Fluorescence Intensity Levels Research Articles

Liver cell-membrane specific antibodies in autoimmune hepatitis patients detected by flow cytometry

Abstract In this study, we identified autoantibody in patients with autoimmune hepatitis against liver cell membrane using flow cytometry. After incubation of one of the hepatoblastoma cell lines, Hep G2, with serum from a patient, and the addition of FITC-labeled anti-human Ig antibody, anti-membrane antibodies were analyzed by flow cytometry. By our method, the antibody in serum can react only with autoantigens on the cell surface. Furthermore, propidium iodide staining enabled us to exclude the possibility of crossreaction of antibodies against dead cells. The relative fluorescence intensity in 12 patients with autoimmune chronic active hepatitis was significantly higher than that in 20 normal subjects, six primary biliary cirrhosis (PBC), 18 chronic viral hepatitis, and 11 systemic lupus erythematosus (SLE). The normal level of fluorescence intensity provided by sera from the SLE patients indicated that antibody binding to the liver cell membrane was not derived from Fc-mediated immune complex capture. These findings demonstrated that this flow cytometric technique provides a simple and accurate method for the detection of autoantibodies against liver cell membrane.

Antibodies to tubulin and actin bind to the surface of a human monocytic cell line, U937.

Intermittent reports of cytoskeleton proteins (actin and tubulin) on the cell surface have appeared over the last 13 years. Whereas most have concentrated on lymphocytes, this study provides evidence for the presence of these proteins on the surface of a human cultured monocyte-like cell line, U937. Both actin and tubulin were detected on the surface of U937 cells by flow cytometry, using an indirect staining procedure based on biotin-streptavidin-phycoerythrin, chosen for greater sensitivity. By use of this procedure, the majority of viable unstimulated U937 cells stained positively for actin and tubulin, although the level of fluorescence intensity was low. With an antibody specific for tyrosine-tubulin, most of the surface tubulin was also found to be tyrosinylated. For vimentin, an intermediate filament protein abundantly present in the cytoplasm of U937 cells, no staining could be detected. Confirmation of the flow cytometry data for surface actin and tubulin on unstimulated U937 cells was achieved by direct vesualization using a confocal laser scanning microscope. When U937 cells were activated with PMA and LPS, a marked reduction in the level of cell surface actin and tubulin occurred. The role of cell surface actin and tubulin on unstimulated U937 cells, in terms of monocyte function, remains to be elucidated.

Use of fluorescence microscopy in the detection of low level oxidation in bituminous coals

Oxidation has a deleterious effect on the coking properties of bituminous coals. Interaction with the atmosphere markedly alters both the physical and chemical attributes of coal, thus reducing its value. Fluorescence microscopy offers a means by which these effects can be readily detected and quantified. Blue light excitation (450–490 nm) has been used to produce autofluorescence in vitrinite and inertinite macerals. Their fluorescence intensities, measured at 650 nm in both Australian Permian and German Carboniferous coals, were found to decrease with duration of exposure to air. This was observed in coals of different rank but was most pronounced in high volatile bituminous coals. By establishing a profile of fluorescence intensity levels in unoxidized coals over a wide rank range, it is possible using this technique to detect whether even minimal oxidation has occurred. A comparison with various carbonization tests showed that only Gieseler fluidity and dilatation were of similar sensitivity.

Hydrophobic interactions in human casein micelle formation: beta-casein aggregation.

The association of non-phosphorylated (0-P) and fully phosphorylated (5-P) human beta-caseins was studied by fluorescence spectroscopy and laser light scattering. The tryptophan fluorescence intensity (FI) level increased between 20 and 35 degrees C, indicating a change in the environment of that residue. A similar transition occurred when ANS was used as a probe. Transition temperatures were slightly lower in 10 mM-CaCl2 but were not affected by an equivalent increase in ionic strength caused by NaCl. The magnitude of the FI change was less for the 5-P than the 0-P protein but was increased for both by CaCl2 addition. These FI data were characteristic of a conformational change and this was supported by fluorescence polarization which indicated that with CaCl2, tryptophan and ANS mobility increased at the transition temperature even though the extent of protein association also increased. Light scattering suggested that protein association proceeded with the primary formation of submicellar aggregates containing 20-30 monomers which then associated further to form particles of minimum micelle size (12-15 submicelles), and eventually larger. The temperature of precipitation of the 5-P form in the presence of CaCl2 was lower than the conformational transition and suggested that both hydrophobic interactions and Ca bridges between phosphate esters on adjacent molecules are important in micelle formation.

