Constraints on Elastic Neutrino Nucleus Scattering in the Fully Coherent Regime from the CONUS Experiment.

We report the best limit on coherent elastic scattering of electron antineutrinos emitted from a nuclear reactor off germanium nuclei. The measurement was performed with the CONUS detectors positioned at 17.1 m from the 3.9 GW_{th} reactor core of the nuclear power plant in Brokdorf, Germany. The antineutrino energies of less than 10 MeV assure interactions in the fully coherent regime. The analyzed dataset includes 248.7 kg d with the reactor turned on and background data of 58.8 kg d with the reactor off. With a quenching parameter of k=0.18 for germanium, we determined an upper limit on the number of neutrino events of 85 in the region of interest at 90% confidence level. This new CONUS dataset disfavors quenching parameters above k=0.27, under the assumption of standard-model-like coherent scattering of the reactor antineutrinos.

The broken circle method: a novel technique that enhances detection of Hill-Sachs lesions on internal rotation shoulder radiographs.

AIM: To describe and test a new method that increases the conspicuity of a Hill-Sachs lesion on internal rotation (IR) radiographs. MATERIALS AND METHODS: This study had institutional review board approval. A retrospective search for patients with a prior shoulder dislocation and a Hill-Sachs lesion documented on magnetic resonance imaging (MRI) was performed over a 10-year period identifying 256 test patients. In Part 1, the IR radiographs from test cases were randomised with controls, and three readers scored them independently for the defect. The readers were then taught the Broken Circle (BC) method and re-scored the radiographs. In Part 2, 15 cases of Hill-Sachs lesions that were missed by all readers in Part 1 were randomised with controls, and were shown to 25 radiology residents before (pre-test) and after (post-test) learning the BC method. A paired t-test was used to compare the differences in sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV). RESULTS: In Part 1, the sensitivity increased 19.7% (54.1%-73.8%; p<0.05) and NPV increased 10.8% (62.5%-73.3%; p<0.01). In Part 2, post-test sensitivity for residents increased 16.3% (55.2%-71.5%; p<0.0001), accuracy increased 13.4% (64%-77.4%; p<0.0001), and NPV increased 13.3% (40.8%-54.1%; p<0.0001) independent of the level of training. The change in accuracy was also statistically significant for every individual class. CONCLUSION: The BC method was an effective technique that facilitated detection of a Hill-Sachs lesion at all levels of training, and was useful as a teaching tool.

Detection and localisation of hip fractures on anteroposterior radiographs with artificial intelligence: proof of concept.

AIM: To investigate the feasibility of applying a deep convolutional neural network (CNN) for detection/localisation of acute proximal femoral fractures (APFFs) on hip radiographs. MATERIALS AND METHODS: This study had institutional review board approval. Radiographs of 307 patients with APFFs and 310 normal patients were identified. A split ratio of 3/1/1 was used to create training, validation, and test datasets. To test the validity of the proposed model, a 20-fold cross-validation was performed. The anonymised images from the test cohort were shown to two groups of radiologists: musculoskeletal radiologists and diagnostic radiology residents. Each reader was asked to assess if there was a fracture and localise it if one was detected. The area under the receiver operator characteristics curve (AUC), sensitivity, and specificity were calculated for the CNN and readers. RESULTS: The mean AUC was 0.9944 with a standard deviation of 0.0036. Mean sensitivity and specificity for fracture detection was 97.1% (81.5/84) and 96.7% (118/122), respectively. There was good concordance with saliency maps for lesion identification, but sensitivity was lower for characterising location (subcapital/transcervical, 84.1%; basicervical/intertrochanteric, 77%; subtrochanteric, 20%). Musculoskeletal radiologists showed a sensitivity and specificity for fracture detection of 100% and 100% respectively, while residents showed 100% and 96.8%, respectively. For fracture localisation, the performance decreased slightly for human readers. CONCLUSION: The proposed CNN algorithm showed high accuracy for detection of APFFs, but the performance was lower for fracture localisation. Overall performance of the CNN was lower than that of radiologists, especially in localizing fracture location.

Platelet activating factor raises intracellular calcium ion concentration in macrophages.

