Measuring the Three-Dimensional Kinematics of a Free-Swimming Koi Carp by Video Tracking Method

A video tracking system for measuring three-dimensional kinematics of a free-swimming fish is presented. The tracking is accomplished by simultaneously taking images from the ventral view and the lateral view of the fish with two cameras mounted on two computer-controlled and mutually orthogonal translation stages. Compared to the previous system we reported, the time resolution is greatly improved. A koi carp is selected for the experiment. By processing the images caught by the video tracking system, the three-dimensional kinematics of the koi carp during a continuous swimming containing several moderate maneuvers are obtained. In particular, the pitching motion of fish body and the tail motion, including lateral excursion, variation in tail height and torsion, are revealed for burst-and-coast swimming and turning maneuver. The error analysis is also provided for the measurement results.     

Analysis of three-dimensional ciliary beating by means of high-speed stereomicroscopy.

Results are presented on the analysis of three-dimensional motion of compound cilia or cirri in voltage-clamped specimens of the protozoan Stylonychia mytilus. Time series of three-dimensional data were obtained by using the anaxial illumination method for simultaneous recording of stereoscopic video images. Data processing involved the following steps: determination of a reference coordinate system based solely on features present in each stereo-pair; tracing of cirral axes in digitized images, conversion to parameter curves by means of least-squares polynomial approximation, conversion of pairs of two-dimensional data to a series of three-dimensional data; correction for distortion due to projective shortening and conversion to a series of polynomial triplets, and analysis of the periodical components of the motion pattern in the frequency domain. Reconstructed beating cycles show typical differences between hyperpolarization-induced ciliary activity and depolarization-induced ciliary activity. Reconstructions of the motion of the basal segment of a cirrus are in agreement with existing data. Analysis of the curvature and torsion of a cirral axis during beating does not reveal any simple pattern of propagated activity within the axoneme. The return stroke may be subdivided into two phases. First, a curvature peak develops proximally. Secondly, a region with increased torsion arises more distally and spreads out in proximal direction. Both curvature and torsion return to minimal values by the beginning of the power stroke.     

Determination of ciliary polarity precedes differentiation in the epithelial cells of quail oviduct.

In quail oviduct epithelium, as in all metazoan and protozoan ciliated cells, cilia beat in a coordinated cycle. They are arranged in a polarized pattern oriented according to the anteroposterior axis of the oviduct and are most likely responsible for transport of the ovum and egg white proteins from the infundibulum toward the uterus. Orientation of ciliary beating is related to that of the basal bodies, indicated by the location of the lateral basal foot, which points in the direction of the active stroke of ciliary beating. This arrangement of the ciliary cortex occurs as the ultimate step in ciliogenesis and following the oviduct development. Cilia first develop in a random orientation and reorient later, simultaneously with the development of the cortical cytoskeleton. In order to know when the final orientation of basal bodies and cilia is determined in the course of oviduct development, microsurgical reversal of a segment of the immature oviduct was performed. Then, after hormone-induced development and ciliogenesis, ciliary orientation was examined in the inverted segment and in normal parts of the ciliated epithelium. In the inverted segment, orientation was reversed, as shown by a video recording of the direction of effective flow produced by beating cilia, by the three-dimensional bending forms of cilia immobilized during the beating cycle and screened by scanning electron microscopy, and by the position of basal body appendages as seen in thin sections by transmission electron microscopy. These results demonstrate that basal body and ciliary orientation are irreversibly determined prior to development by an endogenous signal present early in the cells of the immature oviduct, transmitted to daughter cells during the proliferative phase and expressed at the end of ciliogenesis.     