Flow cytometric analysis of oxidative product formation in phytohemagglutinin-stimulated ethanol-treated immune mononuclear cells

Oxidative products formed by immune mononuclear cells were studied by flow cytometry. JURKAT T cells and peripheral blood mononuclear cells were incubated with 2,7-dichlorofluorescin diacetate. This substance was hydrolysed in the cells, leading to a non-fluorescent product which was oxidized into highly fluorescent 2,7-dichlorofluorescein by oxygen reactive species. These latter products were analysed by flow cytometry in phytohemagglutinin (PHA)-stimulated ethanol (ETH)-treated mononuclear cells. The level of fluorescence intensity (FI) was found higher in stimulated cells than in non-stimulated cells. ETH displayed two different effects on the cells: either a decrease of FI associated with a decrease of the number of fluorescent cells (FC) or an increase in FI. Both effects were dose-dependent. ETH is an effective scavenger of .OH radicals, but it is also oxidized by the microsomal ETH oxidizing system with production of oxygen reactive species, which probably explains the opposite effects of ETH. In the presence of desferal, an iron-chelating agent, and nordihydroguaiaretic acid, an inhibitor of the lipooxygenase pathway, the cells showed a decrease of FI and FC. These results suggest that .OH and other oxygen reactive species are involved in stimulation by PHA of ETH-treated immune mononuclear cells.

Cyclooxygenase and lipoxygenase inhibitors act differently on oxidative product formation by immune mononuclear cells: A flow cytometric investigation

The action of indomethacin, a cyclooxygenase inhibitor, and nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, on oxidative products formed by immune mononuclear cells was studied by flow cytometry. Jurkat T-cells and peripheral blood mononuclear cells were incubated with 2′,7′-dichlorofluorescin diacetate: this substance is hydrolyzed in the cells leading to non-fluorescent 2′,7′-dichlorofluorescin which is oxidized by oxygen reactive species into highly fluorescent 2,7′-dichloroflourescein. Using this fluorescent probe, the formation of oxygen reactive species in phytohemagglutinin-stimulated mononuclear cells treated by NDGA or indomethacin was followed by flow cytometry. We observed that NDGA caused a marked decrease, both in the level of fluorescence intensity and in the number of fluorescent cells, whereas indomethacin caused a small increase in fluorescence intensity. On the other hand, NDGA inhibited and indomethacin increased the incorporation of tritiated thymidine by phytohemagglutinin-stimulated lymphocytes. These results suggest that oxygen reactive species are involved in the stimulation of immune mononuclear cells.

Fluorescein labeling of Fab′ while preserving single thiol

We determined the conditions for labeling rabbit Fab′ with fluorescein isothiocyanate to provide maximal fluorescence intensity compatible with the preservation of integrity of its single thiol residue. We found that we could reproducibly substitute Fab′ with two fluorescein moieties in 30 min at room temperature at pH 8.9. We achieved a high level of fluorescence intensity while we preserved the integrity of more than half of the existing thiols.

Carboxyfluorescein transfer across the blood-retinal barrier evaluated by quantitative fluorescence microscopy: Comparison with fluorescein

Carboxyfluorescein levels in ocular tissues of normal rats were measured using quantitative fluorescence microscopy and compared with fluorescein levels to determine the extent to which blood-retinal barrier permeability is affected by the difference in lipid solubility of these two dyes. Retinal fluorescence intensity measurements at 2 min after i.v. dye injection were very much lower for carboxyfluorescein than for fluorescein despite similar plasma free dye concentrations. Marked leakage of dye from the optic disc into peripapillary retina was identified. At 1- and 2 hr, retinal levels of the two dyes became more similar, because fluorescein was removed from retina faster than carboxyfluorescein. After sodium-iodate-induced damage of the pigment epithelium, high levels of both dyes were evident in retina, but carboxyfluorescein was localized chiefly within extracellular space whereas fluorescein also densely stained cell somata. The fluorescence intensity levels recorded, which are proportional to the total mass of dye in the tissue, were correspondingly lower for carboxyfluorescein than for fluorescein, indicating that they were markedly affected by the different distribution of the two dyes. To relate tissue fluorescence intensity directly to dye concentration in the extracellular fluid, measurements were obtained from isolated retinas incubated in dye solutions of known concentration. Log-log plots demonstrated a linear relation between fluorescence intensity and medium concentration for both dyes, but retinal fluorescence of carboxyfluorescein, in correspondence with its limited distribution in the tissue space, was consistently less than that of fluorescein. The ratio of carboxyfluorescein to fluorescein fluorescence varied with the retinal layers but was constant for each layer over the concentration range tested. These fluorescence intensity ratios then were used to adjust the in vivo data so that comparison between the two dyes more closely reflected their extracellular dye concentration. With this correction the amount of carboxyfluorescein present in outer retina shortly after dye injection was approx. 1/10 that of fluorescein, indicating that carboxyfluorescein penetrates the pigment epithelium less readily than fluorescein, as expected from the difference in lipid solubility of the two dyes. However, fluorescence of both dyes in retina and presumably in vitreous humor eventually reached similar levels. This is attributed to entry of the dyes at sites of barrier discontinuity, as at the optic disc, and by a difference in their rates of removal from the intraocular compartment.

