Radiation Detectors

Over the last 30 years or so the detection of radioactivity on TLC plates has taken dramatic leaps forward. Prior to the introduction of radiation detectors, the classical method used for the detection and quantification of radioactivity on a plate involved firstly exposure to X-ray film. This could take from a few hours up to 1-2 months and this technique only located the radioactivity. The second step after location was quantification which was achieved by removing the zone of interest, either by scraping off the silica gel or by cutting away if the plates were aluminium or plastic, followed by liquid scintillation counting. Such a procedure is extremely labour-intensive and is limited in terms of accuracy and resolution (see above).

The first radiation detectors were called radioscanners and these were developed and introduced in the early 1960s. This was a major step forward in the automatic detection and subsequent quantification of radioactive components on TLC plates. The sensitivity and resolution of the instruments were not very high but peaks could be detected and their relative amounts subsequently quantified. At around the same time, spark chambers were also developed for use with TLC plates. Although, these detectors could locate individual components on TLC plates, quantification was not possible.

Another major step forward for radio-TLC came in the early 1980s when the so-called linear analyser was introduced. This instrument was easier to use and more sensitive than the old scanners and was automated to the extent that up to four plates could be run overnight. As a consequence, improved quantitative results were obtained and analysis time was shortened. However, resolution was still not as good as that obtained by using autoradiography and two-dimensional plates could not easily be evaluated.

Currently there are a number of instruments available which have equal resolution to that obtained with autoradiography or are at least approaching it. These instruments include those using the new phosphor imaging technology, the multi-wire system, or the multi-detector system (micro-channel array detector).

The basic functioning of all these detectors is outlined below and a comparison of the advantages and disadvantages of each detector is given later.

Spark chambers The spark chamber is an easy-to-use, low cost technique for photographically locating areas of radioactivity on TLC plates. Exposure times are relatively short and the images obtained on Polaroid film can be quickly transferred back to the original chromatogram using an inbuilt episcope print projector. This means that the areas of radioactivity can then be removed for efficient counting using a liquid scintillation counter. The spark chamber can also be used for the rapid qualitative screening of plant and tissue sections to assess the degree of uptake.

Reviews of spark chambers and their uses have been published previously. Essentially, the spark chamber consists of electrodes contained in a chamber filled with a mixture of argon containing 10% methane, and this gives a high sensitivity to ^-radi-ation. The gas is ionized by radioactive emissions and these emissions are recorded on film with a camera. The Polaroid film integrates the individual flashes produced over a suitable exposure period. Due to the intensity of the sparks the film is rapidly saturated, leading to blackening of the film, and hence direct quantification is not possible.

Radioscanners These instruments were developed and first sold commercially in the early 1960s and utilize a mechanically driven windowless gas-flow Geiger counter. These counters have an interchangeable aperture plate (collimator slit) which controls the size of the area being measured. The TLC plate is scanned by the moving detector head and the signal obtained from the radioactivity source is amplified and recorded. The resultant chromatogram can then be printed on a suitable recorder or integrator-plotter. When the speed of the scanner and recorder are synchronized the exact location of the radioactivity on the TLC plate can be obtained by aligning the chromatogram with the TLC plate. Some manufacturers continue to produce radioscanners but, due to the increasing number of new detection systems (described below) which have better sensitivity and resolution, the number of radioscanners available for quantitative TLC has decreased.

Linear analysers The introduction of the linear analyser provided a great boost for the users of radio-TLC since these detectors brought with them not only improved sensitivity and resolution but also much-improved automation. For example, up to four plates, each with several tracks, can be measured overnight and the chromatograms and accompanying quantitative tables automatically printed out. For the first time in this field the resultant data can be stored and reprocessed at a later date. With the development of new desktop publishing software, the chromato-grams and quantitative results can be directly transferred into reports or publications.

The linear analysers currently used are based on imaging counters developed for high energy physics and medical imaging in the late 1960s and early 1970s. Essentially, the detector head moves automatically to any track on the TLC plate. Once in position the head is gently lowered on to the surface of this track of the TLC plate and the instrument is then ready to begin measurement. At this point the detector has formed a counting chamber since the TLC plate itself has closed the opening of the detector, making the counting chamber gastight. Immediately the detector is resting on the plate the flow of counting gas (argon/methane) is automatically activated and within a few seconds the counting chamber is purged of air and filled with the counting gas.

