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Direct Beta Autoradiography using MCP Detectors

Nuclear Instruments and Methods in Physics Research A, 392 (1997) 349-353.

Authors

J.E. Leesa, G.W. Frasera, and D.Dinsdaleb
aX-ray Astronomy Group, Dept. of Physics and Astronomy, University of Leicester, University Road, Leicester LE1 7RH, UK. bMRC Toxicology Unit, Lancaster Road, Leicester LE1 9HN, UK

Abstract

We describe a new form of detector for digital autoradiography which combines high sensitivity and good spatial resolution (<100 microns). The detector is based on proximity registration of betas by radioisotope-free low noise microchannel plates (MCPs) developed for photon counting X-ray astronomy. Low dark count rates (<0.1 cm-2 s-1) are combined with the high (>50%) electron detection efficiency of small pore MCPs for common beta emitting isotopes (3H, 14C, 35S...).

In particular, the MCP detector is highly sensitive to the biologically important but previously difficult to detect low energy (average 6~keV, endpoint 18.6~keV) beta emission from tritium. We report 3H sensitivities and linearity derived from images of a 3H standard, together with images of 3H-Putrescine doped semi-issue sections of rat lung and isolated single cells from rabbit lung. We compare these results with those of previous attemps to digitally image tritium.

Introduction

We have recently investigated the potential of a detector using "low noise" microchannel plates (MCPs) for beta autoradiography, especially in tritium imaging. Existing methods for digital autoradiography have difficulty in detecting the low energy betas (average energy 6~keV) from tritium sources. Efficient imaging of tritium using gas proportional counters, for example, requires the sample to be "built-in" to the chamber since the low energy betas cannot penetrate the counter window [1-3]. Avalanche photodiode arrays have efficiencies [4] limited by their surface dead layer. Other forms of digital autoradiography detectors - image intensifiers [5,6] photostimulable phosphor [7] ("image plates") and fibre-optic coupled CCDs [8] tend to have low 3H efficiency because of the small optical signal developed in the input scintillator layer. Further loss of signal occurs if a fibre optic taper or lens is also required to demagnifiy the sample to match the output sensor. Tritium imaging using the classical medium of X-ray film requires exposures of duration weeks-months.

The present approach [9] utilises the high electron detection efficiency of MCPs [10,11] for the direct imaging of the betas from samples placed in close proximity to the input MCP in vacuo (10-6mbar).

Detectors

Two chevron MCP detectors, one with a large active area (93mm x 93mm) and the other with a smaller active area (30mm diameter) to match sample size and resolution requirements (see below), were used in our investigation of tritium imaging. Each detector uses two Philips [12] low noise glass MCPs, originally developed for photon counting X-ray astronomy, with resistive anode readout [13]. Table 1 describes the MCPs.

The main source of detector background in "standard" lead silicate glass MCPs is from the 40K beta-emission in the potassium, a major glass consituent [14]. Low noise MCPs, however, are manufactured from proprietary potassium and rubidium-free glass. All detector body parts were manufactured from PCTFE [15], rather than such materials as Macor, which itself contains potassium [16]. Dark noise rates for both the present detectors were 0.12 counts cm-2s-1 above a discriminator level of 0.05 times the peak detector gain [13].

The ultimate sensitivity of any beta detector depends, for a given electron detection efficiency, on reducing the background noise to a minimum. As most of the residual low noise detector background is due to 1.46 MeV 40K gamma rays from concrete in the laboratory walls and floor, the detector background can be reduced to cosmic ray levels (0.015 counts cm-2 s-1 [17]) by surrounding the detector with lead shielding [13].

Table 1: Microchannel plate parameters
Parameter  Value
Thickness (L)  1.5mm
Channel diameter (D) 12.5 microns
Channel Pitch (p) 15.0 microns
L:D 120:1
Channel Bias Angle 6°

Results

i) Tritium Standard

A tritium standard source [18] was mounted 0.5mm from the large area MCP input surface. Detector plus standard were then placed under vacuum (<10-6mbar operating pressure). Figure 1 shows an image of the tritium standard accumulated in 20 hours. The standard has 14 7x5mm2 tritium-loaded wax "cells" with logarithmically decreasing activities (originally 466.5-0.0 micro-Ci per gram) of which only 11 are visible above background in figure 1. We have therefore measured the sensitivity of the MCP detector to 3H to be 0.39 micro-Ci per gram in 20 hours.
[Tritium standard]
Figure 1 show an image of Tritium standard. The activity (micro Ci per gram) corrected for the age of the standard is listed for each detected cell. Note that the image intensity (right hand bar) has been displayed on a log10 scale to improve the contrast between the strongest and weakest cells.

The present configuration of our detector, designed for X-ray detection, operates with a potential of -4.5kV on the front plate, effectively repelling a large fraction of the the low energy 3H betas. We would therefore anticipate a factor of two increase in beta count rate were the detector operated with zero potential on the input MCP, and with the readout element at high positive potential.