The fluorescence intensity of the lipophilic probe N-phenyl-1-naphthylamine responds to the oxidation-reduction state of purified Escherichia coli cytochrome o incorporated into phospholipid vesicles

N-Phenyl-1-naphthylamine (NPN), a reagent which has been used previously to probe the fluidity or microviscosity of the membrane lipids of intact cells of Escherichia coli, was found to respond to the redox state of purified cytochrome o incorporated into lipid vesicles formed from purified or E. coli phospholipids. NPN was bound to the proteoliposomes to produce a steady-state level of fluorescence intensity. Addition of the substrate ascorbate, in the presence of phenazine methosulfate as an electron donor, did not alter the fluorescence. However, following complete removal of oxygen from the medium by oxidation of the substrate by molecular oxygen catalyzed by cytochrome o, there was an increase in the fluorescence of NPN. This coincided with the reduction of cytochrome o. Reoxidation of the cytochrome by addition of oxygen decreased the fluorescence to steady-state levels until the oxidant had been completely reduced. The fluorescence changes were dependent on the incorporation of cytochrome o into phospholipid vesicles but were insensitive to the state of energization of the vesicle membrane.

Fluorescent labeling of cysteinyl residues. Application to extensive primary structure analysis of proteins on a microscale.

The specificity and efficiency of fluorescent labeling of proteins by reduction and subsequent alkylation with 5-N-[(iodoacetamidoethyl)amino]naphthalene-1-sulfonic acid (5-I-AEDANS) [J.J. Gorman, (1987) Anal. Biochem. 160, 376-387] has been investigated. Proteins studied include porcine insulin, chicken ovalbumin and bovine serum albumin. Amino acid analysis of the B-chain derivative of insulin revealed quantitative recovery of cysteine in its S-carboxymethyl form and no other carboxymethylated amino acid derivatives. Fast-atom-bombardment mass spectrometric (FAB-MS) analysis of this derivative also indicated specific labeling of cysteine residues and automated stepwise protein sequence analysis of the derivative was performed to completion with initial and average repetitive yields of 73% and 96%, respectively. Tryptic peptides produced from the ovalbumin and serum albumin derivatives were fractionated by HPLC and subsequently analysed by amino acid analysis, FAB-MS and automated stepwise protein sequence analysis. These analyses have revealed that the labeling procedure exhibits a high degree of efficiency and is specifically directed towards S-alkylation of cysteine residues. The high level of fluorescence intensity of the label enabled specific detection of trace quantities of cysteine-containing peptides derived from contaminating protein(s). It is apparent that in addition to facilitating isolation of small quantities of proteins the labeling procedure is compatible with standard protein chemistry techniques involved in obtaining extensive structural data on isolated proteins.

The fluorescence intensity of the lipophilic probe N-phenyl-1-naphthylamine responds to the oxidation-reduction state of the respiratory chain in everted membrane vesicles of Escherichia coli

N-Phenyl-1-naphthylamine (NPN), a reagent which has been used previously to probe the fluidity or microviscosity of the membrane lipids of intact cells of Escherichia coli, was found to respond to metabolic changes in everted inner membrane vesicles from this organism. NPN as bound to the vesicles to produce a steady-state level of fluorescence intensity. Addition of substrate or ATP did not alter the fluorescence. However, following complete removal of oxygen from the medium by oxidation of substrate through the respiratory chain, there was an increase in the fluorescence of NPN. Reoxidation of the components of the respiratory chain by the addition of oxygen, ferricyanide, fumarate or nitrate decreased fluorescence to the steady-state level until the oxidant had been completely reduced. The fluorescence changes were insensitive to the state of energization of the membrane. It is proposed that NPN responds to the state of reduction of components of the respiratory chain either directly by reacting with a component of the chain or indirectly through an effect transmitted to the membrane by a change in the conformation of respiratory chain components.

