B. Vojnovic

A charged-particle microbeam: I. Development of an experimental system for targeting cells individually with counted particles

Charged-particle microbeams provide a unique opportunity to control precisely, the dose to individual cells and the localization of dose within the cell. The Gray Laboratory is now routinely operating a charged-particle microbeam capable of delivering targeted and counted particles to individual cells, at a dose-rate sufficient to permit a number of single-cell assays of radiation damage to be implemented. By this means, it is possible to study a number of important radiobiological processes in ways that cannot be achieved using conventional methods. This report describes the rationale, development and current capabilities of the Gray Laboratory microbeam.

A charged-particle microbeam: II. A single-particle micro-collimation and detection system.

The use of a charged-particle microbeam provides a unique opportunity to control precisely, the number of particles traversing individual cells and the localization of dose within the cell. The accuracy of aiming and of delivering a precise number of particles crucially depends on the design and implementation of the collimation and detection system. This report describes the methods available for collimating and detecting energetic particles in the context of a radiobiological microbeam. The arrangement developed at the Gray Laboratory uses either a V-groove or a thick-walled glass capillary to achieve 2-5 microns spatial resolution. The particle detection system uses an 18 microns thick transmission scintillator and photomultiplier tube to detect particles with > 99% efficiency.

The irradiation of V79 mammalian cells by protons with energies below 2 MeV. Part I: Experimental arrangement and measurements of cell survival.

The relative biological effectiveness (RBE) has been determined for protons with mean energies of 1.9, 1.15 and 0.76 MeV, from measurements of the survival of V79 Chinese hamster cells. The cells are supported as a monolayer and are swept through a beam of scattered protons produced using a 4 MeV Van de Graaff accelerator. An estimation of the dose and unrestricted linear energy transfer (LET) variation within the sensitive volume of the cells is given for the three proton energies. The RBEs for cell survival (relative to 250 kVp X-rays) at the 10 per cent survival level are 1.6, 1.9 and 3.36 for protons with track-average LETs of 17, 24 and 32 keV microns-1 respectively, and the data suggest that protons are most effective at about 40-50 keV microns-1. It is shown that the proton RBEs can be reconciled with those of other light ions if plotted against z*2/beta 2 (where z* is the effective charge and beta is the relative velocity) rather than against LET.

The effects of counter-ion condensation and co-ion depletion upon the rates of chemical repair of poly(U)radicals by thiols

Bimolecular rate constants for reactions of poly(U) radicals with a series of thiols of varying net charge (Z) were measured by pulse radiolysis with conductivity detection at low ionic strength. At pH 7 and 18 degrees C the values of k2 (M-1s-1) were: reduced glutathione (Z = -1), less than 500; 2-mercaptoethanesulphonic acid (Z = -1), 1.5 x 10(3); 2-mercaptoethanol (Z = 0), 1.8 x 10(5); cysteine (Z = 0), 2.0 x 10(5); cysteamine (Z = +1), 4.1 x 10(7). Values determined at pH 4 were: 2-mercaptoethanol, 6.1 x 10(5); cysteamine 2.2 x 10(8); N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065, Z = +2), 4.6 x 10(8). The variation in rate with structure could not reasonably be attributed to inherent reactivity differences in the thiols and was ascribed to inhomogeneous distributions of the thiols in solution resulting from electrostatic interactions. Thus, cationic thiols are concentrated approximately 100-fold near poly(U), relative to neutral thiols, as a consequence of counter-ion condensation, whereas anionic thiols have approximately 100-fold lower concentration near poly(U) than neutral thiols as a result of co-ion depletion. These results show that the ability of a thiol to repair radical sites in a polyanion is dramatically influenced by its net charge as a consequence of the counter-ion condensation and co-ion depletion phenomena.

An arrangement for irradiating cultured mammalian cells with aluminium characteristic ultrasoft x-rays.

Ultrasoft x-rays are useful for testing the validity of mechanistic models of biological damage caused by radiation. Described here is the construction and operation of a cold-cathode transmission-target discharge tube for irradiating mammalian cells in vitro with aluminium characteristic x-rays (1.487 keV). Particular attention is given to the problems of sample preparation and dosimetry for this shallowly penetrating radiation. The proportion of contaminating bremsstrahlung radiation is measured to establish the optimum operating conditions. Preliminary data from experiments using V79 Chinese hamster cells show that aluminium characteristic x-rays are about twice as effective at inactivating the cells as 250 kVp x-rays.

Action Spectra for Single- and Double-strand Break Induction in Plasmid DNA: Studies Using Synchrotron Radiation

Ionizing radiations deposit a wide range of energies in and around DNA and this leads to a corresponding spectrum of complexity of the lesions induced. The relationships between the amount of energy deposited and the yields and types of damage induced are important in modelling the physical and chemical stages of radiation effect and linking them to biological outcome. To study these relationships experimentally, plasmids were mounted as a monolayer and exposed in vacuum to near-monoenergetic photons from the Daresbury Synchrotron. After irradiation, the DNA was washed off and assayed for single-(ssb) and double-strand breaks (dsb) using agarose gel electrophoresis. Dose-effect relationships for ssb and dsb induction were obtained at various energies in the range 8-25 eV. The initial responses in the low-dose region allowed damage yields to be estimated. However, a common feature is that the responses showed energy-dependent plateaus at higher doses as if a fraction of the DNA were shielded. Various measures were taken both to minimize and to correct for this effect. The data appear to show that the yields of ssb and dsb increase only slowly with photon energies > 10 eV, with a suggestion of similar threshold energies for both lesions. In the energy range covered, the yield of ssb is 12-20-fold greater than that of dsb. The data indicate that ssb and dsb may have a common precursor in this system. Earlier work with low-energy electrons showed that at 25 eV ssb were induced but no dsb were detected.

Status of Charged Particle Microbeams for Radiation Biology

The Gray Cancer Institute is one of a small number of laboratories worldwide routinely using particle microbeam techniques for radiobiological applications. Cellular micro-irradiation methods have been used to provide experimental opportunities not possible with typical broad-field irradiation methods. Using microbeams, it is possible to deliver precise doses of radiation to selected individual cells, or sub-cellular targets in vitro. This technique continues to be applied to the investigation of a number of phenomena currently of great interest to the radiobiological community. In particular, it is the study of so-called non-targeted effects (where cells are seen to respond indirectly to ionizing radiation) that are benefiting most from the use of microbeam approaches. One important non-targeted effect is the bystander-effect where it is observed that unirradiated cells exhibit damage in response to signals transmitted by irradiated neighbours.