MULTIWIRE DETECTOR AND DATA ACQUISITION SYSTEM FOR TIME RESOLVED EXPERIMENTS

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MULTIWIRE DETECTOR AND DATA

ACQUISITION SYSTEM FOR TIME RESOLVED EXPERIMENTS

A. Faruqi

To cite this version:

A. Faruqi. MULTIWIRE DETECTOR AND DATA ACQUISITION SYSTEM FOR TIME RE- SOLVED EXPERIMENTS. Journal de Physique Colloques, 1986, 47 (C5), pp.C5-149-C5-156.

�10.1051/jphyscol:1986520�. �jpa-00225837�

JOURNAL DE PHYSIQUE

Colloque C5, supplbment au no 8, Tome 47, aoOt 1986

MULTIWIRE DETECTOR AND DATA ACQUISITION SYSTEM FOR TIME RESOLVED EXPERIMENTS

A.R. FARUQI

MRC Laboratory of Molecular Biology, Hills Road, GB-Cambridge CB2 ZQH, Great-Britain

R6sum6 - Un systsme pour effectuer des mesures cinetiques aux rayons X sur des muscles est decrit, de mzme que le logiciel d'acquisition de donnkes et d'analyse des rksultats. Plusieurs exemples d'application sont donnks.

Abstract

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The technique of time resolved X-ray diffraction is a powerful tool for studying dynamic processes in structural biology where ordered structures are involved, and has proven of particular value in the study of vertebrate skeletal muscle. The principal ideas behind the technique are relatively simple

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The basic contractile muscle unit, the sarcomere, shown in Fig 1, consists of two types of inter-digitating protein filaments 1 8 1 . The 'thick' filaments, consisting mainly of myosin are about 1.6 pm in length and have an array of 'projections' along most of its length arranged in a helical fashion. The thin filaments are about 1 pm in length and consist mainly of actin with smaller quantities of the regulatory proteins tropomyosin and troponin. The thin filaments are attached to the Z-lines, the distance between two Z-lines being approximately 2.3 pm at normal body length. It is believed that during contraction the projections on the thick filament form cross-bridges with the thin filament and go through a

'conformational' change which produces a relative sliding movement between the two filaments. The polarity of the filaments is reversed half-way along the sarcomere so that opposing forces are generated in the two halves. A large number of sarcomeres generate force in series and in parallel which is transmitted to the skeleton through the tendons to allow external work to be performed.

There is a great deal of ordered structure within the contractile proteins in a living muscle which makes it suitable for X-ray diffraction studies. A brief discussion on the X-ray patterns from muscle is given to serve as an introduction to hardware and software requirements for time resolved studies. As an example, a small angle X-ray pattern from resting frog sartorius muscle, recorded on the EMBL

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:1986520

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Fig 1 Schematic daigram showing thick and thin filament organisation within a sarcomere.

MERIDIAN

LAYER Lnvts (SPACINGS

PSD SET FOR LAYER LNE

RECORMNG

Fig 2 Small Angle X-ray pattern from resting frog sartorius muscle.

outstation at Hamburg, is shown in Fig 2 /lo/. The main features of the pattern are a series of layer lines, with a p e r i o d w 4 2 9 h , g i v q the helical repeat of cross-bridges on the thick filaments with a strong meridional reflection at 143h given by the axial repeat of the crossbridges. There is a dramatic reduction in the intensity of the myosin related layer lines during contraction /11/ due to the loss of helical symmetry on the thick filaments as the cross-bridges get attached to binding sites on actin. Considerably weaker layer lines are produced by the helical arrangement of actin in the thin filament at 59A and 51h and according to recent reports 112,141 both the 59& and 51h layer lines get stronger during contraction due to labelling by cross-bridges. In addition, the second actin layer line at 180A gets considerably stronger during contraction, presumably due to the movement of tropomyosin in the actin helical groove; the position of tropomyosin plays a vital role in the 'steric blocking mechanism' which regulates muscle activity.

The side-to-side packing of the thick and thin filaments on a hexagonal lattice results in the equatorial pattern shown in Fig 3. The (1.0) reflection is generated by the thick filaments while the (1,l) reflection is generated by a combination of the thick and thin filaments. Early experiments indicated a dramatic increase in the (1,O) intensity and a decrease in the (Z,I) intensity during isometric contractions.