Peritoneal cells from thioglycollate-stimulated mice were allowed to adhere to coverglasses for 2 h to give a dense monolayer of adherent cells greater than 95% of which were macrophages. After incubation with the tetra-acetoxymethyl ester of quin2, coverglasses were rinsed with Ca2+-free saline, oriented at a 45 degree angle in square cuvettes containing a magnetically driven stir bar, and analyzed for changes in quin2 fluorescence in a spectrofluorimeter. Such fluorescence, taken as an indication of intracellular calcium ion concentration ([Ca2+]i), increased as exogenous calcium ion concentration ([Ca2+]o) was raised to 1 mM. At [Ca2+]o approximately equal to 10 microM, [Ca2+]i = 72 +/- 14 nM (n = 26); at [Ca2+]o = 1 mM, [Ca2+]i = 140-220 nM, levels not increased by N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine, a membrane-permeant chelator of heavy metals than can quench quin2. Addition of mouse alpha + beta fibroblast interferon, lipopolysaccharide, thrombin, collagen, vasopressin, ADP, compound 48/80, or U46619 did not change [Ca2+]i. However, addition of platelet activating factor (PAF) (2-20 ng/ml) raised [Ca2+]i by 480 nM within 1 min if [Ca2+]o = 1 mM. In the presence of 5 mM EGTA, PAF raised [Ca2+]i by 25 nM. This suggests that PAF causes influx of exogenous Ca2+, as well as releasing some Ca2+ from intracellular stores. Consistent with these results, when PAF was added to 1 mM Ca2+ in the presence of 100 microM Cd2+ or Mn2+ to block Ca2+ influx, [Ca2+]i increased by only intermediate amounts; at the times of such dampened peak response, [Ca2+]i could be raised within 1 min to normal PAF-stimulated levels by chelation of the exogenous heavy metals with diethylenetriaminepentaacetic acid. Normal PAF responses were observed in the presence of indomethacin. The lowest dose of PAF observed to raise [Ca2+]i was 0.1 ng/ml. Response of [Ca2+]i to 2-20 ng/ml PAF was transient, and second applications had no effect. The PAF response also was seen in cell suspensions. These results suggest that an increase in [Ca2+]i may be an early event in PAF activation of macrophages.

Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator.

A new, fluorescent, highly selective Ca2+ indicator , "quin2", has been trapped inside intact mouse and pig lymphocytes, to measure and manipulate cytoplasmic free Ca2+ concentrations, [Ca2+]i. Quin2 is a tetracarboxylic acid which binds Ca2+ with 1:1 stoichiometry and an effective dissociation constant of 115 nM in a cationic background mimicking cytoplasm. Its fluorescence signal (excitation 339 nm, emission 492 nm) increases about fivefold going from Ca-free to CA-saturated forms. Cells are loaded with quin2 by incubation with its acetoxymethyl ester, which readily permeates the membrane and is hydrolyzed in the cytoplasm, thus trapping the impermeant quin2 there. The intracellular quin2 appears to be free in cytoplasm, not bound to membranes and not sequestered inside organelles. The fluorescence signal from resting cells indicates a [Ca2+]i of near 120 nM. The millimolar loadings of quin2 needed for accurately calibrated signals do not seem to perturb steady-state [Ca2+]i, but do somewhat slow or blunt [Ca2+]i transients. Loadings of up to 2mM are without serious toxic effects, though above this level some lowering of cellular ATP is observed. [Ca2+]i was well stabilized in the face of large changes in external Ca2+. Alterations of Na+ gradients, membrane potential, or intracellular pH had little effect. Mitochondrial poisons produced a small increase in [Ca2+]i, probably due mostly to the effects of severe ATP depletion on the plasma membrane. Thus intracellulary trapped chelators like quin2 offer a method to measure or buffer [Ca2+]i in hitherto intractable cell types.

Cytoplasmic pH and free Mg2+ in lymphocytes.

Measurements have been made of cytoplasmic pH, (pHi) and free Mg2+ concentration, ( [Mg2+]i), in pig and mouse lymphocytes. pHi was measured in four ways: by a digitonin null-point technique; by direct measurement of the pH of freeze-thawed cell pellets; from the 31P nuclear magnetic resonance (NMR) spectrum of intracellular inorganic phosphate; and by the use of a newly synthesized, intracellularly-trappable fluorescent pH indicator. In HEPES buffered physiological saline with pH 7.4 at 37 degrees C, pHi was close to 7.0. Addition of physiological levels of HCO3- and CO2 transiently acidified the cells by approximately 0.1 U. Mitogenic concentrations of concanavalin A (Con A) had no measurable effect on pH in the first hour. [Mg2+]i was assessed in three ways: (a) from the external Mg2+ null-point at which the ionophore A23187 produced no net movement of Mg2+ or H+; (b) by Mg-sensitive electrode measurements in freeze-thawed pellets; and (c) from the 31P nuclear magnetic resonance spectrum of the gamma-phosphate of intracellular ATP. Total cell Mg2+ was approximately 12 mmol per liter cell water. The NMR data indicated [Mg2+]i greater than 0.5 mM. The null-point method gave [Mg2+]i approximately 0.9 nM. The electrode measurements gave 1.35 mM, which was thought to be an overestimate. Exposure to mitogenic doses of Con A for 1 h gave no detectable change in total or free Mg2+.

