Optically stimulated luminescence techniques in retrospective dosimetry

Optically stimulated luminescence signals from natural quartz and feldspar are now used routinely in dating geological and archaeological materials. More recently they have also been employed in accident dosimetry, i.e. the retrospective assessment of doses received as a result of a nuclear accident. Since 1990 the exploration of this wide variety of applications has driven an intensive investigation and development programme at Risø, in measurement facilities and techniques. This paper reviews some of the outcomes of this programme, including (i) optimisation of stimulation and emission windows, and detection sensitivity, (ii) experience with various stimulation light sources, including filtered incandescent lamps (420–550 nm) and high intensity light emitting diodes (470 nm) and laser diodes (830–850 nm). We also discuss recently developed high-precision single-aliquot measurement protocols. These offer exciting possibilities in dating and accident dosimetry, and are already leading to new demands on measurement techniques and facilities.     

The use of ESR spectroscopy for the investigation of dosimetric properties of egg shells

Electron spin resonance (ESR) spectroscopy was used to investigate the dosimetric properties of chicken egg shells. The ESR spectra of the irradiated egg shell were found to have an asymmetric absorption characterized by a major resonance at g=2.0019 and a minor resonance at g_||=1.9980. The study was carried out on g=2.0019 signal because of the accuracy of measurements and the possibility of using it as ESR dosimeter. The activation energy (E), frequency factor (k₀) and mean-life (τ) were calculated to be 1.50±0.10 eV, 2×10¹³ s⁻¹ and (4.4±0.4)×10⁴ year respectively. Dose–response was investigated between dose ranges of 1 Gy and 10 kGy for ⁶⁰Co γ-rays. Dose–response was found to be appropriate for dosimetry in the range 3 Gy to 10 kGy. The lower limit of observable doses for egg shell sample was about 3 Gy. The other ESR dosimetric parameters of egg shell samples, fading characteristic, light effect, dose-rate dependence and energy dependence, have also been studied in detail. Apart from its non-tissue equivalence, egg shell has very good dosimetric properties with insignificant fading, light independence, linearity in dose–response (3 Gy–10 kGy), dose-rate independence and independence from energy above 500 keV. It suggests that egg shell may be used as a retrospective γ radiation dosimetry after nuclear accidents or other short accidental radiation events.     

Synthesis of water-soluble, ring-substituted squaraine dyes and their evaluation as fluorescent probes and labels

A series of ring-substituted squaraines absorbing and emitting in the red and NIR spectral region was synthesized and their spectral and photophysical properties (quantum yields, fluorescence lifetimes) and photostabilities were measured and compared to Cy5, a commonly used fluorescent label. The absorption maxima in aqueous media were found to be between 628 and 667 nm and the emission maxima are between 642 and 685 nm. Squaraine dyes exhibit high extinction coefficients (163,000–265,000 M⁻¹ cm⁻¹) and lower quantum yields (2–7%) in aqueous buffer but high quantum yields (up to 45%) and long fluorescence lifetimes (up to 3.3 ns) in presence of BSA. Dicyanomethylene- and thio-substituted squaraines exhibit an additional absorption around 400 nm with extinction coefficients between 21,500 and 44,500 M⁻¹ cm⁻¹. These dyes are excitable not only with red but also with blue diode lasers or light emitting diodes. Due to the favourable spectral and photophysical properties these dyes can be used as fluorescent probes and labels for intensity- and fluorescence lifetime-based biomedical applications.     

