Computer-assisted Motion Analysis System Research Articles

Live cell tracking based on cellular state recognition from microscopic images

The analysis of cell motion is an essential process in fundamental medical studies because most active cellular functions involve motion. In this paper, a computer-assisted motion analysis system is proposed for cell tracking. In the proposed tracking process, unlike in conventional tracking methods, cellular states referring to the cellular life cycle are defined and appropriate strategies are adopted for cells at different states. The use of cellular state recognition allows detection of possible cell division and hence can improve the robustness of cell tracking. Experimental results show that cells can be successfully segmented and tracked over a long period of time, and the proposed system is found to be as accurate as manual tracking. Various quantitative analyses and visualizations are used to represent cell motion, which demonstrates the usefulness of the proposed system in the study of cell dynamics.

The internal phosphodiesterase RegA is essential for the suppression of lateral pseudopods during Dictyostelium chemotaxis.

Dictyostelium strains in which the gene encoding the cytoplasmic cAMP phosphodiesterase RegA is inactivated form small aggregates. This defect was corrected by introducing copies of the wild-type regA gene, indicating that the defect was solely the consequence of the loss of the phosphodiesterase. Using a computer-assisted motion analysis system, regA(-) mutant cells were found to show little sense of direction during aggregation. When labeled wild-type cells were followed in a field of aggregating regA(-) cells, they also failed to move in an orderly direction, indicating that signaling was impaired in mutant cell cultures. However, when labeled regA(-) cells were followed in a field of aggregating wild-type cells, they again failed to move in an orderly manner, primarily in the deduced fronts of waves, indicating that the chemotactic response was also impaired. Since wild-type cells must assess both the increasing spatial gradient and the increasing temporal gradient of cAMP in the front of a natural wave, the behavior of regA(-) cells was motion analyzed first in simulated temporal waves in the absence of spatial gradients and then was analyzed in spatial gradients in the absence of temporal waves. Our results demonstrate that RegA is involved neither in assessing the direction of a spatial gradient of cAMP nor in distinguishing between increasing and decreasing temporal gradients of cAMP. However, RegA is essential for specifically suppressing lateral pseudopod formation during the response to an increasing temporal gradient of cAMP, a necessary component of natural chemotaxis. We discuss the possibility that RegA functions in a network that regulates myosin phosphorylation by controlling internal cAMP levels, and, in support of that hypothesis, we demonstrate that myosin II does not localize in a normal manner to the cortex of regA(-) cells in an increasing temporal gradient of cAMP.

Hip and ankle walking strategies: effect on peak plantar pressures and implications for neuropathic ulceration.

Treatment of neuropathic plantar ulcers often is directed at reducing excessive, repeated peak plantar pressures (PPP). The purposes of this study were to determine whether instructing a subject to walk using a hip strategy would reduce forefoot PPP and change the kinematics of walking during a single session of testing. Thirteen subjects, 7 with peripheral neuropathy and a history of a recent plantar ulcer, and 6 controls participated. PPPs were measured with an in-shoe pressure monitoring system. Kinematics were measured with a computer-assisted motion analysis system. After data were collected as subjects walked using their normal walking pattern, subjects were instructed to walk using the hip strategy by decreasing their push-off, pulling their leg forward from their hips, decreasing step length, and maintaining their normal walking velocity. Compared with using the normal (ankle) strategy, using the hip strategy showed a significant 27% decrease in forefoot PPP and a 24% increase in heel PPP. Kinematic changes were decreased plantar flexion angular velocity, hip extension range-of motion (ROM), and step length, increased dorsiflexion ROM, and hip flexion ROM, but no change in walking velocity. These findings indicate that a change in walking pattern can result in lower forefoot PPP during a single session. Assuming patients can maintain the alterations in their walking pattern, these adaptations may help to heal plantar ulcers in some patients with peripheral neuropathy.