A demonstration of validity of 3-D video motion analysis method for measuring finger flexion and extension

This study demonstrates the validity of using 3-D video motion analysis to measure hand motion. Several researchers have devised ingenious methods to study normal and abnormal hand movements. Although very helpful, these earlier studies are static representations of a dynamic phenomenon. Despite the many studies of hand motion using scientifically impeccable techniques, little is known about digital motion, and there are still few researchers investigating dynamic three-dimensional motion of the hand. Results from a three-camera video motion analysis system were compared to those from the "gold standard", 2-D lateral view fluoroscopy. We used these two methods to record hand motion simultaneously during unrestricted flexion and extension of the index finger of the dominant hand in 6 neurologically normal, healthy volunteers. After collection and post-processing, the waveforms of the PIP, DIP and MCP joint angles were compared using the adjusted coefficient of multiple determination (R2(a), or CMD). The mean CMD values for the MCP, PIP and DIP joint angle waveforms were 0.96, 0.98 and 0.94, respectively, suggesting a close similarity between motion of comparable joints analyzed by the 2-D and 3-D methods. This shows that the method of 3-D motion analysis is capable of accurately quantifying digital joint motion. It is anticipated that 3-D motion analysis, in addition to being used as a research tool, will also have clinical applications such as surgical planning in neuromuscular disorders and the documentation of abnormal motion in many other pathological hand conditions.     

Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination

Structured illumination microscopy is a method that can increase the spatial resolution of wide-field fluorescence microscopy beyond its classical limit by using spatially structured illumination light. Here we describe how this method can be applied in three dimensions to double the axial as well as the lateral resolution, with true optical sectioning. A grating is used to generate three mutually coherent light beams, which interfere in the specimen to form an illumination pattern that varies both laterally and axially. The spatially structured excitation intensity causes normally unreachable high-resolution information to become encoded into the observed images through spatial frequency mixing. This new information is computationally extracted and used to generate a three-dimensional reconstruction with twice as high resolution, in all three dimensions, as is possible in a conventional wide-field microscope. The method has been demonstrated on both test objects and biological specimens, and has produced the first light microscopy images of the synaptonemal complex in which the lateral elements are clearly resolved.     

A demonstration of the validity of a 3-D video motion analysis method for measuring finger flexion and extension

This study demonstrates the validity of using 3-D video motion analysis to measure hand motion. Several researchers have devised ingenious methods to study normal and abnormal hand movements. Although very helpful, these earlier studies are static representations of a dynamic phenomenon. Despite the many studies of hand motion using scientifically impeccable techniques, little is known about digital motion, and there are still few researchers investigating dynamic three-dimensional motion of the hand. Results from a three-camera video motion analysis system were compared to those from the “gold standard”, 2-D lateral view fluoroscopy. We used these two methods to record hand motion simultaneously during unrestricted flexion and extension of the index finger of the dominant hand in 6 neurologically normal, healthy volunteers. After collection and post-processing, the waveforms of the PIP, DIP and MCP joint angles were compared using the adjusted coefficient of multiple determination (R²_a, or CMD). The mean CMD values for the MCP, PIP and DIP joint angle waveforms were 0.96, 0.98 and 0.94, respectively, suggesting a close similarity between motion of comparable joints analyzed by the 2-D and 3-D methods. This shows that the method of 3-D motion analysis is capable of accurately quantifying digital joint motion.

It is anticipated that 3-D motion analysis, in addition to being used as a research tool, will also have clinical applications such as surgical planning in neuromuscular disorders and the documentation of abnormal motion in many other pathological hand conditions.     

Automated measurement of ciliary beat frequency

Measurements of ciliary beat frequency using video images are dependent on observer interpretation. To obtain objective estimates of ciliary beat frequency from video-image sequences, a computer-based method was developed. Regions of interest of video-image sequences were selected and digitized. Variations in numerical values representing light intensity resulting from cilia beating were extracted and analyzed using autocorrelation techniques. The ciliary beat frequencies obtained for 14 in vitro experiments on ciliated cells or epithelium from the frog palate (Rana catesbeiana) over the range of frequencies 2-25 Hz correlated well with independent observer measurements (r = 0.979). The addition of such computer-based methods to video observer-based systems allows more objective and efficient determinations of ciliary beat frequency.     

Regulation of airway ciliary activity by Ca2+: simultaneous measurement of beat frequency and intracellular Ca2+.

