- Email: [email protected]
A new gaseous imaging detector for the assay of lymphocyte cultures
I
INSTRUMENTS &METHODS IN PHYSICS RESEARCH
Nuclear Instruments and Methods in Physics R~settreh A310 (1991) 354-358 North.Holland
i
ii
ii
A new gaseous imaging detector for the assay of lymphocyte cultures J.E. B a t e m a n
a n d A. J o y c e Rutlwr/ord Appleton LatnJratory, Chilton. Dich'ot. Oxen O X I I OQX. U K S . C . K n i g h t a n d P. B e d f o r d Climca! Research Centre. Watford Road. Harrow, Midd.x IIAI 3UJ. UK
Tritium-labelled cell cultures used in studies of lymphocyte proliferation at the Clinical Research Centre are blotted in arrays of 10×6 spots spaced at 6 ram. An imaging detector based on the differential induction signals produced at a central amplifying electrode has been developed for the imaging and assay of these blots. A spatial resolution - 2.5 mm FWHM attained over the aperture of 60 mm× 36 mm enables the individual spots to be reliably counted. Data is captured in a PC/AT at rates which permit an assay to be completed in typically 30-60 min. The simplicity of both the detector and the readout electronics leads to a low cost system. Images and assay results are presented.
I. Introduction In studies of the human immune system at the Clinical Research Centre (CRC) a key technique involves the measurement of lymphocyte proliferation by means of the uptake of tritium into the D N A of the multiplying cells from the precursor 3H-thymidine. The cell clones are grown in a miniaturised well system developed at the CRC (20 !sl hanging drop cultures) [1] and blotted onto a matrix in an array of 6 x 10 spots spaced 6 mm apart. Assay of the tritium uptake in each welt is currently performed irk a liquid scintillation counter (LSC), each of the 60 blots being cut out and counted separately. While the LSC is the most efficient counter of tritium activity the high cost of the instrument makes across difficult and the complicated sample preparation reduces the system throughput significantly. Internal counting of tritium in a gas proportional counter is less efficient than the LSC: however, with an imaging gas detector the possibility exists to count all 60 wells simultaneously without any difficult sample preparation so enabling a useful productivity level to be attained. (See ref. [2] for a detailed discussion of the application of gas detectors in this context.) Although several sophisticated (and expensive) imaging gas detectors exist [2], the limited demands of this application in terms of active area, spatial resolution and rate capability led us to consider the use of the imaging pin detector. This very simple device (described in detail in ref. [3]) appeared capable of providing a reliable and economic solution to the problem of tritium assay. As fig. 1 shows, the pin detector is a gas counter in which the amplification takes place on a single central
electrode. The electron cloud released by a tritium beta near the surface of the blot follows the field lines down into the high field region near the charged central
!
\/ i
,i=.
C.ni
\
7
r
I
i,
i
i
IC~
L Fig. 1. Schematic layout of the imaging pin detector in plan and section.
0168-9002/91/$03.50 ~ 1991 - Elsevier Science Publishers B.V. All rights reserved
J.E. ltatemun et ul / A dete~-tor for the g~.;al" o/h'mph~'vte cuhure~
electrode (anode) where amplification takes place. The
proximity of the anode shadows the electrostatic induction pulses picked up on the surrounding pairs of or. thogonal cathodes. The asymmetry of these pulses can be used to calculate the point of origin of the tritium beta in the plane of the sample. The device has two significant advantages for this application, (i) the pin is insensitive to dust contamination (and in any case, easy to clean) which is essential in a counter which will be exposed to the ambient air regularly and (it) the readout electronics required are relatively simple, being required simply to capture four pulse heights for each event and perform two simple sums to arrive at the coordinates of the event, it is thus expected that the device will possess the virtues of robustness, reliability and low cost. The performance of a prototype instrument is described. The readout is controlled by a P C / A T compatible computer under a specially written software package which provides all the facilities required by the user.