Anti-immunoglobulin, cytoplasmic free calcium, and capping in B lymphocytes.

This paper examines, in mouse spleen lymphocytes, the effect of anti-immunoglobulin (anti-Ig) on the cytoplasmic free calcium concentration, [Ca2+]i, measured with the fluorescent indicator quin2, and the relationship of [Ca2+]i to the capping of surface Ig. Anti-Ig causes a rapid rise of [Ca2+], which precedes capping. Assuming that only those 40-50% of the cells which can bind anti-Ig (the B cells) undergo a [Ca2+]i response, [Ca2+]i in those cells approaches 500 nM. It declines to resting levels over many minutes, roughly paralleling the formation of caps. Part of the [Ca2+]i signal is due to stimulated influx across the plasma membrane, since in Ca2+-free medium, anti-Ig gives a smaller and shorter [Ca2+]i rise. The amplitude of this reduced transient now varies inversely with quin2 content, as if some 0.25 mmol Ca per liter of cells was released into the cytoplasm from internal stores. These stores are probably sequestered in organelles since A23187 in Ca2+-free medium also causes a transient [Ca2+]i rise after which anti-Ig has no effect. These organelles seem not to be mitochondria because uncouplers have hardly any effect on [Ca2+]i. Though anti-Ig normally raises [Ca2+]i before causing capping, there seems to be no causal link between the two events. Cells in Ca2+-free medium whose stores have been emptied by A23187, still cap with anti-Ig even though there is no [Ca2+]i rise. Cells loaded with quin2 in the absence of external Ca2+ still cap anti-Ig normally even though their [Ca2+]i remains steady at below 30 nM, four times lower than normal resting [Ca2+]i.

Dicarbocyanine fluorescent probes of membrane potential block lymphocyte capping, deplete cellular ATP and inhibit respiration of isolated mitochondria.

3,3'-Dipropylthiodicarbocyanine iodide, a widely used fluorescent probe of membrane potential, was found to inhibit anti-Ig antibody, induced capping of mouse lymphocytes. The dye also lowered the cell ATP content. Experiments with isolated mitochondria revealed that the probe had a potent inhibitory action at site I of the respiratory chain. This mitochondrial blockade helps to explain the ATP depletion and blockade of capping, and gives cause for caution in the use of this dye as a probe of cell membrane potential. Three related dicarbocyanine dyes had similar toxic effects, but two cyanine dyes with much longer alkyl side chains, which have been used as probes of membrane fluidity, did not.

Neutral carrier ion-selective microelectrodes for measurement of intracellular free calcium.

This paper describes the making and testing of calcium-selective microelectrodes for measurement of intracellular [free Ca2+] levels. Pipettes of tip outer diameter down to 0.4 micron were siliconized by a novel and easy method of vapor treatment. The tips were filled with a sensor mixture using the neutral ligand and solvent of Oehme et al. (Oehme, M., Kessler, M. and Simon, W. (1976) Chimia (Aarau) 30, 204-206) but with very hydrophobic cations replacing Na+ in the salt component. This change improved electrode stability and greatly reduced hysteresis in the responses to changing [Ca2+] levels. Lowering the Ca2+ concentration in the internal electrolyte also increased electrode lifetime. Electrodes showed a Nernstian response to [Ca2+] down to 1 micro M free concentration in 0.1 M KCl, and usually a useful response to below 100 nM Ca2+. Selectivity for Ca2+ over Mg2+ and H+ was sufficiently high that typical free intracellular levels of Mg2+ and H+ caused negligible interference. Selectivity for Ca2+ over Na+ was adequate for cells with 10(-2) M free Na+, but higher levels could raise significantly the detection limit for Ca2+. Preliminary measurements of [free Ca2+] have been made in frog skeletal muscle, ferret ventricular myocardium, and early embryos of Xenopus laevis. Relative merits of Ca2+ microelectrodes and optical indicators are discussed.

