Automated regional analysis of B-mode ultrasound images of skeletal muscle movement

To understand the functional significance of skeletal muscle anatomy, a method of quantifying local shape changes in different tissue structures during dynamic tasks is required. Taking advantage of the good spatial and temporal resolution of B-mode ultrasound imaging, we describe a method of automatically segmenting images into fascicle and aponeurosis regions and tracking movement of features, independently, in localized portions of each tissue. Ultrasound images (25 Hz) of the medial gastrocnemius muscle were collected from eight participants during ankle joint rotation (2° and 20°), isometric contractions (1, 5, and 50 Nm), and deep knee bends. A Kanade-Lucas-Tomasi feature tracker was used to identify and track any distinctive and persistent features within the image sequences. A velocity field representation of local movement was then found and subdivided between fascicle and aponeurosis regions using segmentations from a multiresolution active shape model (ASM). Movement in each region was quantified by interpolating the effect of the fields on a set of probes. ASM segmentation results were compared with hand-labeled data, while aponeurosis and fascicle movement were compared with results from a previously documented cross-correlation approach. ASM provided good image segmentations (<1 mm average error), with fully automatic initialization possible in sequences from seven participants. Feature tracking provided similar length change results to the cross-correlation approach for small movements, while outperforming it in larger movements. The proposed method provides the potential to distinguish between active and passive changes in muscle shape and model strain distributions during different movements/conditions and quantify nonhomogeneous strain along aponeuroses.     

Computational methods for quantifying in vivo muscle fascicle curvature from ultrasound images

Muscle fascicles curve during contraction, and this has been seen using B-mode ultrasound. Curvature can vary along a fascicle, and amongst the fascicles within a muscle. The purpose of this study was to develop an automated method for quantifying curvature across the entirety of an imaged muscle, to test the accuracy of the method against synthetic images of known curvature and noise, and to test the sensitivity of the method to ultrasound probe placement. Both synthetic and ultrasound images were processed using multiscale vessel enhancement filtering to accentuate the muscle fascicles, wavelet-based methods were used to quantify fascicle orientations and curvature distribution grids were produced by quantifying local curvatures for each point within the image. Ultrasound images of ramped isometric contractions of the human medial gastrocnemius were acquired in a test–retest study.The methods enabled distinct curvatures to be determined in different regions of the muscle. The methods were sensitive to kernel sizes during image processing, noise within the image and the variability of probe placements during retesting. Across the physiological range of curvatures and noise, curvatures calculated from validation grids were quantified with a typical standard error of less than 0.026 m^?1, and this is about 1% of the maximum curvatures observed in fascicles of contracting muscle.     

In vivo fascicle length measurements via B-mode ultrasound imaging with single vs dual transducer arrangements

Ultrasonography is a useful technique to study muscle contractions in vivo, however larger muscles like vastus lateralis may be difficult to visualise with smaller, commonly used transducers. Fascicle length is often estimated using linear trigonometry to extrapolate fascicle length to regions where the fascicle is not visible. However, this approach has not been compared to measurements made with a larger field of view for dynamic muscle contractions. Here we compared two different single-transducer extrapolation methods to measure VL muscle fascicle length to a direct measurement made using two synchronised, in-series transducers. The first method used pennation angle and muscle thickness to extrapolate fascicle length outside the image (extrapolate method). The second method determined fascicle length based on the extrapolated intercept between a fascicle and the aponeurosis (intercept method). Nine participants performed maximal effort, isometric, knee extension contractions on a dynamometer at 10° increments from 50 to 100° of knee flexion. Fascicle length and torque were simultaneously recorded for offline analysis. The dual transducer method showed similar patterns of fascicle length change (overall mean coefficient of multiple correlation was 0.76 and 0.71 compared to extrapolate and intercept methods respectively), but reached different absolute lengths during the contractions. This had the effect of producing force–length curves of the same shape, but each curve was shifted in terms of absolute length. We concluded that dual transducers are beneficial for studies that examine absolute fascicle lengths, whereas either of the single transducer methods may produce similar results for normalised length changes, and repeated measures experimental designs.     