2. The basic structure and operation of the pin detector The essential features of the imaging pin detector are illustrated in plan and section in fig. 1. A standard aluminium box is adapted as the gas containment vessel. The anode (pin) electrode consists of spherically tipped circuit board connector (tip radius 0.75 ram) mounted in the coned end of a brass rod. Four copper foil cathode electrodes sample the induction pulse emitted by the positive ion cloud as it retreats from the anode in the aftermath of an avalanche and feeds external amplifiers via coaxial connectors. The tritiumlabelled blot is fixed to the lid of the box directly above the pin and covered by a fine stainless steel mesh (10 cycles/mm) of 50% transparency in order to discharge the positive ion current associated with the avalanches. With a gas atmosphere of argon + 7.5~ methane (flow rate 300 cm3/min) and a pin of 1.5 mm diameter an operating EHT of 3.5 kV is required. The "'production" version of the chamber has some minor refinements not illustrated in fig. I. namely, a proper "'O" ring seal on the box lid with spring-loaded catches to guarantee a good seal and a proper blot holder which can be loaded in seconds with automatic registration of the well positions. When the counter is operating the electron packet from a tritium beta drifts down to cause an anode avalanche of = 5 × 106 electrons delivered in - 2 ItS. Something less than 5% of this signal appears on each of the cathodes with an a~ymmetry in the pulses of _+ 15% (depending on the beta position}. Thus fairly low noise charge amplifiers are required for successful operation.
.~s S
l'hc beta coordinates are ~zmpl~ calculated u,,mg the
algorithms = (right - left }/( rtght + left} and .r = (top - b o t t o m ) / ( t o p + bottom). This leads to a moderate amount of barrel/pin cushion distortion in the image which can be rectified by the usual correction formulae. It is clear from the geometry of the pin electrode that the electric field (and hence the gain) must d~rease as the electron cloud impacts further and further away from the central position. This leads to a requirement to impose pulse height selection on accepted events in order to achieve an approximately uniform response over the active area.
3. The electronic readout system The basic aim of the electronic design is to carry out as many functions as possible in software in the controlling computer. Thus the four cathode signals aft,;r suitable amplification (see fig. 2) are held on standard ana-
i
/,
"-.\.
~
,
L
i ¥1
~YB . IX-BISC 7
i DISC
1
[ It! ~-2~__.I'°
S/HIllO(
t I
,o~o___~i ,,~o i
.
|!
.
I ;
,
D~S-8abe
[
PCleI. Fig. 2. Schematic of the clectronic readout s~stem required to capture data from the imaging pin detector.
V. BIOLOGY/CONDENSED MATTER
356
J,E, Bateman et al. / A deteclor /or the us,~ay of lymphmyte cultures
Iogue sample and hold modules and passed down an analogue bus (screened ribbon cable) to a Metrabyte DAS-8 multiplexed ADC which resides on the extension bus of the P C / A T computer. The timing of this sequence is determined by a zero-cross discriminator triggering on the anode pulse. The low noise preamplifiers are the charge amplifiers developed at RAL for the OPAL lead-glass calorimeter [4]. With the loading of the cathodes and the connecting cables a noise level of 1100 electrons is achieved. The next stage of amplification is achieved using fast video amplifiers (Elantec EL2020) and the peaks of the pulses are held for 1.5 ms on National Semiconductor LF398 sample and hold modules. Video line drivers (EL2003) drive the 2.5 m long analogue bus to the computer. Data capture to the P C / A T is arranged through the Metrabyte DAS-8, an 8-channel high speed A / D converter card. One bit of the 3-bit digital input port is used as a polling flag to initiate data conversion under soltware control. Setting up procedures involve timing the sample and holds and the data capture loops and carefully calibrating the gains and offsets of the cathode channels using a precision pulse generator in conjunction with the pulse height analyser facility provided in the software package. The dead-time correction is made by calibrating the system and applying an appropriate correction factor at the processing stage.
Fig. 3. (a) Typical screen image of a blot assay as performed by .the imaging pin detector system and (b) with the boxes defining the wells for counting purposes.