There are two kinds of systems available today which function in a similar way: each utilizes a different design to locate the exact position of the radioactivity on the plate. One system uses the resistive anode technique and a schematic diagram of this detector is shown in Figure 1. High voltage is applied to a 25 cm anode wire fixed along the length of a windowless detector (1cm wide) and positioned directly above the TLC plate. This wire is constructed of carbon-coated quartz and has a high electrical resistance. When a radioactive emission enters the detector, the gas is ionized and electrons are produced along the particle track. The free electrons are accelerated towards the anode wire by the electric field produced by the high voltage. The electrons continue to ionize more gas as they approach the wire, and the

Figure 1 Schematic diagram of a linear analyser detector with a resistive anode wire. (Reproduced with permission from Clark and Klein, 1996.)

resulting number of electrons becomes large enough to be detected electronically. The pulse of electrons is collected on the anode wire near the position of the initial ionization. The charge divides in the wire, and pulses appear in the amplifiers located at both ends of the wire. The amplitude of the pulse measured by each amplifier is proportional to the resistance between that end of the wire and the position where the electrons were collected. The ratio of these two pulses is linearly related to the original position of the event on the wire. The position of each event is calculated and stored in a computer memory to provide a digital image of the distribution of radioactivity on the plate.

The second type of detection system uses the delay wire technique; a schematic diagram of this detector is shown in Figure 2.

The ^-radiation (fast electrons) emitted from the radioactive source on the plate ionizes the counting gas which has been specifically chosen so that this process can freely take place. This is the primary mode of ionization and the resultant charged particles, free electrons and positive ions, are then accelerated towards the anode wire and cathode, respectively. In this primary mode of ionization the free electrons are accelerated to such an extent that they themselves cause ionization of the counting gas, producing further free electrons and ions and this is the secondary ionization mode. This continues, causing an avalanche of ions from the primary point of ionization towards the anode wire.

Concurrently, the positive ions produced move relatively slowly towards the cathode. These positive

Figure 2 Schematic diagram of a linear analyser detector utilizing a delay wire technique. TA, Time-to-amplitude; ADC, analog-to-digital converter. (Reproduced with permission from Clark and Klein, 1996.)

ions sometimes combine with electrons, producing ultraviolet radiation of sufficient energy to cause further ionizations in a process known as the photoelectric effect. Once sufficient ionization has taken place, a spark is produced, which gives rise to a pulse in both the anode and cathode. The amplitude of the pulse is proportional to the number of ions produced and hence this type of detector is generally called a proportional counter.

The above is a description of the principle of detection. The location of the source of the ionizations is obtained by making use of a delay wire. The delay wire is a very thin wire which is wound over the cathode and pulses pass along this wire in both directions. The pulses are detected by amplifiers at each end of the wire. The arrival of a pulse at one end starts the time-to-amplitude (TA) circuit, while the other pulse is delayed and provides a stop signal in the circuit. The difference between the time of arrival at the two ends of the wire can thus be measured and is proportional to the position of the initial ionization. An analog-to-digital converter (ADC) converts the TA signal to a digital position value that is processed by the data system.

Using this method of detection, the whole of the delay line remains active and thereby the entire length of the chromatogram can be measured at the same time. Once one track of a TLC plate has been measured according to the pre-set time, it automatically moves to the next and the measuring process is repeated.

Radioanalytic imaging system (Ambis) When this instrument was introduced in about 1988, a description of its functioning was reported. The Ambis 4000 directly detects ^-particles from a wide variety of isotopes and is suitable for gels, blots, TLC plates and any sample type of the dimensions 20 x 20 cm. It is reported in the company literature that this instrument can be 100 times faster than X-ray film.

The detector consists of 3696 individual detector elements (each giving a data point) configured in a hexagonal array. Image quality is improved by increasing the number of data points and this is achieved by moving the sample through 72, 144 or 288 discrete positions. Therefore, counts are recorded in 266 112, 532 224 or 1 064 448 data points (i.e. 3696 x number of discrete positions) from which an image is obtained. This image can then be displayed on a monitor and the areas of radioactivity quantified. A background detector which operates concurrently and in a similar way is located above the main detector, and compensates for background radiation.

Different resolution plates, which have different size and shape apertures, can be inserted into the instrument and these plates control the resolution and efficiency (i.e. sensitivity) of the instrument. In general, this means that, using the correct aperture, the detector can be tuned to obtain maximum resolution (at the expense of sensitivity). Conversely, when the instrument is tuned for maximum sensitivity, this is at the expense of resolution. Therefore, aperture choice is governed by sample size and the number and resolution of components required within the sample.

Multiwire Proportional Counters (MWPC)

Digital Autoradiograph (Berthold) This two-dimensional detector is reported to be 100 times more sensitive than the linear analyser and measures all areas of radiation from a 20 x 20 cm surface simultaneously.

The radio-TLC plate is placed on the measuring table and is then automatically loaded into the detector, which also controls the flow of the P-10 counting gas (90% argon + 10% methane). The detector is principally a two-dimensional position-sensitive MWPC. Essentially, it consists of three parallel wire planes, X, Y and Z, each with 100 wires. The spacing between the planes and the wires is only a few millimetres. The central plane (Z) is maintained at a positive potential of 1800 V and the counting chamber is filled with P-10 gas. Charged pulses are generated on the Z plane wires by ionizing particles (^-particles). The orthogonally crossed wire planes X and Y, below and above Z, pick up the charge signals from the Z plane at their position of origin, hence the position of the radioactivity on the TLC plate can be located.