Airway ciliary activity is influenced by [Ca2+]i, but this mechanism is not fully understood. To investigate this relationship, ciliary activity and [Ca2+]i were measured simultaneously from airway epithelial ciliated cells. Ciliary beat frequency was determined, for each beat cycle, with phase-contrast optics and high-speed video imaging (at 240 images s-1) and correlated with [Ca2+]i determined, at the ciliary base, by fast imaging (30 images s-1) of fura-2 fluorescence. As a mechanically induced intercellular Ca2+ wave propagated through adjacent cells, [Ca2+]i was elevated from a baseline concentration of 45 to 100 nM, to a peak level of up to 650 nM. When the Ca2+ wave reached the ciliary base, the beat frequency rapidly increased, within a few beat cycles, from a basal rate of 6.4 to 11.6 Hz at 20-23 degrees C, and from 17.2 to 26.7 Hz at 37 degrees C. Changes in [Ca2+]i, above 350 nM, had no effect on the maximum beat frequency. We suggest that airway ciliary beat frequency is 1) controlled by a low range of [Ca2+]i acting directly at an axonemal site at the ciliary base and 2) that a maximum frequency is induced by a change in [Ca2+]i of approximately 250-300 nM.     

Mapping of the functional microcirculation in vital organs using contrast-enhanced in vivo video microscopy

A functional microcirculation is vital to the survival of mammalian tissues. In vivo video microscopy is often used in animal models to assess microvascular function, providing real-time observation of blood flow in normal and diseased tissues. To extend the capabilities of in vivo video microscopy, we have developed a contrast-enhanced system with postprocessing video analysis tools that permit quantitative assessment of microvascular geometry and function in vital organs and tissues. FITC-labeled dextran (250 kDa) was injected intravenously into anesthetized mice to provide intravascular fluorescence contrast with darker red blood cell (RBC) motion. Digitized video images of microcirculation in a variety of internal organs (e.g., lung, liver, ovary, and kidney) were processed using computer-based motion correction to remove background respiratory and cardiac movement. Stabilized videos were analyzed to generate a series of functional images revealing microhemodynamic parameters, such as plasma perfusion, RBC perfusion, and RBC supply rate. Fluorescence contrast revealed characteristic microvascular arrangements within different organs, and images generated from video sequences of liver metastases showed a marked reduction in the proportion of tumor vessels that were functional. Analysis of processed video sequences showed large reductions in vessel volume, length, and branch-point density, with a near doubling in vessel segment length. This study demonstrates that postprocessing of fluorescence contrast video sequences of the microcirculation can provide quantitative images useful for studies in a wide range of model systems.     

Apical sensory organ in larvae of the patellogastropod Tectura scutum

The apical sensory organ in veliger larvae of a patellogastropod, a basal clade of gastropod molluscs, was studied using ultrastructural and immunohistochemical techniques. Immediately before veligers of Tectura scutum undergo ontogenetic torsion, the apical sensory organ consists of three large cells that generate a very long apical ciliary tuft, two cells that generate a bilateral pair of shorter ciliary tufts, and a neural ganglion (apical ganglion). Putative sensory neurons forming the ganglion give rise to dendrites that extend to the apical surface of the larva and to basal neurites that contribute to a neuropil. The ganglion includes only one ampullary neuron, a distinctive neuronal type found in the apical ganglion of other gastropod veligers. Serotonin immunoreactivity is expressed by a medial and two lateral neurons, all having an apical dendrite, and also by neurites within the neuropil and by peripheral neurites that run beneath the ciliated prototrochal cells that power larval swimming. The three cells generating the long apical ciliary tuft are lost soon after ontogenetic torsion, and the medial serotonergic cell stops expressing serotonin antigenicity in late-stage veligers. The lateral ciliary tuft cells of T. scutum may be homologs of lateral ciliary tuft cells in planktotrophic opisthobranch veligers. A tripartite arrangement of sensory dendrites, as described previously for veligers of other gastropod clades, can be recognized in T. scutum after loss of the apical ciliary tuft cells.     