Skin Necrosis due to Photodynamic Action of Benzoporphyrin Depends on Circulating Rather than Tissue Drug Levels: Implications for Control of Photodynamic Therapy

In an ideal world, photodynamic therapy (PDT) of abnormal tissue would reliably spare the surrounding normal tissue. Normal tissue responses set the limits for light and drug dosimetry. The threshold fluence for necrosis (TFN) was measured in normal skin following intravenous infusion with a photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA) Verteporfin as a function of drug dose (0.25-2.0 mg/kg), wavelength of irradiation (458 and 690 nm) and time interval (0–5h) between drug administration and irradiation. The BPD-MA levels were measured in plasma and skin tissue to elucidate the relationship between TFN, drug kinetics and biodistribution. The PDT response of normal skin was highly reproducible. The TFN for 458 and 690 nm wavelengths was nearly identical and the estimated quantum efficiency for skin response was equal at these two wavelengths. Skin phototoxicity, quantified in terms of 1/ TFN, closely correlated with the plasma pharmacokinetics rather than the tissue pharmacokinetics and was quadratically dependent on the plasma drug concentration regardless of the administered drug dose or time interval between drug and light exposure. This study strongly suggests that noninvasive measurements of the circulating drug level at the time of light treatment will be important for setting optimal light dosimetry for PDT with liposomal BPD-MA, a vascular photosensitizer.     

Tetra chlorocopper(II) chemistry: delineation of optical, thermal properties

The thermal decomposition paths of pyridinium tetra chlorocopper(II) (A) differs from the anilinium tetra chlorocopper(II) (B). In the case of pyridinium tetra chlorocopper(II) it loses two molecules of pyridinium hydrochloride and CuCl₂ is formed in the temperature range of 140–350 °C. Whereas the anilinium tetra chlorocopper(II) (B) loses one mole of anilinum hydrochoride along with one molecule of aniline. The compound B has a well define structural change taking place in the region of 70–110 °C which is reflected in DSC as well as resistance measurements. The p-methoxy anilinium tetra chlorocopper(II) monohydrate (C) shows solvatochromicity. The absorption maximum of p-methoxy anilinium tetra chlorocopper(II)monohydrate in acetonitrile (460 nm) changes to 570 nm on addition of methanol. The absorption at 570 nm can be switched back to 460 nm by suppression of ligand exchange with addition of hydrochloric acid. Thus, chemically driven optical switching properties can be observed.     

Two-photon excitation of a phytofluor protein

Phytofluors are highly fluorescent proteins in which the chromophore in a phytochrome is replaced with phycoerythrobilin (PEB), the pigment precursor of the cyanobacterial light harvesting protein phycoerythrin. We examined the fluorescence spectra of the N-terminal region of the cyanobacterial phytochrome 1 from cyanobacterium Synechocystis sp. Pcc 6803 bound to PEB. This protein, Cph1(N514)-PEB, displayed a good two-photon cross-section of 20–30 GM for excitation at 792 nm. This phytofluor also exhibits a high fundamental anisotropy at most practical two-photon excitation (2PE) wavelengths from 700 to 900 nm. Identical lifetimes and correlation times with one and 2PE indicates that the phytofluor is not adversely affected by the intensities needed for 2PE. The one-photon absorption extends well beyond the absorption spectrum and even beyond the emission spectrum to 700 nm. The phytofluor thus appears to be a suitable probe for 2PE and/or cellular imaging.     

Poly(ethyleneimine) as reducing and stabilizing agent for the formation of gold nanoparticles in w/o microemulsions

This paper is focused on the use of branched poly(ethyleneimine) (PEI) as reducing as well as stabilizing agent for the formation of gold nanoparticles in different media. The process of nanoparticle formation was investigated, in the absence of any other reducing agents, in microemulsion template phase in comparison to the nucleation process in aqueous polymer solution.

On the one hand, it was shown that the polyelectrolyte can be used for the controlled single-step synthesis and stabilization of gold nanoparticles via a nucleation reaction and particles with an average diameter of 7.1 nm can be produced.

On the other hand, it was demonstrated that the polymer can also act as reducing and stabilizing agent in much more complex systems, i.e. in water-in-oil (w/o) microemulsion droplets. The reverse microemulsion droplets of the quaternary system sodium dodecylsulfate (SDS)/toluene–pentanol (1:1)/water were successfully used for the synthesis of gold nanoparticles. The polymer, incorporated in the droplets, exhibits reducing properties, adsorbs on the surface of the nanoparticles and prevents their aggregation. Consequently, nanoparticles of 8.6 nm can be redispersed after solvent evaporation without a change of their size.