Dose-dependent elevation of cyclic AMP, activation of glycogen phosphorylase, and release of lactate by amylin in rat skeletal muscle.

We report here our investigation of the role of cyclic AMP (cAMP) in amylin signal transduction in isolated strips of soleus muscle. Rat amylin, at 100 nM, increased cAMP levels, from 0.431 +/- 0.047 to a peak of 1.24 +/- 0.01 pmol cAMP/mg wet wt. after 5 min, in the absence of added phosphodiesterase inhibitor. The EC50 of the response was 0.48 nM (+/- 0.12 log units) in the absence of insulin and 0.3 nM (+/- 0.18 log units) in the presence of 7.1 nM insulin. The response seen with a maximally effective concentration of amylin (10 nM) was similar to that seen with a maximally effective concentration of epinephrine (1 microM) under the same conditions. Consistent with the observed rise in cAMP there was an increase in glycogen phosphorylase a (EC50 2.2 nM +/- 0.25 log units), decreased glycogen content (EC50 0.9 nM +/- 0.22 log units) and enhanced production of lactate (EC50 1.5 nM +/- 0.33 log units). These data support the concept that amylin promotes glycogenolysis in skeletal muscle and enhances production of lactate through glycolysis as a result of activation of Gs coupled receptors, stimulation of adenylate cyclase, elevation of cAMP levels and activation of glycogen phosphorylase.

Lymphocyte membrane potential assessed with fluorescent probes.

The membrane potential of mouse spleen lymphocytes has been assessed with two fluorescent probes. 3,3'-Dipropylthiadicarbocyanine (diS-C3-(5)) was used for most of the experiments. Solutions with high K+ concentrations depolarised the cells. Valinomycin, an inophore which adds a highly K+-selective permeability membranes, slightly hyperpolarised cells in standard (6 mM K+) solution, and in 145 mM K+ solution produced a slight additional depolarisation. These findings indicate a membrane whose permeability is relatively selective for K+. Very small changes in potential were seen when choline replaced Na+, or gluconate replaced Cl-, supporting the idea of K+ selectivity. The resting potential could be estimated from the K+ concentration gradient at which valinomycin did not change the potential-the "valinomycin null point" - and under the conditions used the resting potential was approx.-60 mV. B cell-enriched suspensions were prepared either from the spleens of nu/nu mice or by selective destruction of T cells in mixed cell populations. The membrane potential of these cells was similar to that estimated for the mixed cells. In solution with no added K+, diS-C3-(5) itself appeared to depolarise the lymphocytes, in a concentration dependent manner. With the 100 nM dye normally used, the membrane potential in K+-free solution was around -45 mV, and 500 nM dye almost completely depolarised the cells. In standard solution quinine depolarised the cells. Valinomycin could still depolarise these cells indicating that depolarisation had not been due to dissipation of the K+ gradient. Since in K+-free solution diS-C3-(5) blocks the Ca2+-activated K+ channels in human red blood cell ghosts and quinine also blocks this K+ channel it is suggested that the resting lymphocyte membrane may have a similar Ca2+-activated K+ permeability channel. Because of the above mentioned effect of diS-C3-(5) and other biological side effects, such as inhibition of B cell capping, a chemically distinct fluorescent probe of membrane potential, bis(1,3-diethylthiobarbiturate)-trimethineoxonol was used to support the diS-C3-(5) data. This new probe proved satisfactory except that it formed complexes with valinomycin, ruling out the use of this ionophore. Results with the oxonol on both mixed lymphocytes and B cell-enriched suspensions gave confirmation of the conclusions from diS-C3-(5) experiments and indicated that despite its biological side effects, diS-C3-(5) could still give valid assessment of membrane potential.

Triggering of lymphocyte capping appears not to require changes in potential or ion fluxes across the plasma membrane.

Capping induced by anti-Ig antibody on mouse spleen lymphocytes was found to proceed normally over a wide range of membrane potentials from approx. 0 to -65 mV, as estimated with fluorescent probes. The potential was manipulated by ionic substitution in the medium and/or application of gramicidin. Various agents which inhibit capping had differing effects on the membrane potential, some producing no measurable change, others depolarising the cells. In particular valinomycin (10-7 M) was found to inhibit capping in cells both slightly hyperpolarised from the normal resting potential, and fully depolarised. Valinomycin was found to deplete the lymphocytes markedly of ATP and this effect was sufficient to account for the inhibition of capping. Capping occurred in a simplified (sucrose) medium lacking Na+, K+ and Ca2+, suggesting that fluxes across the plasma membrane of these ions are not required. It is concluded that after ligand binding, some reorganisation of receptor protein at the inner face of the membrane is the sufficient stimulus for the intracellular rearrangements involved in capping.