4. The software system The software package is a large modularised program written in Borland Turbo Basic accessed by a menu system. All the necessary factlities for data capture, display and calibration are provided. Data taking can be initiated by the user by specifying the required run time or the required number of events. A delay of 15 min is incorporated so that the counter gas may be purged before data taking commences. Access to the DAS-8 card is via the Basic-callable routines supplied by the manufacturer. The data (in the form of four pulse heights per event) is stored in a buffer of 64 kb (8k events) which is saved to disc when full. At the end of the exposure the pulse heights are processed (using calibration parameters stored in the program) into an image of 256 × 256 16-bit integers (two arrays must be used because of the 64k block size) and displayed using the gray scale facilities of VGA graphics. Fig. 3a shows the image of a typical blot. This image is saved along with the raw pulse height data and a parameter file containing all the essential exposure details. As fig. 3a shows, the blot images do not lie on a perfectly regular array so that a simple subdivision of the field is not feasible. Accordingly, provision has been
made for the user to set up a file with the blot image centres recorded for future use. A subroutine uses these centre values to integrate the counts in each blot by defining a rectangular well as shown in fig. 3b. The well counts are stored on disc with the rest of the data for evaluation of the blot counting rates. The menu also provides various useful facilities: - All the parameters of the current run (name, total counts, exposure time etc.) may be inspected. - A histogram across a specified row of wells may be produced. - A pulse height spectrum may be generated for the total pulse heights in the x- and y-channels. This is an essential feature for setting up the readout and is also useful for monitoring the counter gain. - The pulse height selection thresholds can also be changed from the menu although this should not be necessary after the initial setting up.
5. The control system In order to ensure safe operation in the laboratory environment a small control system is provided. A microswitch on the lid of the counter automatically
J.E. Baleman el ul. / A detectorfor the ax.~'a.ro[ !rmphoo'te culture~ disconnects the EHT and closes the solenoid gas valve on opening the counter. On closing the counter lid the same switch triggers a 15 rain delay (CMOS 555 timer) which inhibits the application of the EHT for this period to allow the gas to purge the counter. A pressure switch in the gas line detects loss of gas flow and switches off the EHT. When the main power to the unit is switched off the gas supply is isolated. A series of lamps indicates to the user the status of the system (e.g. purging, standby, EHT on, etc.).
6. System performance The prototype system has been operating now for some six months while the final touches were applied to both the hardware and the software. During this period the performance parameters of the system have been evaluated as reported below.
Spatial resolution The spatial resolution of the individual blots lies between 2.5 and 3 mm F W H M (depending slightly on position). This is adequate to resolve the counts from adjacent bins with negligible contamination if the well size is kept to an edge of 14 pixels. The loss of counts in the well from this limitation is at most a few percent.
Sensitivity As discussed in some detail elsewhere [5], the micron range of tritium betas in solids makes external counting very inefficient and very dependent on the distribution of the activity in the substrate, in the case of the pin detector our absolute efficiency lies in the 0.1% region. However, the only practical concern is whether the samples under assay can be counted accurately enough in an acceptable period of time. The large variability observed in nominally identical blots when counted on the LSC (up to o = 20%) implies that it is pointless to accumulate large statistics and so keeps exposure times down. The blot illustrated in fig. 3 accumulated well counts in the range 7-866 in a 30 min exposure (0.2-29 cpm). Since this blot appears to cover a fairly typical range of activities it is anticipated that exposures in the range of 30-60 min will be adequate.
357
sample and the 15 min purge cycle was implemented. Fig. 4 shows the histograms of the accumulated readings. The standard deviations observed are very satisfactory ranging from 12.5.4 for the lowest rate to 7.1"4 for the highest. If one subtracts the Pois,,,on contribution (in quadrature) in each case one obtains a very rough estimate of the detector contribution of 6-10% to the total standard deviation. The main suspected cause for long term drift is the dependence of the avalanche gain on the ambient pressure. Unfortunately ambient pressure was rather stable during the three weeks of the study and it was impossible to quantify any effect accurately. At worst we may have a variation in the observed rates of i-2~. per 10 mbar change in ambient pressure.
Uniformity Careful pulse height selection can minimise the nonuniformity of response caused by the considerable gain variation around the pin. However, the residual effect is quite significant and must be calibrated out of the data. Two blots were prepared at the CRC with nominally identical cell counts in each well. As already noted, the reproducibility of nominally'identical samples is relatively poor so in order to average out this effect the two blots were counted in two orientations rotated by 180 ° giving four independent .samples of each well position in the counter. The four sets of well counts were aggregated and averaged to produce a correction map for the detector. This calibration exercise was reasonably successful in that the standard deviation of the 60 well samples on one of the "'uniform" blots reduced from 30.2% before calibration to 15.8% afterwards. This is not quite down to the 12¢~ accuracy achieved on repetitive measurements of the same well but is adequate for
-/ll >
Background Background counts in the detector set the limiting sensitivity. A 30 rain exposure with no blot present produced a mean count of 1.5 per well (0.05 cpm).
