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Measurement of intracellular ATP using luminescence offers an alternative method for the routine determination of viable animal cells in a large number of cultures. Optimal conditions of the new technology are described as is its application in various test systems.
Singlet oxygen ((1)O(2)) is widely believed to be the major cytotoxic agent involved in photodynamic therapy (PDT). We showed recently that measurement of the weak near infrared luminescence of (1)O(2) is possible in cells in vitro and tissues in vivo. Here, we investigated the relationship between the integrated luminescence signal and the in vitro PDT response of AML5 leukemia cells sensitized with aminolevulinic acid-induced protoporphyrin IX (PpIX). Sensitized cell suspensions were irradiated with pulsed 523 nm laser light at average fluence rates of 10, 25, or 50 mWcm(-2) and, (1)O(2) luminescence measurements were made throughout the treatment. Cell survival was measured with either propidium iodide-labeled flow cytometry or colony-forming assay. The PpIX concentration in the cells, the photobleaching, and the pO(2) in the cell suspensions were also monitored. There were large variations in cell survival and (1)O(2) generation in different experiments due to different controlled treatment parameters (fluence and fluence rate) and other uncontrolled factors (PpIX synthesis and oxygenation). However, in all of the cases, cell kill correlated strongly with the cumulative (1)O(2) luminescence and allowed direct estimation of the (1)O(2) per cell required to achieve a specific level of cell kill. This study supports the validity and potential utility of (1)O(2) luminescence measurement as a dosimetric tool for PDT, as well as confirming the likely role of (1)O(2) in porphyrin-based PDT.
To develop an index to measure alveolar macrophage activity, the fluorescent technique for detection of calcium flux was paid special attention. In this study, a parallel luminescence measuring system was remodelled for fluorescence measurement using a 96-well microplate. The fluorescence indicators widely used to measure cytosolic free calcium ion concentration require excitation at ultraviolet (UV) wavelengths. Instruments to produce UV wavelengths are expensive compared to those used to produce visible wavelengths, and UV wavelengths are potentially injurious to cells. To avoid these problems, Fluo 3 (excitation wavelength in the visible range) was used as the fluorescent dye for detecting calcium ions. The parallel luminometer was remodelled successfully for fluorescent measurement as assessed by the results obtained from the measurements of a common fluorescent dye, fluorescein. Concentrations of free calcium ions were measured using Fluo 3 at 37 degrees C to consider the measurement of calcium flux in living cells. Although a linear relationship between concentrations of free calcium ions and fluorescence were observed, a diminution of fluorescence over time was also observed. To measure calcium flux in living cells, further instrumental and experimental improvements are thus needed.
An area located in the Southern Urals was contaminated in 1949-1956 as a result of radioactive waste releases into the Techa river by the Mayak Production Association. The external dose reconstruction of the Techa river dosimetry system (TRDS-2000) for the exposed population is based on an assessment of dose rates in air (DRA) obtained by modeling transport and deposition of radionuclides along the river for the time before 1952 and by gamma dose rate measurements since 1952. The aim of this paper is to contribute to a verification of the TRDS-2000 external dose assessment. Absorbed doses in bricks from a 130-year-old building in the heavily exposed Metlino settlement were measured by a luminescence technique. By the autumn of 1956 the population of Metlino had been evacuated, and then a water reservoir was created at the village location, which led to a change in the radioactive source geometry. Radiation transport calculations for assumed environmental sources before and since 1957 were performed with the MCNP Monte Carlo code. In combination with TRDS-2000 estimates for annual dose rates in air at the shore of the Techa river for the period 1949-1956 and contemporary dose rate in air measurements, absorbed doses in bricks were calculated. These calculations were performed deterministically with best estimates of the modeling parameters and stochastically by propagating uncertainty distributions through the calculation scheme. Assessed doses in bricks were found to be consistent with measured values within the uncertainty bounds, while their best estimates were approximately 15% lower than the luminescence measurements.