In vitro analysis of kinematics and elastostatics of the human rib cage during thoracic spinal movement for the validation of numerical models

Neither kinematic nor stiffness properties of the rib cage during thoracic spinal motion were investigated in previous studies, while being essential for the accurate validation of numerical models of the whole thorax. The aim of this in vitro study therefore was to quantify the kinematics and elastostatics of the human rib cage under defined boundary conditions. Eight fresh frozen human thoracic spine specimens (C7-L1, median age 55 years, ranging from 40 to 60 years) including entire rib cages were loaded quasi-statically in flexion/extension, lateral bending, and axial rotation using pure moments of 5 Nm. Relative motions of ribs, thoracic vertebrae, and sternal structures as well as strains on the ribs were measured using optical motion tracking of 150 reflective markers per specimen, while specimens were loaded displacement-controlled with a constant rate of 1°/s for 3.5 cycles. The third full cycle was used to determine relative angles and strains at full loading of the spine for all motion directions. Largest relative angles were found in the main loading directions with only small motions at the mid-thoracic levels. Highest strains of the intercostal spaces were detected in the anterior section of the lowest fourth of the rib cage, showing compressions and elongations of more than 10% in all spinal motion planes. Elastostatic rib deformation was generally less than 1%. Rib-sternum relative motions exhibited complex motion patterns, overall showing relative angles below 2°. The results indicate that rib cage structures are not macroscopically deformed during spinal motion, but exhibit characteristic reproducible kinematics patterns.     

A physical model for galvanotaxis of Paramecium cell

We propose a qualitative physical model of galvanotaxis of Paramecium cells using a bottom-up approach to link the microscopic ciliary motion and the macroscopic behavior of the cells. From the characteristic pattern of ciliary motion called the Ludloff phenomenon, the torque that orients the cell toward the cathode is derived mathematically. Dynamical equations of motion are derived and their stability is discussed. In numerical simulations using our model, cells exhibit realistic behavior, such as U-turns, like real cells.     

Computational modeling of a dynamic knee simulator for reproduction of knee loading

As a first step towards reproducing desired three-dimensional joint loading and motion on a dynamic knee simulator, the goal of this study was to develop and verify a three-dimensional computational model that generated control profiles for the simulator using desired knee loading and motion as model inputs. The developed model was verified by predicting tibio-femoral loading on an instrumented analog knee for given actuator forces and the ability to generate simulator control profiles was demonstrated using a three-dimensional walking profile. The model predicted axial tibia loading for a sagittal-plane dual-limb squat within 1% of measured peak loading. Adding out-of-sagittal-plane forces decreased the accuracy of load prediction. The model generated control profiles to the simulator that produced axial tibia loading within 16% of desired for walking. Discrepancies in predicted and measured quadriceps forces influenced the accuracy of the generated control profiles. Future work will replace the analog knee in both the model and machine with a prosthetic knee.     

High-speed digital microscopy

High-speed imaging is an ideal technique to accurately resolve the temporal and spatial characteristics of rapid events at either the molecular or cellular level. In this article, the digital imaging techniques used to simultaneously acquire transillumination phase-contrast images, at 240 images s(-1) (high-speed), to characterize ciliary beat frequency, and fluorescence images, at 30 images s(-1) (fast), to measure intracellular calcium concentration ([Ca2+]i), are described. With this technique, a precise correlation between the changes in ciliary beat frequency with changes in [Ca2+]i can be made. Simultaneous imaging is achieved by using different wavelengths of light to form the phase-contrast and fluorescent images and selectively directing these light wavelengths to different cameras with dichroic mirrors and bandpass filters. High-speed images compatible with standard video recording equipment are obtained by prematurely resetting the raster scan of a CCD camera with additional vertical synchronization pulses. The fast [Ca2+]i images are determined using the ratiometric dye fura-2 and a recording technique that monitors rapid changes in fluorescence at a single wavelength and uses intermittent reference images for calibration.     