2n
I(| I~t/~StJRF D I ~ T (
StabifiO' Three wells were selected from the blot illustrated in fig. 3 and followed over a three week period during which 54 exposures were taken. Before each exposure the lid was taken off the counter to simulate a change of
~0
I I'PM )
Fig. 4. Histogram of three well counts (mean rates of 5.35 +_ 0.67, 12.11+1.4 and 27.45_+1.94 cpm) accumulated in 54 exposures. The relative standard deviations of 12.5~, 11.5,~ and 7.1~ are to be compared with expected Poisson values of 8.8q~, 5.8'~ and 3.9~. V. BIOLOGY/CONDENSED MATTER
358
J.E. Bateman et al, / A detector for the assay of lymphmTte cultures
the type of experiments for which the device is intended. Clearly this calibration procedure can be iterated using additional "uniform" blots to improve the uniformity of the system further. Practicality and e~,onomy Operation of the pin detector system is very simple. A new blot can be installed in less than a minute. When the lid is sealed the purge timer starts its 15 rain delay automatically. The user switches the EHT on and pushes the reset button. The standby light is now illuminated indicating that the EHT will come on automatically in 15 rain time. The user now selects the data taking option on the computer menu. specifies the exposure length and the file name for the resulting data and walks away from the system. The resulting blot image is presented on the screen at the end of the period represented by the exposure time plus 15 rain. The only consumable of the system is the argonmethane gas mixture. A 10 m3 cylinder provides enough gas for ~ 600 exposures at a cost of about £0.07 per exposure. The simple structure of the pin detector and the relative simplicity of the electronic readout system make it obvious that the equipment costs of this instrument are extremely reasonable compared, for example, with the cost of a liquid scintillation counter.
7. Conclusions By application of the principles of the imaging pin detector we have produced a tritium assay instrument for use in lymphocyte proliferation studies which is simple, robust, reliable and economic to produce and use.
Acknowledgements We gratefully acknowledge the generous support of our colleagues in the development of this system; in particular, at RAL Paul Mackay and at CRC John Baker and his staff in Bioengineering Division and Sharifa lqball.
References [!] S.C. Knight, in: Lymphocytes - a Practical Approach, ed. G.G.B. Klaus (IRL, 1988) p. 189. [2] J.E. Bateman, Electrophoresis I! (1990) 367. [3] J.E. Bateman, Nucl. Instr. and Meth. A240 (1985) 177. [4] R. Stephenson. Report RL-82-082, Rutherford Appleton Laboratory. [5] J.E. Bateman, Nucl. Inslr. and Meth. A241 (1985) 275.
INSTRUMENTS &METHODS IN PHYSICS RESEARCH
Nuclear Instruments and Methods in Physics R~settreh A310 (1991) 354-358 North.Holland
i
ii
ii
A new gaseous imaging detector for the assay of lymphocyte cultures J.E. B a t e m a n
a n d A. J o y c e Rutlwr/ord Appleton LatnJratory, Chilton. Dich'ot. Oxen O X I I OQX. U K S . C . K n i g h t a n d P. B e d f o r d Climca! Research Centre. Watford Road. Harrow, Midd.x IIAI 3UJ. UK
Tritium-labelled cell cultures used in studies of lymphocyte proliferation at the Clinical Research Centre are blotted in arrays of 10×6 spots spaced at 6 ram. An imaging detector based on the differential induction signals produced at a central amplifying electrode has been developed for the imaging and assay of these blots. A spatial resolution - 2.5 mm FWHM attained over the aperture of 60 mm× 36 mm enables the individual spots to be reliably counted. Data is captured in a PC/AT at rates which permit an assay to be completed in typically 30-60 min. The simplicity of both the detector and the readout electronics leads to a low cost system. Images and assay results are presented.