Automated analysis of muscle fibre images

A technique for studying the distribution and size of different fibre types in muscles is proposed for automated analysis of individual fibres in optical density images from ATPase-stained muscle sections. After delineation, fibres may be classified into different histological types (1, 2A, 2B and 2C) using the measurement of their mean optical density (mOD). The densitometric measurements were obtained from three serial histological slides stained under different conditions. The delineation procedure is performed on one of the images: the resulting mask is fitted to the other images using a linear coordinate transform. Along with densitometric measurements, the lesser diameter of the fibres is computed. Both in processing and in analysis, extensive use was made of mathematical morphology tools. All software was implemented on a VICOM digital image processor, extended with a VISIOMORPH morphoprocessor board.     

Reliability and accuracy of an automated tracking algorithm to measure controlled passive and active muscle fascicle length changes from ultrasound

Manual tracking of muscle fascicle length changes from ultrasound images is a subjective and time-consuming process. The purpose of this study was to assess the repeatability and accuracy of an automated algorithm for tracking fascicle length changes in the medial gastrocnemius (MG) muscle during passive length changes and active contractions (isometric, concentric and eccentric) performed on a dynamometer. The freely available, automated tracking algorithm was based on the Lucas–Kanade optical flow algorithm with an affine optic flow extension, which accounts for image translation, dilation, rotation and shear between consecutive frames of an image sequence. Automated tracking was performed by three experienced assessors, and within- and between-examiner repeatability was computed using the coefficient of multiple determination (CMD). Fascicle tracking data were also compared with manual digitisation of the same image sequences, and the level of agreement between the two methods was calculated using the coefficient of multiple correlation (CMC). The CMDs across all test conditions ranged from 0.50 to 0.93 and were all above 0.98 when recomputed after the systematic error due to the estimate of the initial fascicle length on the first ultrasound frame was removed from the individual fascicle length waveforms. The automated and manual tracking approaches produced similar fascicle length waveforms, with an overall CMC of 0.88, which improved to 0.94 when the initial length offset was removed. Overall results indicate that the automated fascicle tracking algorithm was a repeatable, accurate and time-efficient method for estimating fascicle length changes of the MG muscle in controlled passive and active conditions.     

Automatic tracking of medial gastrocnemius fascicle length during human locomotion

During human locomotion lower extremity muscle-tendon units undergo cyclic length changes that were previously assumed to be representative of muscle fascicle length changes. Measurements in cats and humans have since revealed that muscle fascicle length changes can be uncoupled from those of the muscle-tendon unit. Ultrasonography is frequently used to estimate fascicle length changes during human locomotion. Fascicle length analysis requires time consuming manual methods that are prone to human error and experimenter bias. To bypass these limitations, we have developed an automatic fascicle tracking method based on the Lucas-Kanade optical flow algorithm with an affine optic flow extension. The aims of this study were to compare gastrocnemius fascicle length changes during locomotion using the automated and manual approaches and to determine the repeatability of the automated approach. Ultrasound was used to examine gastrocnemius fascicle lengths in eight participants walking at 4, 5, 6, and 7 km/h and jogging at 7 km/h on a treadmill. Ground reaction forces and three dimensional kinematics were recorded simultaneously. The level of agreement between methods and the repeatability of the automated method were quantified using the coefficient of multiple correlation (CMC). Regardless of speed, the level of agreement between methods was high, with overall CMC values of 0.90 ± 0.09 (95% CI: 0.86-0.95). Repeatability of the algorithm was also high, with an overall CMC of 0.88 ± 0.08 (95% CI: 0.79-0.96). The automated fascicle tracking method presented here is a robust, reliable, and time-efficient alternative to the manual analysis of muscle fascicle length during gait.     

The effect of ultrasound probe orientation on muscle architecture measurement.