This was explained as being due to a transfer of mass (in the form of cross-bridges) from the thick to the thin filaments 171. In more recent work, with the availability of more powerful X-ray sources and detectors, the time course of the intensity of these reflections has been investigated with improved time resolution. As described in this paper, one can monitor the sarcomere length changes during isometric twitches and correlate changes in the equatorial pattern with both the generation of tension and sarcomere length.

In order to make time resolved X-ray measurements from muscle one needs a suitable X-ray detector equipped with special electronics and software. The requirements from such a system are briefly summarised below to underline the specific features required for this work /4,9/.

Due to the relatively large .mass of disordered material, there is relatively strong scattering from muscle produking 'unwanted' background, superposed on which is the scattering from the 'ordered' part of the structure producing the signal. The signal-to-background ratio varies between approximately 0.1 for the first myosin layer line at 4298 to % 0.5 for the (1,O) equatorial reflection. Partly due to ti$

high background, the total scattering factor of a typical muscle specimen is %1110 which means that for a synchrotron radiation camera producing a flux of 10 photons/sec, the detector has to be able to cope with overall counting rates of 10 MHz. The counting rates are higher along the equator and the meridian than other parts of the pattern and some rates are given for various interesting parts of the pattern below.

Equatorial Pattern (one-dimensional slice) % 4 MHz (1,O) Equatorial Reflection % 200 KHz

143h Meridional Reflection % 40 KHz

4298 Myosin Layer Line (one quadrant) % 4 KHz The detector most suitable for these measurements should be (i) capable of recording two dimensional X-ray patterns (ii) have a spatial resolution of about 256 x 256 pixels

(iii) be capable of recording data at rates of 10 MHz without serious distortions or inefficiency

(iv) be capable of very fast readout into a memory on a timescale small compared to the smallest time frame required for the experiment (i.e. <<0.5

millisecond). Typically, experiments require 100-250 separate time frames with frame times varying between 0.5 and 10 milliseconds.

There is as yet no single detector which can fulfil all the requirements listed above. We have however, made use of various types of detectors, developed in our laboratory and elsewhere, which though not ideal, allow a number of useful measurements to be made on selected parts of the pattern / 3 , 9 / . We descrIbe briefly the high count rate detector and data acquisition system which has been constructed

JOURNAL DE PHYSIQUE

Channels

Fig 3 Equatorial Pattern from resting frog sartorius muscle.

Multlwlre Ilnear Parolld

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Detector mmorY

Tenston. lsngth

and other

H H

parameters memory

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1

-

CCD Array

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Fig 4 Data Acquisition System.

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in Cambridge to study the equatorial part of the pattern in particular (due to the high counting rate requirement) but is also useful for other parts of the pattern.

Detectors based on similar principles have also been developed at EMBL and LURE 16,151. A considerably simplified outline of the data acquisition system is shown in Fig 4 indicating the main data paths from input sensor to the storage medium. There are essentially three parallel paths of data, the first two carrying X-ray and optical detector readouts, and the third containing physiological information regarding the muscle, etc. The modular nature of the data acquisition system makes it possible to accommodate new types of detectors without a major design effort. For example, we were able to interface the Daresbury Laboratory Multiwire Area Detector, which has its own memory to our system with the addition of a relatively simple interface circuit for use in some measurements 131.

The Multiwire Linear Detector (MWLD) and its readout circuit are mentioned only briefly here as several more detailed reports have been written earlier /1,2/. The optical diffraction part of the system which was added more recently, however, will be described in a little more detail. The MWLD consists of an anode wire plane sandwiched between two solid cathode planes, the plane on the X-ray entrance side being constructed of a 0.4 mm thick sheet of Beryllium. The anode wires are 10 pm in diameter and have a 1 mm pitch (the anode wire plane was constructed in CERN) and act as multiple independent detectors connected to individual amplifiers, discriminators and scalers. The total sensitive depth of the chamber is 10 mms which provides a high detection efficiency for 1.5A X-rays with Xenon mixtures even without applying high pressures.

Data from the scalers is read out by a specially designed module which reads the scalers and stores it in a random access memory. The read-write operation is carried out for the 'previous' frame, with tri-state output latches on the scalers and takes about 400 )IS for the transfer of the complete data. All the main timing parameters used in the experiment are stored in the Timing Controller module prior to the experiment and includes times for frame widths, muscle stimulus, shutter operation, etc. During the experiment most operations are controlled by this module.