I. Introduction In studies of the human immune system at the Clinical Research Centre (CRC) a key technique involves the measurement of lymphocyte proliferation by means of the uptake of tritium into the D N A of the multiplying cells from the precursor 3H-thymidine. The cell clones are grown in a miniaturised well system developed at the CRC (20 !sl hanging drop cultures) [1] and blotted onto a matrix in an array of 6 x 10 spots spaced 6 mm apart. Assay of the tritium uptake in each welt is currently performed irk a liquid scintillation counter (LSC), each of the 60 blots being cut out and counted separately. While the LSC is the most efficient counter of tritium activity the high cost of the instrument makes across difficult and the complicated sample preparation reduces the system throughput significantly. Internal counting of tritium in a gas proportional counter is less efficient than the LSC: however, with an imaging gas detector the possibility exists to count all 60 wells simultaneously without any difficult sample preparation so enabling a useful productivity level to be attained. (See ref. [2] for a detailed discussion of the application of gas detectors in this context.) Although several sophisticated (and expensive) imaging gas detectors exist [2], the limited demands of this application in terms of active area, spatial resolution and rate capability led us to consider the use of the imaging pin detector. This very simple device (described in detail in ref. [3]) appeared capable of providing a reliable and economic solution to the problem of tritium assay. As fig. 1 shows, the pin detector is a gas counter in which the amplification takes place on a single central
electrode. The electron cloud released by a tritium beta near the surface of the blot follows the field lines down into the high field region near the charged central
!
\/ i
,i=.
C.ni
\
7
r
I
i,
i
i
IC~
L Fig. 1. Schematic layout of the imaging pin detector in plan and section.
0168-9002/91/$03.50 ~ 1991 - Elsevier Science Publishers B.V. All rights reserved
J.E. ltatemun et ul / A dete~-tor for the g~.;al" o/h'mph~'vte cuhure~
electrode (anode) where amplification takes place. The
proximity of the anode shadows the electrostatic induction pulses picked up on the surrounding pairs of or. thogonal cathodes. The asymmetry of these pulses can be used to calculate the point of origin of the tritium beta in the plane of the sample. The device has two significant advantages for this application, (i) the pin is insensitive to dust contamination (and in any case, easy to clean) which is essential in a counter which will be exposed to the ambient air regularly and (it) the readout electronics required are relatively simple, being required simply to capture four pulse heights for each event and perform two simple sums to arrive at the coordinates of the event, it is thus expected that the device will possess the virtues of robustness, reliability and low cost. The performance of a prototype instrument is described. The readout is controlled by a P C / A T compatible computer under a specially written software package which provides all the facilities required by the user.
2. The basic structure and operation of the pin detector The essential features of the imaging pin detector are illustrated in plan and section in fig. 1. A standard aluminium box is adapted as the gas containment vessel. The anode (pin) electrode consists of spherically tipped circuit board connector (tip radius 0.75 ram) mounted in the coned end of a brass rod. Four copper foil cathode electrodes sample the induction pulse emitted by the positive ion cloud as it retreats from the anode in the aftermath of an avalanche and feeds external amplifiers via coaxial connectors. The tritiumlabelled blot is fixed to the lid of the box directly above the pin and covered by a fine stainless steel mesh (10 cycles/mm) of 50% transparency in order to discharge the positive ion current associated with the avalanches. With a gas atmosphere of argon + 7.5~ methane (flow rate 300 cm3/min) and a pin of 1.5 mm diameter an operating EHT of 3.5 kV is required. The "'production" version of the chamber has some minor refinements not illustrated in fig. I. namely, a proper "'O" ring seal on the box lid with spring-loaded catches to guarantee a good seal and a proper blot holder which can be loaded in seconds with automatic registration of the well positions. When the counter is operating the electron packet from a tritium beta drifts down to cause an anode avalanche of = 5 × 106 electrons delivered in - 2 ItS. Something less than 5% of this signal appears on each of the cathodes with an a~ymmetry in the pulses of _+ 15% (depending on the beta position}. Thus fairly low noise charge amplifiers are required for successful operation.
.~s S
l'hc beta coordinates are ~zmpl~ calculated u,,mg the
algorithms = (right - left }/( rtght + left} and .r = (top - b o t t o m ) / ( t o p + bottom). This leads to a moderate amount of barrel/pin cushion distortion in the image which can be rectified by the usual correction formulae. It is clear from the geometry of the pin electrode that the electric field (and hence the gain) must d~rease as the electron cloud impacts further and further away from the central position. This leads to a requirement to impose pulse height selection on accepted events in order to achieve an approximately uniform response over the active area.
3. The electronic readout system The basic aim of the electronic design is to carry out as many functions as possible in software in the controlling computer. Thus the four cathode signals aft,;r suitable amplification (see fig. 2) are held on standard ana-
i
/,
"-.\.
~
,
L
i ¥1
~YB . IX-BISC 7
i DISC
1
[ It! ~-2~__.I'°
S/HIllO(
t I
,o~o___~i ,,~o i
.
|!
.
