate localization of the target under open loop conditions and is thus well suited for investigation of the fine structure of the cognitive maps and of their changes induced by lesions or drugs.

Quantitative evaluation of electromyogram activity in rat extensor and flexor muscles immobilized at different lengths.

Because immobilization of muscles in the "long" position mitigates the effects of inactivity and rapid wasting occurs when muscles are immobilized in the "short" position, a study was made of the EMG activity in the soleus (SOL)--an extensor muscle--and the tibialis anterior (TA)--a flexor muscle--in order to clarify the possible role of muscle function in modifying the course of disuse atrophy. EMG activity was recorded in the SOL and TA muscles in adult rats in which the ankle had been immobilized in a plaster cast either in plantar flexion or dorsiflexion. The number of action potentials per minute in samples of the EMG activity from control and immobilized muscles was assessed before, for 10 days during immobilization, and up to 9 days after removal of the cast. Immobilization in the short position (plantar flexion) led to a dramatic reduction in the EMG activity of the SOL (to 10% of the control). On the other hand, fixation of the SOL in the long position was without effect upon resting EMG activity. In the TA, EMG activity was exclusively phasic in character and corresponded to about 3% of that of the SOL. Neither the fixation of the ankle in plantar flexion nor dorsiflexion had any appreciable effect upon EMG activity in the TA. We conclude that, because immobilization in the lengthened position does not increase EMG activity in either extensor or flexor muscles, passive stretch appears to be the factor mainly responsible for mitigating the effects of disuse in this situation. On the other hand, when a typical extensor muscle (SOL) is immobilized in the shortened position and undergoes rapid wasting, an accessory role of decreased activity cannot be excluded.