Consequences of exposure of rats to hyperoxygenation for actomyosin-like protein from synaptosomes of brain.

This paper describes the changes caused by in vivo hyperoxygenation of rats in the brain synaptosomal actomyosin-like protein preparation. We demonstrate that this preparation is composed of protein and phospholipid components attached to each other. Upon hyperoxygenation, a peroxidation of phospholipids in the lipid component proceeds. Simultaneously, with the increase of malondialdehyde, conjugated double bonds, and fluorescence intensity levels, a decrease of protein -SH groups levels, and Mg2+-dependent ATPase activity is observed. The above changes reduce the ability of the protein to superprecipitate, which reflects a contractile reaction in vivo.

Surface Immunoglobulin Light Chain Expression in Pre‐B Cell Leukemiasa

Cytofluorographic analysis of surface immunoglobulin (sIg) light chain clonal excess (CE), defined as (%kappa+ - %lambda+)/(%kappa+ + %lambda+) cells per discrete level of fluorescence intensity, was carried out on mononuclear cells of 32 leukemic patients. Eight demonstrated sIg light chain CE, including four blastic chronic myeloid leukemias (BL-CML), three "null" acute lymphoblastic leukemias (ALL), and one leukemic lymphoblastic lymphoma. Six of the leukemias demonstrated a kappa CE and two had a lambda CE. Sorted kappa+ PB cells from a BL-CML patient were shown to have a diploid DNA stem line and to bear the "common" ALL antigen. To provide further support for our finding of the expression of sIg light chains in ALL, we studied the REH cell line, derived from a "common" ALL patient and found cytoplasmic mu heavy chain and surface Ig lambda CE. Nucleic acid blotting experiments on REH revealed that both kappa genes had been deleted and that lambda genes had been rearranged, as expected in B cells expressing lambda light chains. Moreover, REH cells contained mu and lambda RNA. When REH cells were treated with TPA the amount of mu chain RNA increased by approximately fivefold and the amount of lambda chain RNA increased by approximately twofold. The finding of sIg light chain in pre-B cell leukemias and in the REH cell line, suggests that these leukemic cells are further differentiated along the B-cell lineage than was previously believed.

Phenotypic analysis by flow cytometry of surface immunoglobulin light chains and B and T cell antigens in lymph nodes involved with non-Hodgkin's lymphoma

The objective of this study was to demonstrate the diagnostic usefulness of flow cytometric analysis of surface membrane immunoglobulin light chain and monoclonal antibody reactivities in B cell non-Hodgkin's lymphoma. For this purpose, lymph node cell suspensions from 80 patients (20 normal lymph nodes, 11 lymph nodes with benign lymphoid hyperplasia, and 47 lymph nodes with B cell non-Hodgkin's lymphoma) were studied to detect the expression of surface B and T cell differentiation antigens recognized by a panel of monoclonal antibodies (anti-Leu-1, anti-Leu-5, anti-HLA-DR, J-5, anti-BL-1, anti-BL-2, and anti-BL-7). The clonal excess calculation, percent kappa-positive minus percent lambda-positive/percent kappa-positive plus percent lambda-positive cells per discrete level of fluorescence intensity, was used to study the clonality of surface membrane immunoglobulin light chain expression. Among the BL surface antigens, BL-7 proved to be most consistently expressed in B cell non-Hodgkin's lymphoma (79 percent). It was also present in 57 percent of lymph nodes with benign, hyperplasia. No significant relationships were detected between the patterns of reactivity with the anti-BL monoclonal antibodies and histologic subtypes, although the small number of cases tested in each category precludes any definitive conclusions. Immunophenotypic heterogeneity within subgroups was also observed with expression of the other antigens examined. Monoclonal expression of surface membrane immunoglobulin light chain was seen in 43 of 47 (91 percent) of lymph nodes with non-Hodgkin's lymphoma, three of 11 (27 percent) hyperplastic lymph nodes, and one of 22 (4 percent) normal lymph nodes. When the presence of BL-7 and clonal excess was examined as a panel, 83 percent of B cell non-Hodgkin's lymphomas were positively identified, whereas one normal lymph node and no hyperplastic lymph nodes gave positive results. The simultaneous presence of clonal excess and BL-7 can be a useful diagnostic aid in the differentiation of lymphomatous from hyperplastic lymph nodes. Cytofluorimetry provides a rapid, objective, and reproducible technology to confirm the diagnosis of lymph node involvement in B cell non-Hodgkin's lymphoma.

