Video microsurgery: evaluation of standard laparoscopic equipment for the practice of microsurgery

Traditional microsurgery involves the use of bulky and expensive stereo microscopes that have limited portability. Recent advances in video technology have enabled the exploration of alternative visualization methods. The purpose of this study was to evaluate standard laparoscopic equipment for microvascular anastomoses. Eight surgeons completed anastomoses on rat femoral and synthetic vessels using stereo microsurgery and video microsurgery visualization systems. All surgeons had previous experience with stereo microsurgery and none had ever used video microsurgery. Data were collected on overall anastomosis and individual suture times. A sample of completed anastomoses was placed in a video database and evaluated by use of a quality rating scale (8 to 10, excellent; 6 to 7, adequate; less than 6, poor). All surgeons subjectively evaluated the video microsurgery system. A total of 48 anastomoses were completed. The average total anastomosis time for the stereo microsurgery was 1018.9 +/- 463.2 seconds versus 1738.9 +/- 460.1 seconds for the video microsurgery. The average individual suture placement time was 114.6 +/- 60.6 seconds for the stereo microsurgery versus 211.7 +/- 128.4 seconds for the video microsurgery (p < 0.05). Twenty-five of the anastomoses underwent quality review. The overall score of the stereo microsurgery group was 8.1 +/- 1.7, and the video microsurgery group had an overall score of 7.3 +/- 1.6. Survey results revealed that 75 percent of the participants thought that the video microsurgery would be useful for human operations and would improve surgeon comfort, but 87.5 percent would not use the present video microsurgery system over stereo microsurgery in their practice. Although significant differences exist in overall anastomosis and individual suture completion times, no difference was found in the overall quality. Video microsurgery could become a useful tool on the basis of surgeon ergonomics; however, optical parameters require further refinement.     

A continuous noncontact method for measuring in situ vascular diameter with a video camera

A new, continuous, on-line, video diameter-measuring technique, utilizing a video camera mounted on the sidearm of a stereo microscope, is described. Vessel diameter is derived from changes in the video output signal of the camera or a video recorder when the vessel of interest is displayed horizontally on a monitor and well contrasted with its background. A comparator threshold is set on the filtered video output signal and generates an output pulse that is used to gate horizontal video sync pulses to a digital counter-timer. The number of pulses counted for each video field (no. of horizontal video lines) is proportional to the vessel diameter. The video-derived diameter is calibrated using known standards and correlates well with sonomicrometer-derived diameters of the carotid artery and jugular vein during increasing pressure ramps (r greater than 0.999). The diameter update rate is 60 Hz, and the resolution of the system is one horizontal video line, independent of the vessel size. With suitable magnification and contrast both arteries and veins as small as 200 micron have been measured using this system.     

Behaviour of temperate and sub-tropical reef fishes towards a stationary SCUBA diver

Using remote underwater stereo–video systems we examined fish behaviour towards a stationary SCUBA diver at temperate (Rottnest Island) and sub-tropical (Houtman Abrolhos Islands) reefs in Western Australia. Changes in species richness, relative abundance, fish length, and the mean distance of individual fish from stereo–video cameras, in the presence and absence of a SCUBA diver, were assessed to infer changes in behaviour. Results show that a stationary SCUBA diver may obtain accurate measures of species richness and of the composition of fish assemblages in an area. However, the usefulness of these measures to reflect changes in fish behaviour appears limited as responses of fish towards the stationary SCUBA diver were highly species specific. Several species differed in their mean relative abundance (Heterodontus portusjacksoni, Coris auricularis, Thalassoma lunare), length (Ophthalmolepsis lineolatus, C. auricularis), and the distance to which they would approach the stereo–video systems (Kyphosus sydneyanus, Scarus schlegeli) when a SCUBA diver was present. Here, species-specific changes in the behaviour of several common and abundant fish species towards a stationary SCUBA diver advises caution to avoid biases when interpreting results obtained by SCUBA divers.     

Particle detection,number estimation,and feature measurement in gene transfer studies: optical fractionator stereology integrated with digital image processing and analysis

Assessing the efficacy of in vivo gene transfer often requires a quantitative determination of the number, size, shape, or histological visualization characteristics of biological objects. The optical fractionator has become a choice stereological method for estimating the number of objects, such as neurons, in a structure, such as a brain subregion. Digital image processing and analytic methods can increase detection sensitivity and quantify structural and/or spectral features located in histological specimens. We describe a hardware and software system that we have developed for conducting the optical fractionator process. A microscope equipped with a video camera and motorized stage and focus controls is interfaced with a desktop computer. The computer contains a combination live video/computer graphics adapter with a video frame grabber and controls the stage, focus, and video via a commercial imaging software package. Specialized macro programs have been constructed with this software to execute command sequences requisite to the optical fractionator method: defining regions of interest, positioning specimens in a systematic uniform random manner, and stepping through known volumes of tissue for interactive object identification (optical dissectors). The system affords the flexibility to work with count regions that exceed the microscope image field size at low magnifications and to adjust the parameters of the fractionator sampling to best match the demands of particular specimens and object types. Digital image processing can be used to facilitate object detection and identification, and objects that meet criteria for counting can be analyzed for a variety of morphometric and optical properties.     