Analysis of atherectomy specimens

Atherectomy specimens may be regarded as biopsy tissue excised from human vascular target lesions. Proceeding from contrary histologic findings that attribute focal hypercellularity to restenosis, and hypocellularity to chronic lesions, further analysis of atherectomy specimens was performed to study ultrastructural characteristics and functional aspects propagated by both lesion types. Transmission electron microscopy examination showed that intimal smooth muscle cells (SMCs) were the predominant cells in both primary and restenotic lesions. SMCs exhibited variable degrees of metabolic activation, typically higher in SMCs of restenotic lesions. This SMC phenotype was equally expressed when tissue samples were placed in a cell culture model. In an attempt to quantify SMC activity, proliferative as well as migratory activities of cultured cells were measured by growth curves and a computer-assisted motion analysis system, respectively. A 2- to 3-fold increase of both activity determinants was observed with SMCs cultivated from restenotic lesions compared with those from primary lesions, irrespective of their coronary or peripheral origin. Drug-induced interference of human SMC metabolic activation and antagonism to their proliferative and migratory activities may be helpful in evaluation of therapeutic concepts to prevent restenosis. The antitubulin colchicine was studied for its effect on the defined determinants. The data in vitro demonstrate that colchicine decreased proliferative and migratory activity of SMCs and caused disorganization of the cytoplasmic ultrastructure. In conclusion, electron microscopy and cell culture studies may help to shed more light on the structures and mechanisms underlying restenosis and plaque growth. Deliberate counteraction of any of the specific early events implicated in these complex pathobiologic processes may eventually become effective means to suppress restenosis and may thus result in a prophylactic as well as therapeutic treatment of the diseased vascular wall.

Migratory activity of human smooth muscle cells cultivated from coronary and peripheral primary and restenotic lesions removed by percutaneous atherectomy.

The successful cultivation of human smooth muscle cells (SMC) from coronary and peripheral atherosclerotic lesions removed by percutaneous directional atherectomy is described. Sixty-seven patients in whom plaque material was obtained compose the study population. A total of 73 lesions from both coronary (n = 38) and peripheral (n = 35) arteries of primary (n = 50) and restenotic origin (n = 23) were studied. Successful cultivation was significantly (p less than 0.001) dependent on the quantity of plaque material submitted. Fifty-five percent of patients in whom atherectomy specimens were removed from coronary lesions yielded an adequate SMC population in comparison to 89% of those from peripheral arteries (p less than 0.01). Cultivation was not dependent on the age and sex of patients, lesion origin, risk factors, medications, or incidence of unstable angina. In an attempt to quantify SMC activity, migratory velocity was measured with a computer-assisted motion analysis system. SMC migratory velocity was found to be significantly (p less than 0.001) greater in restenotic than in primary plaque material. This finding was confirmed for both coronary and peripheral lesions. Our data suggest that elevated SMC migratory activity may be an important mechanism in the development of restenotic lesions.

Rotation and switching of the flagellar motor assembly in Halobacterium halobium

Halobacterium halobium swims with a polarly inserted motor-driven flagellar bundle. The swimming direction of the cell can be reserved by switching the rotational sense of the bundle. The switch is under the control of photoreceptor and chemoreceptor proteins that act through a branched signal chain. The swimming behavior of the cells and the switching process of the flagellar bundle were investigated with a computer-assisted motion analysis system. The cells were shown to swim faster by clockwise than by counterclockwise rotation of the flagellar bundle. From the small magnitude of speed fluctuations, it is concluded that the majority, if not all, of the individual flagellar motors of a cell rotate in the same direction at any given time. After stimulation with light (blue light pulse or orange light step-down), the cells continued swimming with almost constant speed but then slowed before they reversed direction. The cells passed through a pausing state during the change of the rotational sense of the flagellar bundle and then exhibited a transient acceleration. Both the average length of the pausing period and the transient acceleration were independent of the stimulus size and thus represent intrinsic properties of the flagellar motor assembly. The average length of the pausing period of individual cells, however, was not constant. The time course of the probability for spontaneous motor switching was calculated from frequency distribution and shown to be independent of the rotational sense. The time course further characterizes spontaneous switching as a stochastic rather than an oscillator-triggered event.