Nevertheless, the polymer acts already as a “template” during the formation of the nanoparticles in water and in microemulsion, so that an additional template effect of the microemulsion is not observed.

The particle formation for both methods is checked by means of UV–vis spectroscopy and the particle size and size distribution are investigated via dynamic light scattering and transmission electron microscopy (TEM).     

Heteroblastic Development and Shade-avoidance in Response to Blue and Red Light Signals in Acacia implexa

Information from blue (400–500 nm) and red (660–730 nm) wavelengths is used by plants to determine proximity of neighbors or actual shading. Plants undergo trait changes in order to out-compete neighbors or accommodate shading. Heteroblasty, the dramatic shift from one leaf type to another during juvenility, can be influenced by the light environment although it is unknown whether cues from blue or red (or both) are driving the developmental process. Seedlings of three populations of Acacia implexa (Mimosaceae) collected from low, medium and high rainfall habitats were grown in a factorial design of high/low blue and red light to determine how light signals affect heteroblasty and patterns of biomass allocation. Low blue light significantly delayed heteroblasty in the low rainfall population and low red light significantly delayed in the low and high rainfall populations. Low blue light increased stem elongation and decreased root biomass whereas low red light induced a strong shade-avoidance response. These results were consistent across populations although the low rainfall population showed greater trait variability in response to red light signals. We conclude that red light conveys a greater information signal than blue light that affects heteroblasty and seedling development in A. implexa .     

Aqueous-ethanol nitro blue tetrazolium solutions for high dose dosimetry

The ions of nitro blue tetrazolium (NBT²⁺) in aqueous ethanol solution are readily reduced radiolytically first to monoformazan (MF⁺) and then to diformazan (DF) having high linear molar extinction coefficients at the absorption maxima of 522 and 570–610 nm. Spectrophotometric analysis then provides potential use in high-dose dosimetry over the dose ranges of 0.1–1 and 1–30 kGy. The solution is stable for the first 24 h after the irradiation.     

Sensitive absorption detection for micro-column liquid chromatography by ultraviolet forward-scattering degenerate four-wave mixing

The performance of forward-scattering degenerate four-wave mixing (F-D4WM) in the mid-ultraviolet (UV) region (351 nm) as a detection technique for micro-column liquid chromatography (μLC) is studied, using nitro-substituted polycyclic aromatic hydrocarbons (NO₂-PAHs) and amino-substituted anthraquinones (AAQs) as test compounds. When using round capillaries, the concentration limits of detection (LOD) for the NO₂-PAHs were 20–70-fold better compared with absorption detection using a U-shaped detector cell. The final result is influenced by photochemical degradation during 351 nm F-D4WM detection. Furthermore, it is demonstrated that the use of recently commercially available square capillaries (i.e., capillaries with both a square cross section and a square bore) instead of round ones is quite suitable for F-D4WM detection. The square capillary (internal dimensions, 75×75 μm²; external dimensions 300×300 μm²) did not cause significant chromatographic band broadening in μLC. The angle of incidence of the laser light should be 56°, thus fulfilling the Brewster condition at the air–quartz boundary. Using this approach for the AAQs, compounds not prone to significant photodegradation under the experimental conditions, a further 4-fold improvement was achieved. As a result, the overall gain in concentration LOD for 2-amino-9,10-anthraquinone (molar absorptivity 4000 M⁻¹ cm⁻¹ at 351 nm) was from 4 to 0.05 μM, and baseline irregulations were reduced.     

A new spectrophotometric readout for the alanine-triphenyl tetrazolium chloride system for high-dose dosimetry

The electron spin resonance (ESR) readout of radical concentration in irradiated L-alanine is well known as a transfer dosimetry system, however, the high cost of the ESR equipment is a serious handicap for large-scale routine application. In this study the reaction between irradiated L-alanine powder and aqueous solution of triphenyl tetrazolium chloride (TTC) was studied. The radical produced by irradiation of alanine reacts during the dissolution with TTC forming pink-colored formazans with absorption maxima around 401 and 487 nm. This readout method can be used in the 2–200 kGy dose range.     