Function of dorsal fins in bamboo shark during steady swimming

To gain insight into the function of the dorsal fins in white-spotted bamboo sharks (Orectolobiformes: Hemiscyillidae) during steady swimming, data on three-dimensional kinematics and electromyographic recordings were collected. Bamboo sharks were induced to swim at 0.5 and 0.75 body lengths per second in a laminar flow tank. Displacement, lag and angles were analyzed from high-speed video images. Onset, offset, duration, duty cycle and asynchrony index were calculated from three muscle implants on each side of each dorsal fin. The dorsal fins were displaced more laterally than the undulating body. In addition, the dorsal tips had larger lateral displacement than the trailing edges. Increased speed was accompanied by an increase in tail beat frequency with constant tail beat amplitude. However, lateral displacement of the fins and duration of muscle bursts remained relatively constant with increased speed. The range of lateral motion was greater for the second dorsal fin (mean 33.3°) than for the first dorsal fin (mean 28.4°). Bending within the fin was greater for the second dorsal fin (mean 43.8°) than for the first dorsal fin (mean 30.8°). Muscle onset and offset among implants on the same side of each dorsal fin was similar. Three-dimensional conformation of the dorsal fins was caused by interactions between muscle activity, material properties, and incident flow. Alternating bilateral activity occurred in both dorsal fins, further supporting the active role of these hydrofoils in thrust production during steady swimming. The dorsal fins in bamboo sharks are capable of thrust production during steady swimming and do not appear to function as stabilizing structures.     

Dynamic motion of paxillin on actin filaments in living endothelial cells

Our three-dimensional (3-D) images showed that paxillin co-localized on actin filaments as fibrous structures, as well as clusters, in endothelial cells (ECs). In living ECs under flow condition, we monitored concurrently the intracellular dynamics of DsRed2-paxillin and GFP-actin by time-lapse video recording and dual-color fluorescence imaging. The results showed that the dynamic motion of paxillin as fibrous structures was associated with actin filaments, but not with microtubules. Our findings suggest that the actin network plays an important role not only in the assembly/disassembly of paxillin at focal adhesions, but also as a track for the intracellular transport of paxillin, which is involved in signaling pathway.     

Morphogenetic movements during axolotl neural tube formation tracked by digital imaging

During neurulation in vertebrate embryos, epithelial cells of the neural plate undergo complex morphogenetic movements that culminate in rolling of the plate into a tube. Resolution of the determinants of this process requires an understanding of the precise movements of cells within the epithelial sheet. A computer algorithm that allows automated tracking of epithelial cells visible in digitized video images is presented. It is used to quantify the displacement field associated with morphogenetic movements in the axolotl (Ambystoma mexicanum) neural plate during normal neural tube formation. Movements from lateral to medial, axial elongations and area changes are calculated from the displacement field data and plotted as functions of time. Regional and temporal differences are identified. The approach presented is suitable for analyzing a wide variety of morphogenetic movements.     

An electro-optic monitor of the behavior of Chlamydomonas reinhardtii cilia

The unicellular green alga Chlamydomonas reinhardtii steers through water with a pair of cilia (eukaryotic flagella). Long-term observation of the beating of its cilia with controlled stimulation is improving our understanding of how a cell responds to sensory inputs. Here we describe how to record ciliary motion continuously for long periods. We also report experiments on the network of intracellular signaling that connects the environment inputs with response outputs. Local spatial changes in ciliary response on the time scale of the underlying biochemical dynamics are observed. Near-infrared light monitors the cells held by a micropipette. This condition is tolerated well for hours, not interfering with ciliary beating or sensory transduction. A computer integrates the light stimulation of the eye of Chlamydomonas with the ciliary motion making possible long-term correlations. Measures of ciliary responses include the beating frequency, stroke velocity, and stroke duration of each cilium, and the relative phase of the cis and trans cilia. The stationarity and dependence of the system on light intensity was investigated. About 150,000,000 total beat cycles and up to 8 h on one cell have been recorded. Each beat cycle is resolved so that each asynchronous beat is detected. Responses extend only a few hundred milliseconds, but there is a persistence of momentary changes that last much longer. Interestingly, we see a response that is linear with absolute light intensity as well as different kinds of response that are clearly nonlinear, implying two signaling pathways from the cell body to the cilia.