Stereo vision and isoluminance.

Recent experiments have shown that stereo depth is given by fusion of illusory ('cognitive') contours. They occur across quite large gaps in figures, when these gaps are unlikely and form the shape of a probable (nearer) masking object or masking feature. Implications are that: (a) clearly defined contours and regions of brightness difference can be produced as postulates from sensory evidence, which may be surprising absence of stimulation; (b) each eye-system can derive its own postulates, or hypotheses, which (c) can be combined to give stereo vision. It has been shown that random-dot stereo depth does not occur when there is colour contrast but no brightness difference between the dots and their background. This we have confirmed by using a new technique for producing isoluminant pictures, of any complexity, with exact registration for any two colours. With this technique, we find large displacements of narrow borders bounding regions that are shifted across isoluminance. These displacements, which are clearly seen as movements, occur with or without colour differences. The direction of shift depends on whether the narrow border is light or dark. It is found that these dramatic shifts do not - when produced in opposite directions to the two eyes and fused - produce stereo depth. It is concluded, following Julesz's paradigm, that these contour displacements have their neural orgin not retinally, but after stereo fusion. Experiments combining the 'cognitive contours' stereo depth with isoluminance are described.     

Stereo and eye movement

We describe a method to solve stereo correspondence using controlled eye (or camera) movements. Eye movements supply additional image frames and monocular depth estimate, which can be used to constrain stereo matching. Because the eye movements are small, traditional stereo techniques of stereo with multiple frame will not work. We develop an alternative approach using a systematic analysis to define a probability distribution for the errors. Our matching strategy then matches the most probable points first, thereby reducing the ambiguity for the remaining matches. We demonstrate this algorithms with several examples.     

Video on the Internet: An introduction to the digital encoding, compression, and transmission of moving image data

In this paper, we seek to provide an introduction to the fast-moving field of digital video on the Internet, from the viewpoint of the biological microscopist who might wish to store or access videos, for instance in image databases such as the BioImage Database (http://www.bioimage.org). We describe and evaluate the principal methods used for encoding and compressing moving image data for digital storage and transmission over the Internet, which involve compromises between compression efficiency and retention of image fidelity, and describe the existing alternate software technologies for downloading or streaming compressed digitized videos using a Web browser. We report the results of experiments on video microscopy recordings and three-dimensional confocal animations of biological specimens to evaluate the compression efficiencies of the principal video compression-decompression algorithms (codecs) and to document the artefacts associated with each of them. Because MPEG-1 gives very high compression while yet retaining reasonable image quality, these studies lead us to recommend that video databases should store both a high-resolution original version of each video, ideally either uncompressed or losslessly compressed, and a separate edited and highly compressed MPEG-1 preview version that can be rapidly downloaded for interactive viewing by the database user.     

Biplane videoroentgenographic analysis of dynamic regional lung strains in dogs.

A method is described for determining the spatial distribution of pulmonary parenchymal strains in the intact canine thorax, using measurements of displacement of metallic (1-mm-diam)) markers percutaneously implanted throughout the parenchyma of the right lung. Dogs are supported head up or head down in a water-immersion respirator with the animal's airway connected to ambient air. Tracking of the parenchymal markers is accomplished by stereo biplane videoroentgenographic recordings, which allow high temporal (60/S) and spatial (+/- 1.5 mm) resolution measurements of the "tagged" lungs during various respiratory maneuvers. After transferring the video information to a stop-action video disc, an operator-interactive computer program is used to input the geometric coordinates of the markers into the computer. The true spatial coordinates are then determined after correction for pincushion and magnification distortions. Spatial and temporal distributions of regional parenchymal strains are obtained by determining the distance between markers on a frame-by-frame basis over the extent of the respiratory cycle. Data indicate nonuniformity in regional lung parenchymal strains.     

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software. Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT. To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images. To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time. The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion. Download video file.^{(111M, mp4)}     

Experimental measurements and design guidelines for real-time software encryption in multimedia wireless LANs

To secure interactive multimedia applications in wireless LANs (WLANs), it is pertinent to implement real time cryptographic services. In this paper we evaluate the use of software based encryption algorithms that are implemented in the layer service provider as defined by WinSock 2 for Windows 95/NT. Our measurements show that software implementation of various encryptors can sustain the throughput requirements of interactive multimedia applications for WLANs such as telephone-quality audio, video conferencing, and MPEG video. We present a design methodology that includes guidelines for a secure multimedia system design in terms of the encryption method chosen as a function of required application throughput, system configuration, protocol layers overhead and wireless LAN throughput. This revised version was published online in July 2006 with corrections to the Cover Date.     