Photodynamic actinometry using microspheres: concept, development and responsivity

Photodynamic therapy (PDT) relies on three main ingredients, oxygen, light and photoactivating compounds, although the PDT response is definitively contingent on the site and level of reactive oxygen species (ROS) generation. This study describes the development of a novel, fluorescent-based actinometer microsphere system as a means of discerning spatially resolved dosimetry of total fluence and ROS production. Providing a high resolution, localized, in situ measurement of fluence and ROS generation is critical for developing in vivo PDT protocols. Alginate-poly-L-lysine-alginate microspheres were produced using ionotropic gelation of sodium alginate droplets, ranging from 80 to 200 microm in diameter, incorporating two dyes, ADS680WS (ADS) and Rhodophyta-phycoerythrin (RPE), attached to the spheres' inside and outside layers, respectively. To test the responsivity and dynamic range of RPE for ROS detection, the production of ROS was initiated either chemically using increasing concentrations of potassium perchromate or photochemically using aluminum tetrasulphonated phthalocyanine. The generation of singlet oxygen was confirmed by phosphorescence at 1270 nm. The resulting photodegradation and decrease in fluorescence of RPE was found to correlate with increased perchromate or PDT treatment fluence, respectively. This effect was independent of pH (6.5-8) and could be inhibited using sodium azide. RPE was not susceptible to photobleaching with light alone (670 nm; 150 Jcm(-2)). ADS, which absorbs light between 600 and 750 nm, showed a direct correlation between radiant exposure (670 nm; 0-100 Jcm(-2)) and diminished fluorescence. Photobleaching was independent of irradiance (10-40 mW cm(-2)). We propose that actinometer microspheres may provide a means for obtaining high spatial resolution information regarding delivered PDT dose within model systems during investigational PDT development and dosimetric information for clinical extracorporeal PDT as in the case of ex vivo bone marrow purging.     

Thermal stability of chemically denatured green fluorescent protein (GFP): A preliminary study

Green fluorescent protein (GFP) is a light emitter in the bioluminescence reaction of the jellyfish Aequorea victoria. The protein consist of 238 amino acids and produces green fluorescent light (λ_max=508 nm), when irradiated with near ultraviolet light. The fluorescence is due to the presence of chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide –Ser⁶⁵–Tyr⁶⁶–Gly⁶⁷–, which buried into β-barrel.

GFP is extremely compact and heat stable molecule. In this work, we present data for the effect of chemical denaturing agent on the thermal stability of GFP. When denaturing agent is applied, global thermal stability and the melting point of the molecule is decreases, that can be monitored with differential scanning calorimetry. The results indicate, that in 1–6 M range of GuHCl the melting temperature is decreasing continuously from 83 to 38 °C. Interesting finding, that the calculated calorimetric enthalpy decreases with GuHCl concentration up to 3 M (5.6–0.2 kJ mol⁻¹), but at 4 M it jumps to 8.4 and at greater concentration it is falling down to 1.1 kJ mol⁻¹. First phenomena, i.e. the decrease of melting point with increasing GuHCl concentration can be easily explained by the effect of the extended chemical denaturation, when less and less amount of heat required to diminish the remaining hydrogen bonds in β-barrel. The surprising increase of calorimetric enthalpy at 4 M concentration of GuHCl could be the consequence of a dimerization or a formation of stable complex between GFP and denaturing agent as well as a precipitation at an extreme GuHCl concentration. We are planning further experiments to elucidate fluorescent consequence of these processes.     