The purpose of this paper was to examine how muscle architecture parameter (MAP) measurements made using brightness-mode ultrasonography (BMU) differ based on probe orientation. The human tibialis anterior muscle was imaged from nine different probe orientations during concentric contractions at four joint angles to determine the effect of probe orientation on the measurement of muscle architecture parameters. Ankle dorsi-flexion torque, tibialis anterior electromyography (EMG), and measures of MAP including fascicle length (FL), pennation angle (PA) and muscle thickness (MT) were collected. Statistically significant differences were found between joint angles for measures of FL and PA and between probe orientations for measures of FL and MT. A comparison of actual MAP values to a geometric model used by researchers to determine an ideal probe orientation show that the actual values vary to a greater extent and do not follow the trend predicted by the model. The results suggest that ultrasound probe orientation affects measures of MAP but the effect either cannot be predicted from a geometric model and/or the error in the measurement technique does not allow a comparison.     

Gastrocnemius muscle fascicle behavior during stair negotiation in humans.

The aim of the present study was to establish the behavior of human medial gastrocnemius (GM) muscle fascicles during stair negotiation. Ten healthy male subjects performed normal stair ascent and descent at their own comfortable speed on a standard-dimension four-step staircase with embedded force platforms in each step. Kinematic, kinetic, and electromyographic data of the lower limbs were collected. Real-time ultrasound scanning was used to determine GM muscle fascicle length changes. Musculotendon complex (MTC) length changes were estimated from ankle and knee joint kinematics. The GM muscle was mainly active during the push-off phase in stair ascent, and the muscle fascicles contracted nearly isometrically. The GM muscle was mainly active during the touch-down phase of stair descent where the MTC was lengthened; however, the GM muscle fascicles shortened by approximately 7 mm. These findings show that the behavior and function of GM muscle fascicles in stair negotiation is different from that expected on the basis of length changes of the MTC as derived from joint kinematics.     

Ultrasound-based subject-specific parameters improve fascicle behaviour estimation in Hill-type muscle model

The estimation of muscle fascicle behaviour is decisive in a Hill-type model as they are related to muscle force by the force–length–velocity relationship and the tendon force–strain relationship. This study was aimed at investigating the influence of subject-specific tendon force–strain relationship and initial fascicle geometry (IFG) on the estimation of muscle forces and fascicle behaviour during isometric contractions. Ultrasonography was used to estimate the in vivo muscle fascicle behaviour and compare the muscle fascicle length and pennation angle estimated from the Hill-type model. The calibration–prediction process of the electromyography-driven model was performed using generic or subject-specific tendon definition with or without IFG as constraint. The combination of subject-specific tendon definition and IFG led to muscle fascicle behaviour closer to ultrasound data and significant lower forces of the ankle dorsiflexor and plantarflexor muscles compared to the other conditions. Thus, subject-specific ultrasound measurements improve the accuracy of Hill-type models on muscle fascicle behaviour.     

Relationship between sprint performance and muscle fascicle length in female sprinters

The purpose of this study was to investigate the relationship between sprint performance and architectural characteristics of leg muscles in 26 female 100-m sprinters. Pennation angle and muscle thickness of the vastus lateralis (VL) and gastrocnemius medialis (GM) and lateralis (GL) muscles were measured by B-mode ultrasonography, and fascicle length was estimated. Sprinters had a significantly lower VL pennation angle, but GM and GL pennation angle was similar between sprinters and female control subjects (N = 22). There was no significant correlation between pennation angle and 100-m personal best performance. Sprinters had significantly greater absolute fascicle length in VL and GL than controls, which significantly correlated to 100-m best-record (r = -0.51 and r = -0.44, respectively). Relative fascicle length (VL and GL) were also significantly greater in sprinters than controls. However, there were no significant correlation between relative fascicle length and 100-m best-record (r = -0.36 and r = -0.29, respectively). No relationship was found between the sprint performance and fat-free mass (r = -0.26) or body mass index (r = -0.03). However, there was a significant correlation between percent (%) body fat and 100-m best-record (r = 0.62, p < 0.01). Adjusting the confounding effect of % fat, significant correlations were seen between relative fascicle length and 100-m best-record (VL; r = -0.39 and GL; r = -0.40). Absolute and relative fascicle length were similar in elite female sprinters compared with previous reported values for elite male sprinters (Kumagai et al., 2000). It was concluded that longer fascicle length is associated with greater sprinting performance in sprinters, but there is no gender differences in fascicle length for elite sprinters.     