Real-time multi-wavelength fluorescence imaging of living cells

We describe a new real-time fluorescence video microscope design for capturing intensified images of cells containing dual wavelength "ratio" dyes or multiple dyes. The microscope will perform real-time capture of two separate fluorescence emission images simultaneously, improving the time resolution of spatial distribution of fluorescence to video frame rates (30 frames/s or faster). Each emission wavelength is imaged simultaneously by one of two cameras, then digitized, background corrected and appropriately combined at standard video frame rates to be stored at high resolution on tape or digital video disk for further off-line analysis. Use of low noise, high sensitivity image intensifiers, coupled to CCD cameras produce stable, high contrast images using ultra low light levels with little persistence or bloom. The design has no moving parts in its optical train, which overcomes a number of technical difficulties encountered in the present filter wheel designs for multiple imaging. Coupled to compatible image processing software utilizing PC-AT computers, the new design can be built for a significantly lower cost than many presently available commercial machines. The system is ideal for ratio imaging applications; the software can calculate the ratio of fluorescence intensities pixel by pixel and provide the information to generate false-color images of the intensity data as well as other calculations based on the two images. Thus, it provides a powerful, inexpensive tool for studying the real-time kinetics of changes in living cells. Examples are presented for the kinetics of rapidly changing intracellular calcium detected by the calcium indicator probe indo-1 and the redistribution kinetics of multiple vital dyes placed in cells undergoing cell fusion.     

Mapping 3-D functional capillary geometry in rat skeletal muscle in vivo

We have developed a novel mapping software package to reconstruct microvascular networks in three dimensions (3-D) from in vivo video images for use in blood flow and O2 transport modeling. An intravital optical imaging system was used to collect video sequences of blood flow in microvessels at different depths in the tissue. Functional images of vessels were produced from the video sequences and were processed using automated edge tracking software to yield location and geometry data for construction of the 3-D network. The same video sequences were analyzed for hemodynamic and O2 saturation data from individual capillaries in the network. Simple user-driven commands allowed the connection of vessel segments at bifurcations, and semiautomated registration enabled the tracking of vessels across multiple focal planes and fields of view. The reconstructed networks can be rotated and manipulated in 3-D to verify vessel connections and continuity. Hemodynamic and O2 saturation measurements made in vivo can be indexed to corresponding vessels and visualized using colorized maps of the vascular geometry. Vessels in each reconstruction are saved as text-based files that can be easily imported into flow or O2 transport models with complete geometry, hemodynamic, and O2 transport conditions. The results of digital morphometric analysis of seven microvascular networks showed mean capillary diameters and overall capillary density consistent with previous findings using histology and corrosion cast techniques. The described mapping software is a valuable tool for the quantification of in vivo microvascular geometry, hemodynamics, and oxygenation, thus providing rich data sets for experiment-based computational models.     

Design of the hapten for the induction of antibodies catalyzing aldol reaction

The transition states of the aldol reaction and their analogues are reported here for the design of the haptens generating catalytic antibodies. The structural and the electrostatic properties were calculated by an ab initio molecular orbital method and were compared using a graphic software. Two transition states that lead to the corresponding stereo isomers of the aldol products were characterized. Also, the suitable transition state analogues were found. It is suggested that the stereo selectivity can be controlled using the catalytic antibodies elicited against the haptens designed here.     

Digital registration and analysis of visual information in behavioral experiment

A new software for recording, registration and computer analysis of behavior of laboratory animals, EthoStudio, has been developed. The software allows the latency, number and duration of up to 10 behavioral patterns to be simultaneously registered with parallel recording of video on a hard disk. The video records can be played back to track animal locomotion using a computer. EthoStudio was applied to study mouse behavior in the open field test. The numbers of rearings and groomings as well as accumulated time of grooming were interactively assayed by pressing and holding buttons on a computer keyboard. The number of squares crossed, the number of entries into the center and the time spend therein were assayed by computer analysis. EthoStudio can be applied to objectively study various kinds of animal behavior with reliability and accuracy necessary for modern ethological, physiological and pharmacological experiment.     

