A quantitative method for the detection and localization of quantum-limited events from radionuclides in cells and tissue sections by computer-enhanced video microscopy

Cellular dynamics often involve extremely low concentrations of biologically active substances, which can be radiolabeled and detected, localized and quantitated by autoradiography. The latter may require exposures from a few days to many months. The objective of this research was to demonstrate the feasibility of reducing this long period of data collection by one to two orders of magnitude, while maintaining or improving the spatial resolution and localization in tissues and the quantitative characteristics inherent in autoradiography. A mathematical model describing the complete system was generated using energy partition calculations to estimate photon production via scintillant per H3 beta particle emission and to estimate the subsequent photon capture based upon imaging system parameters and microscope geometry. Calculations showed that, typically, a single tritium beta particle produces a maximum of 5.8 X 10(3) photons. A photon-limited camera and microscope imaging system were selected and optimized in conjunction with a specially developed physical scintillation model. Results showed that the number of detected photoevents increases monotonically with both signal integration time and, independently, with the concentration of the radionuclide. Consequently, this work demonstrates that video microscopy imaging methods can spatially and temporally quantify very low concentrations of radiolabeled substances and can reduce data acquisition times.     

A new system for ATP-metric immunoanalysis

Treatment of amino-group-containing antigens with adenosine-5'-trimetaphosphate results in their chemical modification by -pppA residues. An immunoanalytical system is proposed based upon competition of these ATP-labelled antigens with those of the sample for immobilized antibodies. Mild acidic treatment of complexes of ATP-labelled antigens with immobilized antibodies results in quantitative liberation of intact ATP. The latter may be determined by the ultrosenstive bioluminescent techniques based upon emission of light with firefly luciferase. The validity of the system has been studied with two clinically important antigens, thyroxine and myoglobin.     

Automatic cell identification and enrichment in lung cancer. I. Light scatter and fluorescence parameters.

Two physical parameters were investigated to automatically recognize cells in sputum from human squamous cell carcinoma of the lung and to separate them for preparation by the Papanicolaou methods, for human interactive identification and for automated high resolution image analysis. The two parameters, 0.5-15.0 degrees forward argon-ion laser light scatter to estimate total cell size and 546 nm Acridine orange fluorescence to approximate total cell DNA content, were measured in a flow-through fluorescence activated cell sorting system. Enrichment for neoplastic cells in three cases of squamous cell carcinoma of the lung averaged 7.8-fold over the original sputum when only green fluorescence was used and 10.5-fold using green fluorescence and forward light scatter. The average enrichment for neoplastic cells was 65.6-fold relative to polymorphonuclear deenrichment.     

A structural study of germination in celery (Apium graveolens L.) seed with emphasis on endosperm breakdown

Germination of celery seed occurred after 6 d of imbibition in light. During this time the embryo enlarged at the expense of the adjacent endosperm cells and at the time of germination was 2–3 times as long as in the dry seed. Breakdown of the endosperm cells near the root cap preceeded radicle emergence. None of these changes occurred in darkness.Endosperm digestion began adjacent to the embryo and spread radially. In degrading cells, the aleurone grains often became larger and fewer in number. The cell walls were modified and appeared to undergo partial degradation. Ultimately the cells seemed to lose their contents. In cells adjacent to the root cap, similar changes occurred except there was a transient appearance of starch grains. Radial progression of endosperm breakdown also occurred in isolated endosperm treated with gibberellin A₄₊₇.The results indicate that (1) the stimulus for breakdown of celery endosperm emanates from the embryo in response to light; (2) the stimulus may be a gibberellin because changes in endosperm cells and the sequence of endosperm digestion during germination resemble the responses of isolated endosperm to gibberellin; and (3) the radial progression of endosperm breakdown during germination may be the result of a sequential response of cells to a uniformly applied stimulus rather than the result of gradual embryo expansion.     

Human cell membrane components bound to beta2-microglobulin in T cell-type cell lines.

Cell membrane components bound to beta2-microglobulin were isolated from Renex 30 (a nonionic detergent)-solubilized membrane materials of two human T cell-type cell lines, MOLT-4 and CCRF-CEM, by gel filtration and lectin affinity chromatography. The isolation was carried out by following the beta2-microglobulin activity by radioimmune inhibition assay. The T cell membrane components bound to beta2-microblogulin had a uniform molecular size of about 200,000 daltons and most of them showed an affinity to lentil lectin. The isolated membrane components were radioiodinated and examined for identity to HLA antigens by sequential precipitation with rabbit anti-HLA antiserum (specific to HLA large components) and with rabbit anti-beta2-microblogulin antiserum. In addition to HLA antigens, the beta2-microglobulin-bound components obtained from the MOLT-4 cells were found to contain certain membrane components that are the same in molecular size as the HLA large components but that are different antigenically from the HLA large components. On the other hand, the beta2-microglobulin-bound membrane components obtained from the CCRF-CEM cells were all HLA antigens. No other membrane components were involved in the binding.     

Adsorption of non-membrane proteins on the surface of model phospholipid membranes

1. Two new methods are proposed for enhancement of the binding of hydrophilic proteins by liposomes. 2. An alkylating derivative of phosphatidic acid has been obtained by its reaction with N,N,N′-tris(2-chloroethyl)-N′- (p-formylphenyl)propylene-1,3-diamine. The alkylating activity of this derivative is very low due to the electron-acceptor effect of the formyl residue. Phosphatidylcholine liposomes which contain this alkylating derivative in the lipid bilayer may be obtained. The compound residing in the outer monolayer may be reduced by NaBH₄. Upon reduction, the formyl residue is transformed into a hydroxymethyl residue. Therefore, the alkylating group of the compound is activated, and proteins may be attached covalently to the outer monolayer by alkylation with such chemically reactive liposomes. 3. Reaction of alkylating liposomes with myoglobin results in covalent binding of this hydrophilic protein. Complement-mediated leakage of such myoglobin-carrying liposomes may be induced by antibodies against myoglobin. 4. Modification of hydrophilic proteins with dansyl chloride results, even at small extents of modification, in a dramatic increase of the affinity of such proteins to phosphatidylcholine liposomes.     

Regulation of stem elongation in chinese cabbage by inflorescence removal and application of growth regulators

The effect of inflorescence removal on stem elongation in Chinese cabbage cv. Spring A was studied. Removal of the inflorescence before its visibility, or upon its appearance but before the beginning of bolting (stages 1–3), markedly reduced the stem length. Removal after the beginning of bolting (stage 5) had no effect on stem length.Application of GA₃ to the treated plants partially or fully restored the elongation of the flowering stem, whereas paclobutrazol inhibited the elongation of the treated, as well as the control stems. Indole-3-acetic acid (IAA) or kinetin was ineffective in restoring stem elongation of the plants from which the inflorescence had been removed. Inflorescences at stages 1–2 were found to secrete about 10 times more gibberellic acid (GA)-like activity compared with control apices or inflorescences at stage 5.It is suggested that the developing inflorescence is the major source of GAs which control stem elongation. However, shortly after the appearance of the inflorescence at the onset of bolting, stem elongation is no longer dependent on GAs derived from the apical inflorescence but require GAs from other sources.Contribution from the Agricultural Research Organization, The Volcani Center Bet Dagan, Israel No. 2218-E, 1987 series.