Carboxyfluorescein. A probe of the blood-ocular barriers with lower membrane permeability than fluorescein.

We have examined the permeability of the blood-ocular barriers to carboxyfluorescein, a dye similar in spectral properties but more polar than fluorescein. Octanol-buffer partition ratios of carboxyfluorescein, measured as an indication of lipid solubility, were approximately 1,000 times lower than those of fluorescein at pH values between 6.40 and 8.03. The partition ratios of both dyes show pronounced pH dependence. We also evaluated intraocular dye distribution by fluorescence microscopy after intravenous (IV) injection in rats. Carboxyfluorescein does not penetrate ciliary or iris epithelial cells, whereas fluorescein prominently stains these cells. Quantitative measurement of fluorescence intensity demonstrates that carboxyfluorescein does not enter the retina even when high doses are administered. Fluorescein, in contrast, can be detected throughout the retina with fluorescence intensity levels proportional to the IV dose administered. The relative inability of carboxyfluorescein to penetrate the blood-ocular barriers is not caused by greater binding to plasma proteins, since the plasma concentration of free carboxyfluorescein is greater than that of fluorescein. We conclude that carboxyfluorescein has potential experimental and clinical use as a probe of the blood-ocular barriers. Because of its low membrane permeability, it may yield a better definition of the nature of barrier abnormalities than is now possible with fluorescein.

Hypoxia and the neonatal rabbit lung: neuroendocrine cell numbers, 5-HT fluorescence intensity, and the relationship to arterial thickness.

We assessed the dynamics of neuroendocrine (NE) cell numbers, the intensity of specific 5-HT fluorescence, and the arterial medial thickness in the lungs of neonatal rabbits in normoxia and acute and chronic hypoxia. Hypoxic neonates had significantly higher NE cell numbers and medial thickness of the pulmonary arteries at 5 days of age than did normoxic controls; 1- and 3-day-old young that died in hypoxia also had significantly higher cell numbers and medial thickness than did hypoxic survivors. A decline in these cell numbers was noted between 1 and 5 days of age among normoxic young, whereas there was no significant change among hypoxic young. Medial thickness was unchanged among normoxic young but increased between 1 and 5 days of age among hypoxic survivors. A 1-day exposure to normoxia of hypoxic young four days postpartum caused a decrease in NE cell numbers and medial thickness to more normal values. Serotonin (5-HT) fluorescence intensity levels of groups of NE cells or neuroepithelial bodies (NEBs) in this group were equal to those of normal controls although these levels were decreased in early chronic hypoxia. Medial thickness and NE cell numbers were inversely correlated with serotonin levels, suggesting that serotonin may be associated with medial hypertrophy and presence of argyrophil material. Medial thickness was positively correlated with NE cell numbers. The above findings led to the following summary: pulmonary NE cells respond to changes in airway oxygen levels; hypoxia or decreased oxygen is associated with decreased cellular 5-HT content and an increase in NE cell numbers by argyrophil stain and medial thickness of pulmonary artery walls. The change to normoxia from hypoxia results in higher cellular 5-HT content and decreased NE argyrophil cell numbers along with reduced pulmonary artery wall medial thickness.

Detection of tumor cells in the peripheral blood of nonleukemic patients with B-cell lymphoma: analysis of "clonal excess"

Abstract Patients with malignant lymphocytic lymphoma often respond to therapy with the disappearance of mass disease and achievement of complete clinical remission. Since the disease usually reappears and eventually causes death, it appears that disseminated disease continues to exist undetected by current methods of clinical analysis. Samples of peripheral blood from nonleukemic patients with malignant lymphocytic lymphoma were examined for an excess of cells bearing the same immunoglobulin light-chain type as tumor cells from tissues involved with the lymphoma. Cells from tumor-bearing tissue and from peripheral blood (which appeared normal by the usual laboratory criteria) were stained with the anti-light-chain reagents, and the fluorescence intensity of individual cells was quantified using the fluorescence-activated cell sorter. Comparison of the number of κ+ and λ+ cells at each level of fluorescence intensity detected monoclonal populations representing as little as 0.1% of the lymphocytes. Though increases in the proportion of monocytes or null cells to lymphocytes prevented complete analysis of B-cell staining in some cases, all peripheral blood samples from patients with lymphoma that could be analyzed (11/20) exhibited evidence of “clonal excess.” Thus, the recognition of an excess of cells bearing the same light-chain type in both blood and tumor tissue provides strong evidence that the circulating monoclonal lymphocytes were tumor cells.