Structure and biology of amylin.

Amylin is a recently discovered 37 amino acid peptide secreted into the bloodstream, along with insulin, from pancreatic beta-cells. It is about 50% identical to calcitonin gene-related peptides (CGRP alpha and CGRP beta) and structurally related to the calcitonins. Amylin can elicit the vasodilator effects of CGRP and the hypocalcaemic actions of calcitonin, while these peptides can mimic newly discovered actions of amylin on carbohydrate metabolism. The different relative potencies of these peptides suggest that they act with different selectivities at a family of receptors. Amylin is deficient in insulin-dependent diabetes mellitus, while plasma levels are elevated in insulin-resistant conditions such as obesity and impaired glucose tolerance. In this Viewpoint article, Tim Rink and colleagues propose that amylin is an endocrine partner to insulin and glucagon; deficiency or excess of amylin may therefore contribute to important metabolic diseases.

Site-directed spin-labeling reveals the orientation of the amino acid side-chains in the E-F loop of bacteriorhodopsin.

Due to high temperature factors and the lack of considerable electron density, electron microscopy and X-ray experiments on the cytoplasmic E-F loop of bacteriorhodopsin result in a variety of structural models. As the experimental conditions regarding ionic strength, temperature and the presence of detergents may affect the structure of the E-F loop, we employ electron paramagnetic resonance and site-directed spin-labeling to study the structure of this loop under physiological conditions. The amino acid residues at positions 154 to 171 were replaced by cysteine residues and derivatized with a sulfhydryl-specific nitroxide spin label one by one. The conventional and power saturation electron paramagnetic spectroscopy provide the mobility of the nitroxide and its accessibility to dissolved molecular oxygen and membrane-impermeable chromium oxalate in the respective site. The results show that K159 and A168 are located at the water-lipid interface of helices E and F, respectively. The orientation of the amino acid side-chains in the helical regions from positions 154 to 159 and 166 to 171 were found to agree with published structural data for bacteriorhodopsin. In the residue sequence from positions 160 to 165 the EPR data yield evidence for a turned loop structure with the side-chains of M163 and S162 oriented towards the proton channel and the water phase, respectively.

[Spectrometric assessment of thyroid depth within the radioiodine test]. / Spektrometrisches Verfahren zur Tiefenbestimmung der Schilddrüse im Rahmen des Radioiodtests.

UNLABELLED: Aim of this study is the validation of a simple method for evaluating the depth of the target volume within the radioiodine test by analyzing the emitted iodine-131 energy spectrum. PATIENTS, METHODS: In a total of 250 patients (102 with a solitary autonomous nodule, 66 with multifocal autonomy, 29 with disseminated autonomy, 46 with Graves' disease, 6 for reducing goiter volume and 1 with only partly resectable papillary thyroid carcinoma), simultaneous uptake measurements in the Compton scatter (210 +/- 110 keV) and photopeak (364-45/+55 keV) windows were performed over one minute 24 hours after application of the 3 MBq test dose, with subsequent calculation of the respective count ratios. Measurements with a water-filled plastic neck phantom were carried out to perceive the relationship between these quotients and the average source depth and to get a calibration curve for calculating the depth of the target volume in the 250 patients for comparison with the sonographic reference data. Another calibration curve was obtained by evaluating the results of 125 randomly selected patient measurements to calculate the source depth in the other half of the group. RESULTS: The phantom measurements revealed a highly significant correlation (r = 0,99) between the count ratios and the source depth. Using these calibration data, a good relationship (r = 0,81, average deviation 6 mm corresponding to 22%) between the spectrometric and the sonographic depths was obtained. When using the calibration curve resulting from the 125 patient measurements, the overage deviation in the other half of the group was only 3 mm (12%). There was no difference between the disease groups. CONCLUSION: The described method allows on easy to use depth correction of the uptake measurements providing good results.

TIRADS for sonographic assessment of hypofunctioning and indifferent thyroid nodules.