The optical diffraction pattern, given by the sarcomere repeats, is recorded on a linear CCD Array (Thomson-CSF, Model TH 7831) consisting of 1728 photodiodes with a centre-to-centre spacing of 13 pm. Associated with each photodiode is a capacitor which stores the charge generated-by incident light, the quantity of charge being proportional to the product of incident intensity and integration time. At both ends of the array there are four 'masked' photo-diodes which provide the reference or

"dark" signal. At the end of an integration period the stored charges are transferred into two CCD analog shift registers, even pixels on one side, and odd pixels on the other. The charges are then transferred through to a sample and hold circuit which is connected to a fast analog to digital converter (TRW, Model 1048 converting 8 bits in 20 ns); the ADC conversion is controlled by an auxilliary clock, whose timing is derived from (and is therefore synchronized with) the main timing clock, as shown in Fig 5. Digitised values, corresponding to the optical diffraction pattern are stored in the random access memory using the Direct Memory Modify mode which sums the new data values with the previously stored values (a fuller account of

this scheme will be published later). A typical optical diffraction pattern with a linear background is shown in Fig 7. Due to the relatively slow speed of access to the memory the rate of ADC conversions are restricted to 0.75 MHz and it takes about 2.3 ms to read out a complete frame. This sets a lower limit of 2.5 ms for the frame times which was considered adequate for the present studies. However, improved time resolution can be obtained with either a faster memory or a diode array with fewer pixels.

The CCD array can be used in two modes: a local mode in which it is scanned continuously with its internal clock and an external mode in which the scan is set by the timing clock. During the quiescent period between muscle cycles, which may be 10-60 secs, the CCD array is switched to 'local' mode to prevent excessive charge building up frnm dark current and residual light in the long integration time. At the start of the cycle, the datum (t=O) pulse switches the CCD array to the external

JOURNAL DE PHYSIQUE

C C D Array

Control

A m p l ~ f ~ e r

Converter Sarnple/Hold

C C D Llnear Array

Diffracted Light Muscle Incident Light

Fig 5 Block diagram of the CCD Readout.

Disk Storage

Fig 6 Optical Diffraction Pattern of the sarcomere repeat showing the first order pattern.

mode and for the duration of the cycle. The first frame pulse transfers the accumulated charge into the analog shift register and the readout initiated which occurs while the following frame is being recorded on the photodiodes and associated capgcitors. To rednce the volume of readout it is possible to select two 'windows' set over the regions of interest on the array with software.

The real time data acquisition software is written in Fortran (RTll), including all interrupt handling, with calls to Camac in Macro for optimum speed. All the relevant timing information, such as frame lengths, stimulus length, etc., is pre-loaded into the Timing Control Module which stays in control of the experiment after the start. During the experimental run various forms of diagnostics are- available for checking data validity, one of the most useful of which is a graphks routine plotting out either two chosen 'X-ray' frames or a CCD frame on the VT640 (equivalent to a lower resolution Tektronix 4010) graphics terminal. It is possible to select a peak on the raw data display during the experiment and plot the time course of the integrated intensity during the cycle. This is very useful, for example, in instances where a significant change in intensity is expected and none found. Similarly, integrating over the complete pattern shows how the total counts are varying over the whole cycle. Accumulated data can be transferred to the Daresbury VAX11/750 central computer via an Ethernet link set up by the Computing Division, Daresbury, for more detailed checks and analysis.

COUNTS

SPACING

MILLISECONDS

Fig 7 Intensity and spacing of the 143h meridional reflection along with the tension generated during an isometric twitch.

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The main data reduction program was written originally to run on a VAX 11/780 in Cambridge, but it can also be used on the Daresbury VAX 11/750, as similar graphics terminals are used in both places/3/. Similar programs have also been written at the EMBL /13/. The main function of the program is to fit backgrounds to the data, in an interactive mode, and obtain several parameters of interest e.g. integrated intensities, peak widths and centroids. Data can be normalised for detector non-uniformities, changes in incident beam intensity and specimen thickness variation during the cycle. Due to the large amount of data a degree of automation has been built into the program. To illustrate the use of the hardware and software Fig 7 shows the time course of the integrated intensity and spacing of the 143A meridional reflection along with the tension generated during an isometric twitch. Considerable awunts of equatorial data have also been collected for various speeds of muscle shortening and are currently being analyzed.

REFERENCES

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