The chiral recognition phenomenon observed in enantioselective excited-state energy transfer processes currently requires the use of chiroptical spectroscopic techniques to probe the magnitude and sense of the discriminatory interactions. The use of chiroptical spectroscopic techniques limits the study of chiral recognition to those molecular species with strong absorption or emission dissymmetry factors. This study presents the theoretical and experimental methodology to determine the magnitude of chiral discriminatory interactions with unpolarized, steady-state luminescence measurements. Based on bimolecular luminescence quenching kinetics for a system containing chiral molecules, the Stern-Volmer equation is derived and contains a chiral discriminatory term for a system containing a chiral but racemic luminophore and an enantiomerically resolved quencher species. The utility of this methodology is confirmed by examining the enantioselective excited-state quenching between several Ln(dpa)(3)(3-) complexes (where Ln = Eu(3+), Tb(3+), or Dy(3+) and dpa = pyridine-2,6-dicarboxylate) acting as the energy donor and either racemic or enantiomerically resolved [Co(dach)(3)](3+) (where dach = trans-1R,2R (or 1S,2S)-diaminocyclohexane) acting as the energy acceptor in an aqueous solution. The results of this study confirm the utility of unpolarized, steady-state luminescence measurements as a probe of chiral discriminatory behavior.
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Some applications of chimeric cellular models are presented to study the biological activities of steroid hormones. We have used several chimeric constructs encoding the DNA binding domain of Gal4 yeast protein fused to the hormone binding domain of various steroid receptors (MR, PR, GR and ER). Interactions of these chimeric receptors with a 17-mer DNA sequence, specific for Gal-4, control expression of the firefly luciferase as a reporter gene. Stable transfected cell lines expressing the firefly luciferase under the control of different steroids were established and an efficient and easy sub-cloning was allowed with the help of an imaging system using a single-photon-counting camera. In the cell lines obtained, the bioluminescent response can be easily measured and thus used to measure specific biological activities of steroid agonists or antagonists. We observed that the responses are effector-concentration-dependent and their biological activities will be compared to those of native receptors.
During the last two decades, novel nonclonogenic methods for pretherapeutic chemosensitivity testing have been developed that are likely to overcome major technical limitations of older assays such as low evaluability rates, low degree of standardization and reproducibility, lack of technical robustness, and poor methodological efficacy. Among these, the microplate adenosine triphosphate (ATP)-based tumor chemosensitivity assay (ATP-TCA) has gained particular merits for ex vivo chemosensitivity testing of native nonhematological tumors including cancers of the breast, ovary, gastrointestinal tract, cervix and corpus uteri, and lung; malignant melanomas; gliomas; sarcomas; and mesotheliomas. For this indication, the ATP-TCA can now be considered the best documented and validated technology. This assay, which is now commercially available, provides a highly reproducible, easy-to-handle kit technique; low technical failure rates; and a high methodological efficacy requiring only 1 x 106 tumor cells to test four to six different drugs or combinations. In ovarian and breast carcinomas, the predictive accuracy is > 90%, with a positive predictive value of 85-90% and a negative predictive value near 100%, respectively. In primary ovarian cancers, the ATP-TCA has been found to accurately predict both clinical response and survival. In two prospective clinical trials in patients with heavily pretreated ovarian cancer, chemotherapy individually selected by the ATP-TCA has been found to triple the response rates and nearly double the survival compared to empirically chosen regimens. Consequently, this assay, which is now under phase III evaluation, has successfully been used in new agent development to screen for novel chemotherapy regimens for the treatment of patients with breast and ovarian carcinoma and melanoma, respectively. This chapter highlights the recent preclinical and clinical experience with this promising technology and gives a detailed description of all the technical aspects of the ATP-TCA.
