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Digital data acquisition and analysis of striated muscle diffraction patterns with a direct memory access microprocessor system
Abstract
A microprocessor based data acquisition computing system has been developed to examine dynamic changes in light diffraction patterns from single skeletal muscle fibers. A significant improvement in digital data acquisition rate compared to previous designs has been achieved by utilizing a fast dedicated analog to digital converter and direct memory access data storage. Diffraction patterns from muscle fibers are imaged onto a 256 element charge‐coupled device. The analog output of a full 256 point frame of data may be digitized and stored in 2.2 ms (128 point half frame in 1.1 ms) with a spatial resolution of up to 5.0 nm/sarcomere. This computing system can transfer up to 28 full frames of data as one continuous block directly between the CCD and memory leaving the CPU free for experimental control and closed‐loop processing. The computing system calibrates and analyzes diffraction data under sofware control for sarcomere length, dispersion, and peak intensity. The operation of this data acquisition computing system is superior to previous digital designs with its increased data acquisition rate and to analog designs with its data manipulation, analysis, experimental control, and processing capabilities.
... The technique of laser diffraction has been used widely to assess mean L s of sarcomeres contained in a small volume of the fiber illuminated by the laser ( Rudel and Zite-Ferenczy, 1979). The advantages of the laser diffraction technique are: (i) a superior signal-to-noise ratio and complete utilization of the dynamic range of the light detectors compared with direct optical imaging ( Myers et al., 1982; Roos et al., 1980), (ii) minimal optics and consequently the minimization of the effects of aberrations that pose significant challenges in optical imaging techniques, and (iii) the inexpensive availability of lasers and optical-to-electrical conversion systems. Multiple-sector measurements of L s with laser diffraction) used laser diffraction for multiple-sector estimation of L s in intact single fibers during fixed-end contractions. ...
... Testing the hypothesis that sarcomere extensibility increases with increasing L s requires snapshots of the L s of serially connected sectors of activated fibers before and at the peak of stretch. The laser diffraction technique has been used widely in L s measurements and was selected for our study because this technique: (1) offers superior signal-to-noise ratio and complete utilization of the dynamic range of the detectors, when compared with direct optical imaging ( Roos et al., 1980; Myers et al., 1982;), (2) requires minimal optics and consequently minimizes the effects of aberrations that pose significant challenges in optical imaging techniques, and (3) provides inexpensive availability of the light sources and optical-toelectrical conversion systems used in this technique. To test the hypothesis, we modified the measurement methodology of laser diffraction from a traditional single-point measurement to a multiple-point measurement approach by rapid translation of the laser beam. ...
A contraction-induced injury results when an activated skeletal muscle or a single fiber is stretched in a ‘lengthening contraction’. This type of injury to muscle fibers is a leading cause of the loss of mobility that is associated with old age. Manifestations of the injury revealed by electron microscopy show focally disrupted single sarcomeres, or small groups of sarcomeres, that are surrounded by intact sarcomeres. The purpose of this dissertation was to study the dynamic behavior of sarcomeres during lengthening contractions of single muscle fibers. The working hypothesis was that contraction-induced injury occurs during a lengthening contraction when sarcomeres undergo elongation at varying rates and those lengthened at the highest rates reach longer lengths and are damaged. To test this hypothesis, a laser scanning system was developed that recorded rapidly (500 s−1) the lengths of sarcomeres contained in each of 20 contiguous regions along ~1 mm segments of a fiber. The fiber segments were obtained from soleus muscles of adult male rats and the membranes of the fibers were permeabilized chemically. The experiments performed with the laser scanning apparatus, indicated that: (i) during a lengthening contraction, the regions of fibers that contain the longer sarcomeres at the onset of the lengthening elongate more compared with the regions that contain shorter sarcomeres; (ii) lengthening contractions increased the variability in the lengths of sarcomeres in relaxed fibers; and (iii) during a lengthening contraction, within any given fiber region, the rate of elongation of