AIM: To test the feasibility of the Thyroid Imaging Reporting And Data System (TIRADS) according to Horvath and Kwak for the assessment of thyroid nodules. PATIENTS, METHOD: Retrospective analysis of patients with thyroid nodules applying the following inclusion criteria: B-mode-ultrasound, surgery and histological results. Thyroid nodules were classified as TIRADS 2, 3, 4A, 4B, 4C, 5 and 6. RESULTS: A total of 172 patients were included (133 women, 48 ± 13 years, 39 men, 49 ± 11 years) with 222 thyroid nodules (24.9 ± 11.5 mm). Final histological diagnosis revealed 203 benign nodules (91%) and 19 malignant nodules (9%; 18 papillary thyroid carcinoma, PTC, and one medullary thyroid carcinoma, MTC). One hundred and sixty thyroid nodules were hypofunctioning in 99mTc-pertechnetate-scintigraphy, 14 nodules were hyperfunctioning and 46 nodules were classified as indifferent. In two cases with small carcinoma < 1 cm 99mTc-pertechnetate-scintigraphy was not performed. According to Horvath, the prevalence of malignancy was 6.7% in TIRADS 2, 0% in 3, 1.9% in 4A, 33% in 4B, 12.5% in 5 and 100% in 6; 73 nodules (39%) were not clearly classifiable, including 3 carcinoma (4.1%). According to Kwak, the prevalence of malignancy was 6.9% in TIRADS 2, 0% in 3, 2% in 4A, 4.1% in 4B, 23.1% in 4C, and 100% in 5 and 6, respectively. Notably, in the subgroup of hot nodules, 11 (79%) were graded as TIRADS 4A or higher, and thus advisable for fine-needle aspiration biopsy in both TIRADS. CONCLUSION: The TIRADS described by Horvath is not practicable due to numerous unclassifiable nodules. The revised TIRADS published by Kwak is feasible and suitable to assess the prevalence of malignancy, but it cannot replace scintigraphic imaging. Fine-needle-biopsy is not necessary in nodules categorized as (K)TIRADS 3, 4A and 5.

Calcium signalling in non-excitable cells: notes on oscillations and store refilling.

Technical advances in studying cellular calcium concentrations, and discoveries about many aspects of signal transduction have transformed this field of biology since this Journal was launched a decade ago. At that time monitoring of the key variable, cytoplasmic free Ca2+ concentration [Ca2+]i, was possible with aequorin or arsenazo ill mainly in large invertebrate cells, though pioneering work with aequorin micro-injection into cardiac and smooth muscle had just started. At that time there was also intense activity by a few groups aiming to make Ca-selective micro-electrodes selective and sharp enough to measure [Ca2+]i in small cells. Also the use of electropermeabilized cells had begun to allow the defining of the concentrations of Ca2+ required to activate secretion in mammalian cells. Nearly all this work and all the relevant electrophysiology relating to calcium signalling had been done in excitable cells, basically muscle and nerve, and was aimed at understanding contraction, transmitter release and neurosecretion, and the control of membrane permeability. Recent advances have now allowed [Ca2+]i to be measured in non-excitable cells.

Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths.

Free Ca2+ concentrations in the cytosol of individual small cells can be recorded with a new fluorescent Ca2+ indicator, "fura-2", and a fluorescence microscope modified to chop rapidly between two wavelengths of excitation. Both fura-2 and its Ca2+ complex fluoresce strongly, but their excitation peaks differ in wavelength. Alternation between the two preferred wavelengths allows assessment of the ratio of Ca2+-bound dye to free dye and hence cytosolic free Ca2+. This ratio measurement largely cancels out the effects of cell thickness, dye content, or instrumental efficiency, uncertainties that can jeopardize measurements at single wavelengths. We describe instrumentation that supplies rapidly alternating excitation wavelengths to either a standard cuvet or a fluorescence microscope. Its use is illustrated by experiments showing changes in cytosolic [Ca2+] accompanying activation of human platelets in suspension or single mouse thymocytes on the microscope.

Receptor-mediated calcium entry.

Occupation of membrane receptors can evoke calcium signals by causing depolarisation and activating voltage-operated calcium channels, by triggering internal release, or by stimulating calcium influx processes not gated by membrane potential, receptor-mediated calcium entry, RCME. This brief review considers different possible coupling mechanisms and the proposal that entry can occur from external medium to intracellular store, by-passing the cytosol, and regulated by the state of filling of the store. Recent studies using Mn2+ as a probe for RCME are outlined, as are some new electrophysiologic measurements with human platelets and investigations of a novel blocker of RMCE, SK&F 96365.