Gamma-ray dosimetry by spectrofluorimetry of phenylacetic acid solution

Aqueous solutions of phenylacetic acid have been evaluated for possible use in γ-ray dosimetry. When aerated aqueous phenylacetic acid solutions are irradiated, photoflourescent species are formed and identified as hydroxyphenylacetic acid. When excited by ultraviolet light at 280 nm, the radiation-induced product shows an emission spectrum with a maximum at 307 nm. The intensity of the emission peak at 307 nm (as well as the area under the peak from 290 to 350 nm) is a linear function of absorbed dose from 0.5 to 25 Gy. This aqueous dosimeter is about ten times more sensitive than that of the conventional ferrous sulfate solution (Fricke) dosimeter. The differences in response at dose rates in the range 0.0055–67 Gy/min are negligible. Conversely, at higher dose rates (170 Gy/min), although the response is linear with dose up to 135 Gy and with proper calibration can be used up to 350 Gy, the photofluorescence signal is somewhat greater than in the lower dose rate range. The estimated random uncertainty limits (1σ) of readings of absorbed dose by the dosimeter are approximately ±2% at a dose of 10 Gy. The fluorescence signal is very much affected by the hydrogen ion concentration of the solution and the intensity of the signal is greatest in the pH range 5–9.5. The radiation chemical yield of the fluorescing species is little influenced by moderate changes in the concentration of phenylacetic acid or by deaeration of the solution. The signal is stable up to at least four weeks, if the solution after irradiation is stored at low temperature (ca. 5°C). However, when stored at room temperature, and in room light, the signal is stable only up to about four days.     

Implementation of flow-through multi-sensors with bead injection spectroscopy: fluorimetric renewable surface biparameter sensor for determination of berillium and aluminum

For the first time a new, sensitive, and simple bead injection spectroscopy–flow injection analysis (BIS–FIA) system with spectrofluorimetric detection is described for the sequential determination of two metals. The sensor is based on the alternate use of two carriers and a commercially available flow cell (Hellma 176-QS). The flow cell is filled by injecting in the flow system 500 μl of a homogeneous bead suspension of an appropriate solid support (Sephadex QAE A-25) previously loaded with the fluorogenic reagent morin (2′,3,4′,5,7-pentahydroxyflavone). A sequential reaction of Al(III) and Be(II) with morin (immobilized on beads) to form their fluorescent complexes is performed on the bead sensing support and their respective fluorescence emission monitored, after doing two successive injections from the mixture solution. Firstly, Al(III) could be determined in the sample using 0.5 M NaCl/HCl, pH 6 as carrier. Then, the carrier solution was changed (0.3 M NaCl/NaOH, pH 12) making possible the elution of Al(III) and the restoration of the baseline, then allowing the reaction of Be(II). At the end of the analysis, beads are automatically discarded from the flow cell, by reversing the flow, and transported out of the system. The analytical signals are measured at an excitation wavelength of 440 nm and an emission wavelength of 520 nm. Using a sample volume of 600 μl, the analytical signal showed a very good linearity in the range 0.1–8 ng ml⁻¹ and 0.1–1 μg ml⁻¹ with detection limits of 0.024 ng ml⁻¹ and 0.010 μg ml⁻¹ for Be(II) and Al(III), respectively. R.S.D.s (%) lower than 5% were obtained for both analytes and the selectivity was improved using EDTA as masking reagent. The sensor was satisfactorily applied to the determination of these metals in waters and simulated alloy samples.     

Radiochromic gel dosemeter for three-dimensional dosimetry

A gel dosemeter in which ionising radiation causes a colour change was produced by modifying an existing Fricke gel system. This allows a more convenient preparation procedure and gives a better quality dosimetric system for three-dimensional (3-D) dose measurements. The role of three active components of the Ferrous sulphate Xylenol orange Gelatin (FXG) gel dosemeter is quantified with special consideration of their effect on system sensitivity and stability. The optimal composition was found to be 0.5 mM ferrous sulphate, 0.1 mM xylenol orange and 25 mM sulphuric acid. The dose response is linear in the range 0.1–30 Gy. The FXG sensitivity, derived from the gradient of the dose response curve, was found to be ΔA=0.084 cm⁻¹ Gy⁻¹, where A is the optical absorption coefficient at a wavelength of 585 nm, with reproducibility and 24 h stability of better than 5%.     