I ;
,
D~S-8abe
[
PCleI. Fig. 2. Schematic of the clectronic readout s~stem required to capture data from the imaging pin detector.
V. BIOLOGY/CONDENSED MATTER
356
J,E, Bateman et al. / A deteclor /or the us,~ay of lymphmyte cultures
Iogue sample and hold modules and passed down an analogue bus (screened ribbon cable) to a Metrabyte DAS-8 multiplexed ADC which resides on the extension bus of the P C / A T computer. The timing of this sequence is determined by a zero-cross discriminator triggering on the anode pulse. The low noise preamplifiers are the charge amplifiers developed at RAL for the OPAL lead-glass calorimeter [4]. With the loading of the cathodes and the connecting cables a noise level of 1100 electrons is achieved. The next stage of amplification is achieved using fast video amplifiers (Elantec EL2020) and the peaks of the pulses are held for 1.5 ms on National Semiconductor LF398 sample and hold modules. Video line drivers (EL2003) drive the 2.5 m long analogue bus to the computer. Data capture to the P C / A T is arranged through the Metrabyte DAS-8, an 8-channel high speed A / D converter card. One bit of the 3-bit digital input port is used as a polling flag to initiate data conversion under soltware control. Setting up procedures involve timing the sample and holds and the data capture loops and carefully calibrating the gains and offsets of the cathode channels using a precision pulse generator in conjunction with the pulse height analyser facility provided in the software package. The dead-time correction is made by calibrating the system and applying an appropriate correction factor at the processing stage.
Fig. 3. (a) Typical screen image of a blot assay as performed by .the imaging pin detector system and (b) with the boxes defining the wells for counting purposes.
4. The software system The software package is a large modularised program written in Borland Turbo Basic accessed by a menu system. All the necessary factlities for data capture, display and calibration are provided. Data taking can be initiated by the user by specifying the required run time or the required number of events. A delay of 15 min is incorporated so that the counter gas may be purged before data taking commences. Access to the DAS-8 card is via the Basic-callable routines supplied by the manufacturer. The data (in the form of four pulse heights per event) is stored in a buffer of 64 kb (8k events) which is saved to disc when full. At the end of the exposure the pulse heights are processed (using calibration parameters stored in the program) into an image of 256 × 256 16-bit integers (two arrays must be used because of the 64k block size) and displayed using the gray scale facilities of VGA graphics. Fig. 3a shows the image of a typical blot. This image is saved along with the raw pulse height data and a parameter file containing all the essential exposure details. As fig. 3a shows, the blot images do not lie on a perfectly regular array so that a simple subdivision of the field is not feasible. Accordingly, provision has been
made for the user to set up a file with the blot image centres recorded for future use. A subroutine uses these centre values to integrate the counts in each blot by defining a rectangular well as shown in fig. 3b. The well counts are stored on disc with the rest of the data for evaluation of the blot counting rates. The menu also provides various useful facilities: - All the parameters of the current run (name, total counts, exposure time etc.) may be inspected. - A histogram across a specified row of wells may be produced. - A pulse height spectrum may be generated for the total pulse heights in the x- and y-channels. This is an essential feature for setting up the readout and is also useful for monitoring the counter gain. - The pulse height selection thresholds can also be changed from the menu although this should not be necessary after the initial setting up.
5. The control system In order to ensure safe operation in the laboratory environment a small control system is provided. A microswitch on the lid of the counter automatically
J.E. Baleman el ul. / A detectorfor the ax.~'a.ro[ !rmphoo'te culture~ disconnects the EHT and closes the solenoid gas valve on opening the counter. On closing the counter lid the same switch triggers a 15 rain delay (CMOS 555 timer) which inhibits the application of the EHT for this period to allow the gas to purge the counter. A pressure switch in the gas line detects loss of gas flow and switches off the EHT. When the main power to the unit is switched off the gas supply is isolated. A series of lamps indicates to the user the status of the system (e.g. purging, standby, EHT on, etc.).
6. System performance The prototype system has been operating now for some six months while the final touches were applied to both the hardware and the software. During this period the performance parameters of the system have been evaluated as reported below.
Spatial resolution The spatial resolution of the individual blots lies between 2.5 and 3 mm F W H M (depending slightly on position). This is adequate to resolve the counts from adjacent bins with negligible contamination if the well size is kept to an edge of 14 pixels. The loss of counts in the well from this limitation is at most a few percent.