Thermoluminescence (TL) and optically stimulated luminescence (OSL) techniques were applied to quartz grains extracted from various depths in bricks taken from buildings in the village of Dolon', Kazakhstan, to determine the cumulative absorbed dose, DT. The measurements were performed in four laboratories (EU supported Measurement Group). The results obtained using TL and OSL are compared and discussed with reference to known sources of experimental uncertainty and relevant luminescence characteristics that may affect the evaluation of the absorbed dose. The external nature of the irradiation due to gamma rays from artificial radionuclides is verified by the measurement of depth-dose profiles, and these are compared with those obtained in previous studies for bricks from the same region. To produce these profiles, the cumulative dose due to natural background radiation, DBG, was assessed, particularly based on the concentrations of radionuclides of lithogenic origin within the bricks and the surrounding environment. The consistency of these estimates of DBG was assessed using depth-dose data and absorbed dose determinations for bricks collected from shielded locations. The values of cumulative absorbed dose since the onset of fallout, DX, were calculated as the difference between the values of total cumulative dose in bricks since its manufacture, DT, and values of DBG. Furthermore, estimations of the cumulative absorbed doses in air in the reference location near the sampled buildings, RLDX, were obtained using corresponding conversion factors, CRL, estimated on the basis of previous work. In addition to results for samples distributed for the International Intercomparison, reference is made to relevant results from samples that were collected in 1999 from Dolon' village and studied as part of EU-supported research.
The emission spectra of luminescent trivalent europium (Eu3+) and terbium (Tb3+) complexes were measured using a microscope laser Raman spectrometer with a doubled Nd:YAG laser (532 nm) and an Ar laser (488 nm). Excitation at 532 and 488 nm corresponded to wavelengths of the 7F1 --> 5D1 band of Eu3+ and the 7F6 --> 5D4 band of Tb3+, respectively. The Eu3+ and Tb3+ complexes were discriminated by high-resolution emission spectra more distinctly and sensitively than by fluorescence spectrometry, the usual analytical method.
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Resonance energy transfer from tyrosine to tryptophan residues was detected in the phage fd. The magnitude of the transfer efficiency was estimated by both a traditional and an alternative method. The latter involved comparison of the indole acceptor excitation spectrum full-width half-maximum with a set of standard values differing in the amount of absorbance contributed by tyrosine donor. Both methods lead to the same conclusion: essentially all the tyrosine residues of the viral coat are within 0.9 nm of a tryptophan residue. Also, fluorescence lifetime measurements provide additional support for the hypothesis that there are at least two different environments for the coat protein's sole indole side-chain. Little if any DNA phosphorescence was seen, consistent with the nucleic acid bases being stacked in the DNA core.
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New developments in the Risø TL/OSL system includes a software controlled heater fitted underneath the beta source to enable irradiation of samples at an elevated temperature. This allows investigations of competition effects from thermally shallow traps and centres. Significant additional software developments include the facility to vary linearly the stimulation power during stimulation (linearly modulated OSL). The elevated temperature irradiator facility has been further expanded to allow the measurement of radioluminescence (RL) during beta irradiation. This additional facility allows the measurement of TL, OSL and RL in the same software controlled automatic sequence. This paper provides a description of the capabilities of the new combined automatic TL/OSL/RL reader, and illustrates the application of the elevated temperature irradiator facility, and the RL unit. The new measurement facilities have provided preliminary information about possible trap competition during storage, and changes in recombination processes.
We have developed a system to irradiate samples and record radioluminescence (RL), optically stimulated luminescence (OSL), and thermoluminescence (TL) at temperatures ranging from -150 degrees C to 200 degrees C. The system consists of a cryostat, an irradiation/stimulation unit fitted with an X-ray tube (40 kV Moxtek) and a quartz window for optical stimulation, and a detection unit that utilises a photomultiplier tube and an interchangeable filter pack. Experiments have been conducted with quartz and albite (a feldspar). TL and OSL experiments show that several optically sensitive trapping states are stable below -50 degrees C. In addition, an increase in OSL is seen as the OSL stimulation temperature is lowered below -50 degrees C, and an increase in RL is apparent as the temperature is lowered during irradiation. This indicates that not only are optically sensitive low temperature traps present but that luminescence becomes more efficient at low temperatures.
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We have applied the thermoluminescence dosimetry technique to measure the total external dose from all the nuclear explosions in a few locations near the Semipalatinsk nuclear test site in Kazakhstan. The technique was optimized at our laboratory by fundamental study of the method of thermoluminescence dosimetry. The measured values of each sample at 10 mm depth were 248+/-102 mGy (KSD-1), 30+/-76 mGy (2(1-3)), 222+/-63 mGy (2(3-2)), 217+/-55 mGy (2(4-1)). The results are part of an international intercomparison exercise using bricks collected from the areas surrounding the Semipalatinsk nuclear test site.