sarcomeres was stable, but different regions stretched at different rates as the contraction proceeded. The present findings support the hypothesis that, within single fibers, non-uniformities develop in the lengths of sarcomeres that are increased by a lengthening contraction. The finding that all fiber regions elongate at stable rates during lengthening contractions contradicts a previous hypothesis that during lengthening contractions, the longest sarcomeres undergo rapid and sudden elongation to extreme lengths. The conclusion is that during lengthening contractions, the longer sarcomeres in series with shorter sarcomeres undergo elongation at higher velocities and upon return to original length, structural elements within some of these longer sarcomeres are functionally compromised. Ph.D. Biomedical Engineering University of Michigan, Horace H. Rackham School of Graduate Studies http://deepblue.lib.umich.edu/bitstream/2027.42/58375/1/appaji_1.pdf
... This trend reflects the increased availability of low-cost mini-and microcomputer hardware and an explosion in the amount and type of software support available for these systems. We have developed a number of real-time data acquisition systems for studies of skeletal muscle contraction which rely on state-of-the-art hardware implemen- tations5679,10] . However, many biological applications require the use of ordinary hardware in an environment maximizing data acquisition efficiency and enabling the inexperienced computer user to perform sophisticated biological experiments . ...
A data acquisition system is described which acquires data from contracting skeletal muscle. The system is designed to run in a multi-user environment while acquiring contractile data in real-time. Time dedicated solely to laboratory experiments is thus eliminated. A menu-driver is included to allow users to enter experimental commands with or without command arguments. Error monitoring functions prevent operator errors from causing data loss. Data storage in both ASCII and binary formats maximizes file flexibility, readability and accessibility. Finally, an on-line tutorial and help facility is provided for user training. The system developed is applicable to any experimental environment involving data acquisition, storage and analysis.
... It was, therefore, desirable to directly monitor the profile of a diffracted order. We accomplished this using the computer system described by Roos et al. (1980) and Lieber and. This system is a microprocessor that can rapidly acquire diffraction patterns from a linear photodiode array, which performs serial output via a charge-coupled device (CCD). ...
An experimental and theoretical analysis is presented involving the effect of variation in fiber and beam diameter upon the determination of average sarcomere length in isolated single muscle fibers using laser light diffraction. The muscle diffraction phenomenon is simplified by first considering diffraction order position and intensity to be the result of grating and Bragg diffraction. It is the product of the intensity profiles, which results from these types of diffraction, that produces the diffracted order. These simplifying assumptions are then extended to the case of the real muscle. Based on these considerations and the theory that we recently presented, conditions are set forth under which grating information (i.e., sarcomere length) can be maximally expressed to yield accurate average sarcomere length values.
... The higher orders diffracted above and below the zero-order plane are of greater interest, and consequently much work has concentrated on the line widths of the various diffraction orders and their intensities. Various data acquisition techniques have used film (Cleworth, 1972;Sandow, 1936) and highly sophisticated, rapid digital computing systems (Roos et al., 1980). The intensity distributions of the first order, and to a lesser extent the second and third, give information about the mean sarcomere length during rest and activation. ...
Light diffraction patterns from single glycerinated frog semitendinosus muscle fibers were examined photographically and photoelectrically as a function of diffraction angle and fiber rotation. The total intensity diffraction pattern indicates that the order maxima change both position and intensity periodically as a function of rotation angle. The total diffracted light, light diffracted above and below the zero-order plane, and light diffracted into individual orders gives information about the fiber's longitudinal and rotational structure and its noncylindrical symmetry.
Through the use of light diffraction the average length of sarcomeres in a region of striated muscle can be rapidly and accurately determined. Changes in sarcomere length accompanying shortening or those occurring during an isometric tetanus can be precisely resolved.