Detection of tumor cells in the peripheral blood of nonleukemic patients with B-cell lymphoma: analysis of “clonal excess”

Patients with malignant lymphocytic lymphoma often respond to therapy with the disappearance of mass disease and achievement of complete clinical remission. Since the disease usually reappears and eventually causes death, it appears that disseminated disease continues to exist undetected by current methods of clinical analysis. Samples of peripheral blood from nonleukemic patients with malignant lymphocytic lymphoma were examined for an excess of cells bearing the same immunoglobulin light-chain type as tumor cells from tissues involved with the lymphoma. Cells from tumor-bearing tissue and from peripheral blood (which appeared normal by the usual laboratory criteria) were stained with the anti-light-chain reagents, and the fluorescence intensity of individual cells was quantified using the fluorescence-activated cell sorter. Comparison of the number of κ+ and λ+ cells at each level of fluorescence intensity detected monoclonal populations representing as little as 0.1% of the lymphocytes. Though increases in the proportion of monocytes or null cells to lymphocytes prevented complete analysis of B-cell staining in some cases, all peripheral blood samples from patients with lymphoma that could be analyzed (11/20) exhibited evidence of “clonal excess.” Thus, the recognition of an excess of cells bearing the same light-chain type in both blood and tumor tissue provides strong evidence that the circulating monoclonal lymphocytes were tumor cells.

Isolation, properties and cellular distribution of D1, a chromosomal protein of Drosophila.

The protein D1 was obtained from nuclei of Drosophila melanogaster embryos and purified by perchloric acid fractionation and preparative gel electrophoresis. In nuclei its amount is approximately 1% of the amount of DNA by weight. D1 is soluble in 5% perchloric acid and extractable from nuclei by solutions of moderate ionic strength (0.35 M NaCl). Amino acid analysis shows that it is rich in both basic (20%) and acidic (27%) aminoacids. In all these properties D1 resembles HMG proteins (high mobility group; Johns et al., 1975) of vertebrates; however, its apparent molecular weight (approximately 50,000) is much higher. The distribution of D1 in salivary gland polytene chromosomes was investigated by immunofluorescence. Two levels of fluorescence intensity were observed: 1) Very bright fluorescence at chromosomal positions 81F, 83E, 101F, 102C and 102F; these sites are shown, by double labeling techniques, to coincide with quinacrine bright sites. 2) Medium to low fluorescence at many sites widely distributed throughout all chromosomes. In order to interpret these results and to relate them to the in vivo distribution of D1, we have investigated the pattern of immunofluorescence staining as a function of the methods of chromosome preparation and salivary gland fixation. The immunological specificity of the anti-D1 serum was studied by comparing its reactivity with D. melanogaster and D. virilis chromosome spreads and whole salivary glands, and by using reagents that minimize non-specific antibody interactions. We conclude that D1 is widely distributed throughout cytoplasm and nucleus, present in many chromomeres but most abundant in chromosomal sites that contain the AT-rich satellite DNA of density 1.672. This distribution, together with available evidence about the nucleotide sequences present in this satellite, suggests that D1 binds preferentially to chromatin containing sequences AATAT and/or AATATAT.

Kinetic study of e-beam excited Ar–CO2 mixture

Dissociation of the CO2 molecule by collision with metastable argon (3P2) was shown a few years ago to lead to the excitation of the first vibrational levels of the metastable (a3π) state of CO and the aim of the present work is to investigate the possibility of using this transfer reaction to obtain population inversions of the a 3Π→X 1Σ transition of the CO molecule. Because of the low CO* fluorescence intensity level, we only performed an indirect kinetic study of e-beam excited Ar–CO2 mixture. Using N2(c→B) (λ=337 nm) fluorescence emission as an Ar* population tracer in Ar–CO2–N2 mixture, we measured the kinetic constants relevant to Ar–CO2 mixture. It is shown that for certain Ar–CO2 mixtures, 50% of the initial excitation created by the electron gun is transferred to Ar* atoms and then disappears in Ar*+CO2 collisions. A comparative study of the instantaneous fluorescence intensity of CO* (a3π) in Ar–CO2 mixture with that of N2(C) in Ar*–N2 mixture showed that the branching ratio of the Ar*+CO2→CO* (a3π) reaction was, at most, 10%. Consequently, the largest population efficiency (5%) does not permit laser oscillation in our experiment. Furthermore, the feasibility of such a laser depends on eventual direct population of ground state level by Ar*+CO2 reaction.