Spectral studies on the interaction of Amido black 10B with DNA in the presence of cetyltrimethylammonium bromide

The interaction of Amido black 10B (AB) with DNA in basic medium was studied in the presence of cetyltrimethylammonium bromide (CTMAB) based on the measurements of resonance light scattering (RLS), UV–vis, CD spectra, and RLS imaging. The interaction has been proved to give a ternary complex of CTMAB–DNA–AB in Britton–Robinson buffer of pH 11.55, which exhibits strong negative Cotton effect at 233.3 nm and 642.8 nm, and strong RLS signals characterized at 469 nm. Experiments showed that the enhanced RLS intensities (ΔI_RLS) against the mixture of AB and CTMAB are proportional to the concentration of fish sperm DNA (fsDNA) and calf thymus DNA (ctDNA), respectively over the range of 0.03–1.0 and 0.05–1.5 μg ml⁻¹, with the limits of determination (3σ) of 7.3 ng ml⁻¹ for fsDNA and 7.0 ng ml⁻¹ for ctDNA.     

Evidence for two light-induced metastable states in Cl3[Ru(NH3)5NO]H2O

Differential Scanning Calorimetry measurements on irradiated Cl₃[Ru(NH₃)₅NO]H₂O reveal the existence of two light-induced long-lived metastable states SI, SII. Irradiation with light in the spectral range 400–500 nm leads to the excitation of SI. For the first time we report experimental evidence for the state SII in this compound, which can be excited by transferring SI into SII with irradiation of light in the spectral range 1000–1200 nm. The excitation and transfer of the metastable states is described and the exponential decays are evaluated according to Arrhenius' law yielding activation energies of E_A(SI)=0.73(3) eV, E_A(SII)=0.66(3) eV and frequency factors of Z(SI)=1 × 10¹² s⁻¹, Z(SII) = 5 × 10¹² s⁻¹.     

Determination of soluble phosphates in water samples using ytterbium(III) and dynamic measurements of light scattering intensity at long wavelength

A selective and fast method has been developed for the determination of phosphates by measuring the formation of ytterbium(III) phosphate through the variation of the light scattering intensity with time. The low solubility of this compound causes an efficient dispersion of the radiation at 490 nm, which is measured at 980 nm using the second-order grating effect. This approach minimizes potential background signals from the sample matrix. The initial rate of the system is automatically obtained in only 0.5 s by stopped-flow mixing technique. The variable optimization study has been carried out using univariate and multivariate methods. The dynamic range of the calibration graph is 0.09–7.9 mmol L⁻¹ (Pearson's correlation coefficient = 0.9999) and the detection limit is 0.03 mmol L⁻¹. The precision of the method, expressed as relative standard deviation, is 2.3%. The study of the potential interference of different inorganic anions showed that arsenate is the main interferent, although it is tolerated in a molar ratio of 5.5. The method has been satisfactorily applied to the determination of soluble phosphates in tap, ground and river water using a previous preconcentration step with a Dowex 1 (1 × 4–400) anionic resin. A 500-fold concentration has been achieved, which has allowed to decrease the detection limit up to 60 mmol L⁻¹. The recovery range is 97.5–102.5%. The results obtained are consistent with those obtained with the standard molybdenum blue method.     

Photometric detection in flow analysis systems using integrated PEDDs

A novel inexpensive optical-sensing technique has been developed for colorimetric flow analysis. This sensing system employs two LEDs whereby one is used as the light source and the other as a light detector. The LED used as light detector is reverse biased with a 5-V supply so that the photocurrent generated by the incident light discharges the capacitance. Direct digital output is provided by a simple timer circuit that measures the time taken for this discharge process from 5 V (logic 1) to 1.7 V (logic 0).

This sensing concept has been applied in flow analysis by constructing an optical flow cell with a pair of LEDs. Calibration of the integrated optical flow cell using a dye resulted in a linear response that obeys the Beer–Lambert law. The flow rate, dynamic range, sensitivity and limits of detection were investigated. The system was also used for pH determination in the range of pH 2.5–6.8 using bromocresol green (BCG). The pK_a of BCG was successfully determined by this technique.