A microcomputer‐based video analyzer system has been developed to obtain area information from a variety of different sample types. Most programs are written in basic to analyze optical information with a conventional video camera and frame grabber. Intensity information from the image is used to automatically obtain area information, rather than the operator‐directed approach used in many digitizing systems. Real‐time operator modifications of the digitized image are possible, as well as image storage and retrieval and graphic display of the results.
A microcomputer‐based data‐acquisition system has been developed to investigate large scale magnetohydrodynamic (MHD) instabilities in a noncircular tokamak, TNT‐A. Two pairs of multiplexers, sample and hold amplifiers, and fast AD converters, together with DMA data transfer, allow the system to support fast multichannel data acquisition (sampling time≂1.3 μs). Spectral analysis of poloidal magnetic field fluctuations gives mode numbers and the relative level of instabilities. A least‐squares method has been devised to identify poloidal mode numbers, while taking into account the effects of noncircularity and displacement of the plasma column. This method proved to be effective for a noncircular tokamak and it enables us to check mode mixing between different frequencies.
Dissertation (Ph.D.)--University of Michigan
A microprocessor based digital recording system has been developed to study the fine structure and asymmetry of diffraction spectra from striated muscle during contraction. Two linear 256-element photodiode arrays provide analog videosignals of the diffraction lines imaged onto these charged coupled devices. The photodiode arrays are alternately read and the videosignals can be digitized and stored within 1.36 ms (two images of 256 points) with a spatial resolution of 5 nm. (In this paper the spatial resolution is considered to be the standard deviation of the first-order maximum of a monochromatic wave of the He/Ne laser measured from the diode-arrays, using ideal gratings with a spacing between 1.6 and 3.6 microns.) The system's memory with a capacity of 192 pairs of images of 256 points can be optimized by means of a threshold to contain about 2000 images without any loss of information. A transient recording approach makes the system capable of recording long term slow phenomena of up to 5 s as well as fast events and the combination of fast events within slow processes. The system presented here has a significantly improved time resolution and storage capacity when compared to other systems and is more versatile. This is the first system which enables the simultaneous examination of the fine structure and asymmetry of diffraction spectra.
The relationship between the diffraction intensity change of the first order line and tension development was examined in mechanically skinned single fibers from the dorsal head of the semitendinosus of frogs. Passive stretch of the fibers resulted in an increase in intensity over the range of sarcomere lengths from 2.5 to 3.6 microns, indicating that the intensity is a function of sarcomere length. Activation of skinned fibers caused a decrease in the intensity, at all sarcomere lengths, where the thick and thin filaments overlapped. The magnitude of the intensity decrease and that of the tension development depended on the Ca2+ concentration in the medium. The drop of intensity-pCa and the tension-pCa curves showed a similarly steep S-shape within a range of 0.5pCa unit, although the intensity-pCa curve shifted to the left; the pCa for 50% decrease in light signal was 6.48 and that for 50% tension development was 6.40. Caffeine (25 mM) added to the medium produced a decrease in the intensity of skinned fibers with the simultaneous development of tension, thereby indicating that caffeine induces a release of Ca2+ from the sarcoplasmic reticulum and disorder in the filaments ensues. Changes in diffraction intensity with electrical stimulation to the intact single fiber were similar, although a more striking summation was observed in the optical response, as compared to the tension development. These results suggest that tension development upon stimulation can be monitored by assessing the magnitude of diffraction intensity decrease in the first order line, except for some shift in the short fiber.
Optical diffraction measurements during rapid releases of active toad muscle show that the sarcomeres contract within 1 millisecond by an amount up to but not greater than 12 nanometers. This crossbridges immediately start cycling to produce the normal contraction velocity in unloaded muscle.