Sperm from tw32/+ mice: Capacitation is normal, but hyperactivation is premature and nonhyperactivated sperm are slow

The t complex in the mouse is a large group of linked genes that affect sperm function in fertilization. In t/+ males, sperm carrying the t complex (t sperm) have normal fertilizing ability, while sperm carrying the normal homolog (+t sperm) are dysfunctional (P. Olds-Clarke and B. Peitz, 1985, Genet. Res. 47, 49). The specific step in fertilization which is dysfunctional, however, is not known. Two characteristics of fertilizing sperm, capacitation (the process by which sperm become capable of undergoing the acrosome reaction) and hyperactivation (the change in swimming behavior concomitant with capacitation), were assayed by objective methods in epididymal sperm from tw32/+ males of two strains, and compared to sperm from +/+ males of the same strains. Capacitated and acrosome-reacted sperm were identified by a chlortetracycline assay (C.R. Ward and B.T. Storey, 1984, Dev. Biol. 104, 287). Hyperactivated sperm were identified by their path shape and swimming speed, using a computer-assisted motion-analysis system (J.M. Neill and P. Olds-Clarke, 1987, Gamete Res. 18, 121). Hyperactivation occurred significantly sooner among sperm from tw32/+ mice than among sperm from +/+ mice of the same strain, while the rates and maximal levels of capacitation and spontaneous acrosome reactions were normal. Of the nonhyperactivated motile sperm from tw32/+ mice, almost all were slower than sperm from +/+ mice of the same strain. While the effect of premature hyperactivation on fertilization is not clear, slow movements are likely to impair fertilizing ability. These results raise the possibility that the slow sperm are the dysfunctional +t sperm.

Incubation of mouse sperm with lactate delays capacitation and hyperactivation and lowers fertilization levels in vitro.

Mouse sperm were incubated in medium with or without 24 mM lactate and assessed for 1) motility characteristics including hyperactivation--a computer-assisted motion analysis system was used; 2) capacitation--a chlortetracycline fluorescent dye binding assay was used; and 3) ability to penetrate oocytes. Lactate affected all aspects of motility and delayed the rates of both hyperactivation and capacitation. When a concentration of 8 x 10(3) sperm/ml was used for insemination in vitro, sperm preincubated 60-90 minutes in medium with lactate prior to insemination in lactate-free medium fertilized fewer oocytes than did sperm preincubated in lactate-free medium. Use of a calcium-sensitive electrode demonstrated that lactate chelated appreciable amounts of calcium in the medium. Capacitation was assayed in sperm incubated 60 minutes in medium with various concentrations of lactate or CaCl2. When medium containing lactate was compared to medium without lactate but having a similar level of free calcium, the level of capacitation of sperm incubated with lactate was less than half that of sperm incubated without lactate. These results demonstrate that including 24 mM lactate in the medium can have detrimental effects on mouse sperm hyperactivation and capacitation. The detrimental effects on capacitation are partly but not completely due to the chelation of calcium by lactate.

A computer‐assisted assay for mouse sperm hyperactivation demonstrates that bicarbonate but not bovine serum albumin is required

Mammalian sperm hyperactivation (HA) is a change in motility that accompanies capacitation (CAP) and is dependent on calcium (Ca) (Yanagimachi and Usui, Exp Cell Res 89:161, 1974). HA may be important for transport through the female tract and/or for fertilization. To develop an objective and quantitative assay for HA in individual mouse sperm, a computer-assisted motion-analysis system was used to describe sperm translational movements. To determine which movements were characteristic of HA, Ca-dependent motility was identified. This was done by incubating sperm with or without calcium (Ca+ or Ca- sperm, respectively), and determining the range of values for each motility parameter that was present only among Ca+ sperm. To do this, we compared frequency distributions of motility parameter values at the time of maximal CAP (90 min). CAP was monitored by measuring the level of in vitro fertilization and by evaluating the pattern of chlortetracycline binding to individual sperm heads [Ward and Storey, Dev Biol 104:287, 1984]. Two Ca-dependent motility subgroups were apparent: 1) a "slow-speed" subgroup with a curvilinear velocity (Vc) less than 169 microns/sec that had none of the characteristics expected of HA sperm; and 2) a subgroup with higher speeds (Vc greater than 169 microns/sec) and wider-amplitude head movements as measured by curvilinear progressiveness ratio (PRc less than 0.56). The latter subgroup was selected as HA, since the frequencies and time course were similar to those for CAP in the same population. Two media components known to be important for CAP, bicarbonate and bovine serum albumin (BSA) were then tested to determine whether they were necessary for HA. Incubation of sperm without bicarbonate prevented HA, but omitting BSA did not affect HA during the first 3 hrs. These data suggest that HA is not tightly coupled with CAP.