An Aufwuchs Chamber Slide for High-Resolution Confocal Laser Scanning Microscopy and Stereo Imaging of Microbial Communities in Natural Biofilms

Aufwuchs chamber slides were constructed by attaching a silicone rubber gasket to a glass slide with epoxy cement. For biofilm growth, the slides were suspended in Cayuga Lake near Ithaca, NY, for 27 days. Biofilms in the chamber were stained with 0.05% acridine orange. After rinsing, the chamber was filled with molten 1% agarose to stabilize filaments and delicate polymer structures at the biofilm surface. Areas of biofilm ~0.5 mm thick on the inner face of the wall of the chamber were selected for side-on optical sectioning in a confocal laser scanning microscope (CLSM). Stacks of high-resolution optical images captured by the CLSM z-sectioning software, were used to create left-right stereo image pairs. At low magnification the stereo pairs showed 3-D details of the microbial landscape in the mature biofilms. Channels, pores, and other structural features of the biofilm matrix were observed in peripheral regions. Higher magnification images revealed the 3-D distribution of specific biofilm components such as filaments of sheathed bacteria projecting outward into the liquid milieu, and organic coatings, including bacterial cells on the surfaces of mineral particles.     

Standardizing the analysis of conditioned fear in rodents: a multidimensional software approach

Data comparability between different laboratories strongly depends on the individually applied analysis method. This factor is often a critical source of variation in rodent phenotyping and has never been systematically investigated in Pavlovian fear conditioning paradigms. In rodents, fear is typically quantified in terms of freezing duration via manual observation or automated systems. While manual analysis includes biases such as tiredness or inter‐personal scoring variability, computer‐assisted systems are unable to distinguish between freezing and immobility. Consequently, the novel software called MOVE follows a semi‐automatized approach that prefilters video sequences of interest for the final human judgment. Furthermore, MOVE allows integrating additional data sources (e.g. force‐sensitive platform, EEG) to reach the most accurate and precise results. MOVE directly supports multi‐angle video recordings with webcams or standard laboratory equipment. The integrated manual key logger and internal video player complement this all‐in‐one software solution. Calculating the interlaboratory variability of manual freezing evaluation revealed significantly different freezing scores in two out of six laboratories. This difference was minimized when all experiments were analyzed with MOVE. Applied to a genetically modified mouse model, MOVE revealed higher fear responses of CB1 deficient mice compared to their wild‐type littermates after foreground context fear conditioning. Multi‐angle video analysis compared to the single‐camera approach reached up to 15% higher accuracy and two fold higher precision. Multidimensional analysis provided by integration of additional data sources further improved the overall result. We conclude that the widespread usage of MOVE could substantially improve the comparability of results from different laboratories.     

Direct quantification of in vitro cell growth through image analysis

Summary Objective, accurate, non-intrusive measurement of in vitro cell growth was realized through microcomputerized video image analysis. Recently-released video and digitizing hardware and software were incorporated into an analytical system which accurately quantified visual differences between cultures on a cell number or fresh mass basis. Sequential measurements during culture incubation further detected and quantified subtle changes in colony area and density resulting from growth. Each measurement was acquired rapidly, without encroaching on the in vitro environment, so cell growth was undisturbed. Custom software routines coordinated the quantification of this detailed record into precise cumulative growth curves.     

An Active Stereo Vision System Based on Neural Pathways of Human Binocular Motor System

An active stereo vision system based on a model of neural pathways of human binocular motor system is proposed. With this model, it is guaranteed that the two cameras of the active stereo vision system can keep their lines of sight fixed on the same target object during smooth pursuit. This feature is very important for active stereo vision systems, since not only 3D reconstruction needs the two cameras have an overlapping field of vision, but also it can facilitate the 3D reconstruction algorithm. To evaluate the effectiveness of the proposed method, some software simulations are done to demonstrate the same target tracking characteristic in a virtual environment apt to mistracking easily. Here, mistracking means two eyes track two different objects separately. Then the proposed method is implemented in our active stereo vision system to perform real tracking task in a laboratory scene where several persons walk self-determining. Before the proposed model is implemented in the system, mistracking occurred frequently. After it is enabled, mistracking never occurred. The result shows that the vision system based on neural pathways of human binocular motor system can reliably avoid mistracking.     

Modification of physical movement in old C57BL/6 mice by DHEA and melatonin supplementation.

As old age results in reduced physical activity as well as less dehydroepiandrosterone (DHEA) and melatonin (MLT) production, low hormone levels may be a component of inactivity. Therefore, we studied the effects of DHEA and/or MLT supplementation on movement and resting in young and old female C57 black mice. Our results showed for the first time that old female C56BL/6 mice have significantly decreased physical activity. Their average speed and resting time were significantly higher than in young mice, whereas ambulatory time, distance traveled, and body movements when stationary (stereo time) were lower. DHEA supplementation significantly increased stereo time in old mice, while decreasing ambulatory time and distance traveled. MLT supplementation of old mice decreased average speed, resting time, and stereo time compared to untreated, old mice. Supplementation with MLT or DHEA, whose production is reduced in aging, restored physical activity levels in old mice. MLT also increased ambulatory time and distance traveled while reducing body movements of young mice.