A laser diffraction technique has been developed for registering small changes in sarcomere length. The technique is capable of resolving changes as small as 0.2 A in isolated frog skeletal muscle fibers. The small sarcomere lengthening that accompanies the drop in tension in the latent period of contraction was investigated. We suggest this lengthening be named latency elongation (LE). The LE is present in a completely slack fiber and must, therefore, be caused by a forcible lengthening process. Furthermore, the LE is dependent on the existence of an overlap between thin and tick filaments. The rate of elongation and the time interval between stimulation and maximum elongation may vary along the fiber. The maximum elongation was 3-5 A per sarcomere. At any instant the drop in tension is a product of the sum of sarcomere lengthenings along the fiber and the slope stiffness of the series elasticity. The latency relaxation (LR) could be registered in the sarcomere length range from 2.2 mum to 3.6-3.7 mum. The amplitude went through a sharp maximum at 3.0-3.1 mum. In the sarcomere length range from 2.2 to 2.8 mum the delay from onset to maximum LR was nearly proportional to the distance from the Z-line to the overlap zone. A working hypothesis is presented. It is suggested that the LE is caused by a lengthening of the thin filaments.
A simple method is devised to record rapid sarcomere length changes of muscle fibres using a lateral effect diode. In the standard position the diffractometer records length changes between 1.65 and 3.8 m, the output being linear 1 V/m with a frequency response of –3 dB at 1.2 kHz. The absolute error ism between 1.65 and 2.80 m and m between 2.81 and 3.30 m. The resolution of length changes ism over the whole range. By varying the detector position the length range can be extended to either side, and spatial resolution can be improved at the expense of length range.
A position‐sensitive dual‐channel light diffracometer has been developed to allow the real‐time monitoring of light diffraction patterns produced by the myofibrillar sarcomeres of single skeletal muscle fibers and small bundles of fibers during contraction. The diffractometer utilizes two linear image devices to permit the analysis of two or more diffraction lines. The sensors are highly sensitive (1.67×10<sup>-4</sup> lux sec), lag‐free, self‐scanning 256‐element charge‐coupled devices driven by Schottky and standard TTL logic circuitry at a data transfer rate/element of about 20–400 kHz. Video output from the diffractometer can be displayed directly on an oscilloscope screen or can be connected to an ADC and minicomputer system so that diffraction spectra can be corrected for dark signal and element nonuniformities and displayed on‐line by the computer CRT. The computer‐evaluated spacing between diffraction lines provides an accurate measure of mean sarcomere length within the muscle, while line shape and width are indicative of the length distribution among sarcomeres. Sarcomere length resolution attainable with the instrument ranges from about 10 to 100 Å at a nominal length of 2.5 μ, depending on muscle‐to‐sensor spacing and whether one or two channels of the instrument are operated.
1. The diffraction spectrum obtained by illuminating a cross-striated muscle fibre with a narrow beam of monochromatic light generally shows asymmetry of light intensity between left and right spectral lines. 2. The asymmetry of light intensity in left and right spectral lines is reversed when the fibre is rotated by 180 degrees. This indicates that intensities of the spectral lines are determined more by Bragg reflexion than by simple diffraction as in a plane grating. Populations of myofibrils with differing tilt of lattice planes must exist in each illuminated fibre segment to account for simultaneous Bragg reflexion to the left- and right-hand sides. 3. The light intensity of spectral lines shows a complicated dependence on the angle at which the incident beam is directed against the fibre. This 'intensity distribution' seems to reflect the specific myofibrillar arrangement at the site of illumination. 4. The intensity distributions of the left and right first order lines show close correlation if the deflexion of the incident beam differs by twice the Bragg angle. 5. The deflexion angle of the incident beam at which maximum intensity in spectral lines is obtained depends on sarcomere length as predicted by Bragg's formula. 6. Intensities of the first and second order lines are little correlated when recorded at the same beam deflexion, but are well correlated when recorded at beam deflexions calculated from Bragg's formula. 7. When a diffraction line is scanned perpendicularly to the meridional plane, the light intensity shows fluctuations resembling those obtained by beam deflexion within the meridional plane.