Determination of fascicle length and pennation in a contracting human muscle in vivo

Fukunaga, Tetsuo, Yoshiho Ichinose, Masamitsu Ito, YasuoKawakami, and Senshi Fukashiro. Determination of fascicle lengthand pennation in a contracting human muscle in vivo.J. Appl. Physiol. 82(1): 354-358, 1997.We have developed a technique to determine fascicle length inhuman vastus lateralis muscle in vivo by using ultrasonography. Whenthe subjects had the knee fully extended passively from a position of110° flexion (relaxed condition), the fascicle length decreasedfrom 133 to 97 mm on average. During static contractions at 10% ofmaximal voluntary contraction strength (tensed condition), fascicleshortening was more pronounced (from 126 to 67 mm), especially when theknee was closer to full extension. Similarly, as the knee was extended, the angle of pennation (fascicle angle, defined as the angle between fascicles and aponeurosis) increased (relaxed, from 14 to 18°; tensed, from 14 to 21°), and a greater increase in the pennation angle was observed in the tensed than in the relaxed condition when theknee was close to extension (<40°). We conclude that there aredifferences in fascicle lengths and pennation angles when the muscle isin a relaxed and isometrically tensed conditions and that thedifferences are affected by joint angles, at least at thesubmaximal contraction level.

     

B-mode ultrasound observations of bull testes during breeding soundness examinations

The testes of 78 adult Beefmaster bulls (13 to > 31 mo old) were scanned with a 5 MHz, linear array portable ultrasound unit during routine breeding soundness examinations. Videotape recordings were used to measure mediastinum testis width and fluid width between visceral and parietal tunics (fluid width), and to count the number of fibrotic foci. Ultrasound results were compared to breeding soundness score parameters, which included total score, age classification, and percentage of primary and secondary sperm abnormalities. The overall mean mediastinum testis width and fluid width were 0.33 cm ± 0.133 and 0.10 cm ± 0.13, respectively; 15 testes had 1–8 fibrotic foci. There were no significant differences in mediastinum testis width and fluid width between left and right testes when compared by breeding soundness score classification or by age classification. There were no significant differences in mediastinum testis width, fluid width, breeding soundness score, or primary or secondary sperm abnormalities in bulls with fibrotic foci compared to bulls without fibrotic foci. There appears to be no significant information that routine testicular ultrasound examination adds to accepted breeding soundness evaluations performed on the same day.     

Automated tracking of mitotic spindle pole positions shows that LGN is required for spindle rotation but not orientation maintenance

Spindle orientation defines the plane of cell division and, thereby, the spatial position of all daughter cells. Here, we develop a live cell microscopy-based methodology to extract spindle movements in human epithelial cell lines and study how spindles are brought to a pre-defined orientation. We show that spindles undergo two distinct regimes of movements. Spindles are first actively rotated toward the cells’ long-axis and then maintained along this pre-defined axis. By quantifying spindle movements in cells depleted of LGN, we show that the first regime of rotational movements requires LGN that recruits cortical dynein. In contrast, the second regime of movements that maintains spindle orientation does not require LGN, but is sensitive to 2ME2 that suppresses microtubule dynamics. Our study sheds first insight into spatially defined spindle movement regimes in human cells, and supports the presence of LGN and dynein independent cortical anchors for astral microtubules.     

In vivo assessment of muscle fascicle length by extended field-of-view ultrasonography