The spatial resolution was estimated, from the edge reponse function [19] of the brightest cell, to be 400 microns FWHM for the tritium-MCP separation of 0.5mm. However, the intrinsic resolution of the large area detector, as previously determined from X-ray measurements, was ~80 microns [13]. Spatial resolution of detectors using resistive anode readout scales inversely with resistive anode side length. Improved imaging resolution for 3H betas can therefore be achieved by (a) reducing the size of the detector and (b) bringing the sample closer to the surface of the input MCP (below). The elimination of the retarding input electric field would also serve to improve resolution.

Figure 2 shows the measured linearity of the large MCP detector for 3H betas and is derived from the cells of the tritium standard. The dashed horizontal line represents the background noise level. The maximum count rate of the brightest cell corresponds to a count rate per channel of only 7.4x10-5s-1. Fraser et al [20] have shown that MCP detector linearity is preserved up to a count rate of order 0.1 channel-1s-1. Thus, the three orders of linearity demonstrated in figure 2 extends in principle to six orders of dynamic range.

ii) Biological samples

To improve spatial resolution and to achieve a better match to single-organ sample size we used the smaller MCP detector (see "Detectors" above) for imaging 3H labelled biological samples. Rat lung slices which had been labeled with tritiated Putrescine (1,4-diaminobutane) as part of an investigation into the damage of alveolar epithelium [21]. Semi thin tissue sections, 1 micron thick, were embedded, after preprocessing, in Araldite in preparation for conventional contact emulsion autoradiography [21].

The sample mounted on a glass cover slip was placed in direct contact with the MCP input surface. Figure 3(a) shows the image from a lung tissue slice after 76 hours. Comparison with an optical light image (figure 3(b)) obtained using a binocular microscope, shows that we can image the gross features of the sample. The air passages, both perpendicular and in the plane of the sample, where there is no uptake of the radiolabel are clearly visible. Tritium is known to concentrate in certain cell types (< 10 micron in size) [21] which, due to the limitations of the present detector resolution, we could not resolve. We have estimated, from figure 3(a), an upper limit to the image resolution of the small format detector of 100 microns since the edge of the sample, from optical microscope inspection, is only very approximately straight.
[Semi-thin lung tissure slice (rat)] [Optical photograph of the same lung tissure slice]

Figure 3: (a) Semi-thin lung tissure slice (rat) labeled with 3H. (b) Optical photograph of the same lung tissure slice. The bar at the bottom represents the image scale.

The activity of the tissue sample under test was unknown but from the known MCP electron efficiencies (50%), the measured noise subtracted sample count rate (0.059 counts s-1 sample thickness (1 micron) and an assumed sample density of 1g cm-3 we have estimated the sample activity to be 0.52 micro-Ci per gram.

A second sample (isolated macrophage cells from a rabbit lung, not shown) was examined with the small detector, having been labelled, as before, with tritated Putrescine (following the method of Dinsdale et al. [21]). The sample was imaged once, then the detector housing was surrounded by lead (estimated solid angle coverage, 35%) and a second image accumulated. Dark noise measured on the shielded detector was a factor of ~0.3 lower than the unshielded value. Signal-to-noise ratios were 7.64:1 and 10.88:1 for the unshielded and shielded configurations respectively, confirming the improvement in sensitivity predicted above.

Discussion

We now compare the performance of our detector with existing electronic detectors of tritium.

Gas proportional counters can detect tritium betas with essentially 100% efficiency only if the sample is "built-in" into the detector [2,3]. The optical avalanche chamber described by Tribollet et al. [3] exhibits a count rate, for a given activity, a factor of 3.5 higher than our MCP detector in its present (electrically unfavourable) configuration (see Results: Tritium Standard). The MCP detector however, has a much better intrinsic spatial resolution compared to ~500 microns given for the avalanche chamber.

Images plates with specially reduced dead layers can be used for imaging tritium [7]. One manufacturer [22] cites a minimum detectable activity for 3H of 1.67 Bq/mm2/hour which is a factor of ~80 poorer than our measured minimum count rate (0.001 mm-2s-1). Spatial resolution for an image plate is ~150 microns FWHM, comparable to that of the present detector.

A recent paper [4] describes the use of avalanche photodiodes (APDs) for measuring the tritium content of biological specimens. The detection efficiency of such devices are limited by the thickness of the surface dead layer which acts as a barrier to the low energy tritium betas. Gordon et al. reports tritium beta efficiencies of 27% for devices having large 1mm2 pixels.

Our measurements of (a) 35S-labelled DNA electrophoresis gels and (b) 14C-labelled rat whole body tissue slices will be described in a future report.

Acknowledgements

The authors would like to thank Jim Pearson for helpful discussions during the preparation of this manuscript. We also acknowledge the loan of the tritium standard from Mitch Flor-Henry, Biolumonics Ltd, Nottingham, UK. The work of the Leicester X-ray Astronomy Group is supported by the UK PPARC, who provided a Research Associateship for JEL.

References