The signals from the wire planes are transmitted via preamplifiers, pulse shapers, discriminators and logic circuits to ADC which are finally coupled to a data acquisition system.

Instantlmager (Canberra Packard) This microchannel array detector provides direct electronic detection and real-time imaging of radioactivity on flat surfaces such as gels, blots, tissue slices and, of course, TLC plates. The detector consists of an array of 210 420 so-called microchannels (diameter 400 |im) in a 20 x 24 cm multilayer plate. The microchannel array plate is a laminated surface about 3 mm thick with alternating conductive and nonconductive materials. A voltage step gradient is applied to the successive conductive layers to create a high electric field (approximately 600 volts mm-1) in the microchannels. The fi-particle emitted from the radioactive source ionizes a gas (argon with small amounts of carbon dioxide and iso-butane) in one of the microchannels. The electrons produced are accelerated by the high electric field in the microchannel, further ionizing the gas, resulting in a cloud of electrons. In this way the microchannels serve as both collimators and preamplifiers.

The cloud of electrons migrates up an electric field gradient into a multiwire chamber located on top of the multilayer. This chamber consists of an anode plane of thin anode wires and two cathode planes (X and Y), as described above for the Digital Autoradiograph. Further avalanche amplification occurs, resulting in electric pulses in the X and Y cathode tracks. The resultant signals are digitized and then decoded to identify the microchannel in which the primary ionization took place, hence locating the position of the radioactive emission. A schematic representation of the microchannel detector is shown in Figure 3.

BioImaging/phosphor imaging analysers The phosphor imagers make use of an imaging plate which is a two-dimensional sensor formed by a layer of fine crystals of photostimulable phosphor (BaFBr : Eu2 + ). The emitted fi-energy is stored upon exposure. In the reading unit the imaging plate is scanned with a laser beam. The energy of the laser stimulates the stored

Figure 3 Schematic diagram of the microchannels of the In-stantImager. (Courtesy of David Englert, Canberra Packard, Meriden, CT, USA.)

Figure 4 Schematic diagram of the principle of detection of the PhosphoImaging analyser. (Courtesy of Fuji Photo Film Co. Ltd, Tokyo, Japan.)

Figure 3 Schematic diagram of the microchannels of the In-stantImager. (Courtesy of David Englert, Canberra Packard, Meriden, CT, USA.)

Figure 4 Schematic diagram of the principle of detection of the PhosphoImaging analyser. (Courtesy of Fuji Photo Film Co. Ltd, Tokyo, Japan.)

electrons to return to the ground state and to emit luminescence in proportion to the recorded radiation intensity. This luminescence is collected in a photo-multiplier tube and converted into an electrical signal. A schematic diagram of the principle of detection is shown in Figure 4.

Data recording and analysis are carried out at a workstation. After reading, the image data on the imaging plate can be erased by exposure to incandescent light and thus the plate can be reused. Imaging plates for the normal weak fi-emitters are available and a specially designed plate for tritium is available. An illustration of the whole imaging process is given in Figure 5.

A prerequisite for good results is to expose the plates in a lead shielding box, particularly those that require longer than 1-2 h exposure time. In this way the contribution of natural background radiation is reduced.

Over the last few years there has been a significant expansion in the variety of instruments available and in the type of imaging plates on offer. Instrumentation has been improved and targeted as far as applications are concerned. For instance, Fuji now has six Phosphor Imaging plate scanners (BAS 1000, 1500, 2000, 2500, 5000 and the new 1800) and Canberra Packard has brought out the Cyclone. Fuji also now offers the FLA 2000 which combines fluorescent and radioisotope detection. The major improvement in the BAS range of instruments has been in resolution, whereby the BAS 5000 can now operate with a resolution of 25 |im, although when used at this high resolution the storage memory required for each scan is extremely high.

Figure 5 Illustration of the phosphoimaging process. IP, Imaging plate. (Courtesy of Fuji Photo Film Co. Ltd, Tokyo, Japan.)

A further instrument using similar technology, recently introduced by Packard, is the Cycloneâ„¢. In this instrument, a solid-state diode laser and confocal optical system moves down the storage phosphor screen as the screen rotates on a carousel. In this process the laser excitation and light collection optics remain in a fixed position relative to the screen surface, so that laser bleed associated with other detectors is eliminated. Furthermore, light collection is increased compared to that obtained with fibreoptic bundles.

A range of imaging plates is now available and these should be chosen according to instrument and requirement. Currently, Fuji is the leading supplier and offers the BAS III, MP, SR, TR and ND imaging plates: care must be taken when selecting a plate because not all plates can be used with all instruments. A range of cassette sizes is also available from Fuji according to plate size.

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