The present study examined the reliability and validity of in vivo vastus lateralis (VL) fascicle length (L(f)) assessment by extended field-of-view ultrasonography (EFOV US). Intraexperimenter and intersession reliability of EFOV US were tested. Further, L(f) measured from EFOV US images were compared to L(f) measured from static US images (6-cm FOV) where out-of-field fascicle portions were trigonometrically estimated (linear extrapolation). Finally, spatial accuracy of the EFOV technique was assessed by comparing L(f) measured on swine VL by EFOV US to actual measurements from digital photographs. The difference between repeated VL L(f) measurements by the same experimenter was 2.1 ± 1.7% with an intraclass correlation (ICC) of 0.99 [95% confidence interval (CI) = 0.95-1.00]. In terms of intersession reliability, no difference (P = 0.48) was observed between L(f) measured on two different occasions, with ICC = 0.95 (CI = 0.80-0.99). The average absolute difference between L(f) measured by EFOV US and using linear extrapolation was 12.6 ± 8.1% [ICC = 0.76 (CI = -0.20-0.94)]; EFOV L(f) was always longer than extrapolated L(f). The relative error of measurement between L(f) measured by EFOV US and by dissective assessment (digital photographs) in isolated swine VL was 0.84% ± 2.6% with an ICC of 0.99 (CI = 0.94-1.00). These results show that EFOV US is a reliable and valid method for the measurement of long muscle fascicle in vivo. Thus EFOV US analysis was proven more accurate for the assessment of skeletal muscle fascicle length than conventional extrapolation methods.     

Automated computer evaluation of time-varying cryomicroscopical images

A system has been developed to perform automatic computerized recognition, tracking, and quantitative morphological analysis of viable cells in freezing solutions. Cryomicroscopical image sequences of freezing cells are digitized and analyzed by computer. Image-processing techniques are used which are insensitive to contrast fluctuations from image to image, and which perform well even in noisy, cluttered images. The generalized Hough transform is used for shape detection, and a heuristic graph-search boundary completion algorithm is applied for shape extraction. The extracted cell shape may be analyzed for changes in cross-sectional area, perimeter length, shape deformation, and other metrics of interest. Knowledge from the shapeextraction phase is used to form a prediction of what shape the cell will be in the next image frame, and thus what to look for in the next shape-detection phase. This combination of knowledge-feedback with a powerful shape-detection technique produces an automatic, dynamic shape-recognition scheme capable of accurate recognition and analysis of the cells regardless of how deformed they may become during the freezing sequence. Example performance of the system is illustrated for a series of micrographs of freezing granulocytes.     

Influence of gait velocity on gastrocnemius muscle fascicle behaviour during stair negotiation

The gastrocnemius medialis (GM) muscle plays an important role in stair negotiation. The aim of the study was to investigate the influence of cadence on GM muscle fascicle behaviour during stair ascent and descent. Ten male subjects (young adults) walked up and down a four-step staircase (with forceplates embedded in the steps) at three velocities (63, 88 and 116 steps/min). GM muscle fascicle length was measured using ultrasonography. In addition, kinematic and kinetic data of the lower legs, and GM electromyography (EMG) were measured. For both ascent and descent, the amount of fascicular shortening, shortening velocity, knee moment, ground reaction force and EMG activity increased monotonically with gait velocity. The ankle moment increased up to 88 steps/min where it reached a plateau. The lack of increase in ankle moment coinciding with further shortening of the fascicles can be explained by an increased shortening of the GM musculotendon complex (MTC), as calculated from the knee and ankle angle changes, between 88 and 116 steps/min only. For descent, the relative instant of maximum shortening, which occurred during touch down, was delayed at higher gait velocities, even to the extent that this event shifted from the double support to the single support phase.     

Automated recognition of intracellular organelles in confocal microscope images

Recognition of the localisation of intracellular proteins is essential to the understanding of their function. It is usually made through knowledge of and comparison to the distribution of well-characterised intracellular organelles by experts in cell biology. We have automated this process in order to achieve a more objective and quantitative assessment of the protein distribution within the cell, which can be employed by the less experienced cell biologist and may be utilised as a training program for inexperienced users, or as a high throughput localisation program for novel genes in functional analysis. Here we describe the development and testing of a classification system based on a modular neural network trained with sets of confocal sections through cell lines fluorescently stained for markers of key intracellular structures. The system functioned well in spite of the variability in pattern that occurs between individual cells and performed with 97% accuracy, which gives us confidence in the method and in its future development. It is envisaged that this program will aid the design of further experiments utilising colocalisation with known organelle marker proteins, in order to confirm putative trafficking pathways and protein–protein interactions of the protein of interest.