Light diffraction patterns from isolated frog semitendinosus muscle fibers were examined. When transilluminated by laser light, the muscle striations produce a diffraction pattern consisting of a series of lines that are projected as points onto an optical detector by a lens system. Diffraction data may be sequentially stored every 18 ms for later processing by digital computer systems. First- and second-order diffraction line intensities were examined from intact, chemically skinned, and glycerinated single fibers. The diffraction line intensities demonstrated a strong length dependence upon passive stretch from reference length to 3.6 micrometer. The first-order intensity linearly increased an average of 15-fold over the range examined. The magnitude of the second order intensity was less than the first order and showed an exponential rise with increasing length. Both first- and second-order intensities decreased upon muscle activation. Data from chemically skinned and glycerinated single fibers were not significantly different from intact fibers, indicating that the membrane structure has little effect upon the diffraction phenomenon in muscle. Theoretical model systems are examined in an attempt to find the basis of these results. Neither an analysis based on a diffraction grating with variable spacing nor the unit cell model of Fujime provides an explanation for the observed length dependency of intensity. Though the origin of the intensity decrease upon stimulation is not known, we have suggested that it could result from lateral misalignment of myofibrils and can occur upon activation.
DURING the development of a high resolution light diffractometer to study sarcomere dynamics, we have observed an unexpected feature of the pattern of sarcomere shortening. We report here that sarcomere shortening during contraction seems to occur in a series of successive bursts punctuated by short but well defined periods during which motion has virtually ceased. Furthermore, it seems that this stepwise pattern of shortening may occur synchronously over many contiguous sarcomeres.
The length dependence of the laser light diffraction pattern produced by R. pipiens whole sartorius muscle has been examined at rest and during tetanic contraction. The muscle diffraction pattern was scanned by a vidicon camera; camera output was digitized and processed by an on-line digital computer to allow a real line display of a section through the diffraction pattern. Analysis of the first order diffraction line profiles yielded values for line amplitude, intensity, center of gravity, line width and percent dispersion. The calculated dispersion provided a measure of the muscle's sarcomere length distribution. First order line amplitudes and intensities were observed to increase, then decrease, with progressive stretch from 1.0 to 1.3 reference length. The percent dispersion among sarcomeres in resting muscle averaged 11% and was approximately proportional (coefficient = .0341) to length. The amplitude and intensity of zero and first order diffraction lines decreased during tetanus, while the line widths and dispersions increased. First order line intesity during tetanus was maximum at about the same length as during rest, i.e., at approximately 2.5 mu sarcomere length. Sarcomere dispersion increased by about 6% during tetanus.
Light diffraction patterns produced by single skeletal muscle fibers and small fiber bundles of Rana pipiens semitendinosus have been examined at rest and during tetanic contraction. The muscle diffraction patterns were recorded with a vidicon camera interfaced to a minicomputer. Digitized video output was analyzed on-line to determine mean sarcomere length, line intensity, and the distribution of sarcomere lengths. The occurrence of first-order line intensity and peak amplitude maxima at approximately 3.0 mum is interpreted in terms of simple scattering theory. Measurements made along the length of a singel fiber reveal small variations in calculated mean sarcomere length (SD about 1.2%) and its percent dispersion (2.1% +/- 0.8%). Dispersion in small multifiber preparations increases approximately linearly with fiber number (about 0.2% per fiber) to a maximum of 8-10% in large bundles. Dispersion measurements based upon diffraction line analysis are comparable to SDs calculated from length distribution histograms obtained by light micrography of the fiber. First-order line intensity decreases by about 40% during tetanus; larger multifibered bundles exhibit substantial increases in sarcomere dispersion during contraction, but single fibers show no appreciable dispersion change. These results suggest the occurrence of asynchronous static or dynamic axial disordering of thick filaments, with a persistence in long range order of sarcomere spacing during contraction in single fibers.