Sensitivity As discussed in some detail elsewhere [5], the micron range of tritium betas in solids makes external counting very inefficient and very dependent on the distribution of the activity in the substrate, in the case of the pin detector our absolute efficiency lies in the 0.1% region. However, the only practical concern is whether the samples under assay can be counted accurately enough in an acceptable period of time. The large variability observed in nominally identical blots when counted on the LSC (up to o = 20%) implies that it is pointless to accumulate large statistics and so keeps exposure times down. The blot illustrated in fig. 3 accumulated well counts in the range 7-866 in a 30 min exposure (0.2-29 cpm). Since this blot appears to cover a fairly typical range of activities it is anticipated that exposures in the range of 30-60 min will be adequate.
357
sample and the 15 min purge cycle was implemented. Fig. 4 shows the histograms of the accumulated readings. The standard deviations observed are very satisfactory ranging from 12.5.4 for the lowest rate to 7.1"4 for the highest. If one subtracts the Pois,,,on contribution (in quadrature) in each case one obtains a very rough estimate of the detector contribution of 6-10% to the total standard deviation. The main suspected cause for long term drift is the dependence of the avalanche gain on the ambient pressure. Unfortunately ambient pressure was rather stable during the three weeks of the study and it was impossible to quantify any effect accurately. At worst we may have a variation in the observed rates of i-2~. per 10 mbar change in ambient pressure.
Uniformity Careful pulse height selection can minimise the nonuniformity of response caused by the considerable gain variation around the pin. However, the residual effect is quite significant and must be calibrated out of the data. Two blots were prepared at the CRC with nominally identical cell counts in each well. As already noted, the reproducibility of nominally'identical samples is relatively poor so in order to average out this effect the two blots were counted in two orientations rotated by 180 ° giving four independent .samples of each well position in the counter. The four sets of well counts were aggregated and averaged to produce a correction map for the detector. This calibration exercise was reasonably successful in that the standard deviation of the 60 well samples on one of the "'uniform" blots reduced from 30.2% before calibration to 15.8% afterwards. This is not quite down to the 12¢~ accuracy achieved on repetitive measurements of the same well but is adequate for
-/ll >
Background Background counts in the detector set the limiting sensitivity. A 30 rain exposure with no blot present produced a mean count of 1.5 per well (0.05 cpm).
2n
I(| I~t/~StJRF D I ~ T (
StabifiO' Three wells were selected from the blot illustrated in fig. 3 and followed over a three week period during which 54 exposures were taken. Before each exposure the lid was taken off the counter to simulate a change of
~0
I I'PM )
Fig. 4. Histogram of three well counts (mean rates of 5.35 +_ 0.67, 12.11+1.4 and 27.45_+1.94 cpm) accumulated in 54 exposures. The relative standard deviations of 12.5~, 11.5,~ and 7.1~ are to be compared with expected Poisson values of 8.8q~, 5.8'~ and 3.9~. V. BIOLOGY/CONDENSED MATTER
358
J.E. Bateman et al, / A detector for the assay of lymphmTte cultures
the type of experiments for which the device is intended. Clearly this calibration procedure can be iterated using additional "uniform" blots to improve the uniformity of the system further. Practicality and e~,onomy Operation of the pin detector system is very simple. A new blot can be installed in less than a minute. When the lid is sealed the purge timer starts its 15 rain delay automatically. The user switches the EHT on and pushes the reset button. The standby light is now illuminated indicating that the EHT will come on automatically in 15 rain time. The user now selects the data taking option on the computer menu. specifies the exposure length and the file name for the resulting data and walks away from the system. The resulting blot image is presented on the screen at the end of the period represented by the exposure time plus 15 rain. The only consumable of the system is the argonmethane gas mixture. A 10 m3 cylinder provides enough gas for ~ 600 exposures at a cost of about £0.07 per exposure. The simple structure of the pin detector and the relative simplicity of the electronic readout system make it obvious that the equipment costs of this instrument are extremely reasonable compared, for example, with the cost of a liquid scintillation counter.
7. Conclusions By application of the principles of the imaging pin detector we have produced a tritium assay instrument for use in lymphocyte proliferation studies which is simple, robust, reliable and economic to produce and use.
Acknowledgements We gratefully acknowledge the generous support of our colleagues in the development of this system; in particular, at RAL Paul Mackay and at CRC John Baker and his staff in Bioengineering Division and Sharifa lqball.
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