scholarly journals Measurement of Intracellular Calcium

1999 ◽  
Vol 79 (4) ◽  
pp. 1089-1125 ◽  
Author(s):  
Akiyuki Takahashi ◽  
Patricia Camacho ◽  
James D. Lechleiter ◽  
Brian Herman
Keyword(s):  
Laser Scanning ◽  
Cell Injury ◽  
Multiphoton Microscopy ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ion Activity ◽  
Intracellular Ca ◽  
Ionic Changes ◽  
Ion Activities ◽  
Subcellular Level

To a certain extent, all cellular, physiological, and pathological phenomena that occur in cells are accompanied by ionic changes. The development of techniques allowing the measurement of such ion activities has contributed substantially to our understanding of normal and abnormal cellular function. Digital video microscopy, confocal laser scanning microscopy, and more recently multiphoton microscopy have allowed the precise spatial analysis of intracellular ion activity at the subcellular level in addition to measurement of its concentration. It is well known that Ca2+ regulates numerous physiological cellular phenomena as a second messenger as well as triggering pathological events such as cell injury and death. A number of methods have been developed to measure intracellular Ca2+. In this review, we summarize the advantages and pitfalls of a variety of Ca2+ indicators used in both optical and nonoptical techniques employed for measuring intracellular Ca2+ concentration.

Intracellular Ca2+ transients in mouse soleus muscle after hindlimb unloading and reloading

1999 ◽  
Vol 87 (1) ◽  
pp. 386-390 ◽  
Author(s):  
Christopher P. Ingalls ◽  
Gordon L. Warren ◽  
R. B. Armstrong
Keyword(s):  
Soleus Muscle ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Hindlimb Unloading ◽  
Hindlimb Suspension ◽  
Confocal Laser ◽  
Specific Force ◽  
Activity Maximum ◽  
Tetanic Force ◽  
Intracellular Ca

The objective of this study was to determine whether altered intracellular Ca2+ handling contributes to the specific force loss in the soleus muscle after unloading and/or subsequent reloading of mouse hindlimbs. Three groups of female ICR mice were studied: 1) unloaded mice ( n = 11) that were hindlimb suspended for 14 days, 2) reloaded mice ( n = 10) that were returned to their cages for 1 day after 14 days of hindlimb suspension, and 3) control mice ( n = 10) that had normal cage activity. Maximum isometric tetanic force (Po) was determined in the soleus muscle from the left hindlimb, and resting free cytosolic Ca2+ concentration ([Ca2+]i), tetanic [Ca2+]i, and 4-chloro- m-cresol-induced [Ca2+]iwere measured in the contralateral soleus muscle by confocal laser scanning microscopy. Unloading and reloading increased resting [Ca2+]iabove control by 36% and 24%, respectively. Although unloading reduced Po and specific force by 58% and 24%, respectively, compared with control mice, there was no difference in tetanic [Ca2+]i. Po, specific force, and tetanic [Ca2+]iwere reduced by 58%, 23%, and 23%, respectively, in the reloaded animals compared with control mice; however, tetanic [Ca2+]iwas not different between unloaded and reloaded mice. These data indicate that although hindlimb suspension results in disturbed intracellular Ca2+ homeostasis, changes in tetanic [Ca2+]ido not contribute to force deficits. Compared with unloading, 24 h of physiological reloading in the mouse do not result in further changes in maximal strength or tetanic [Ca2+]i.

Intracellular Ca2+ dynamics in response to Ca2+ influx and Ca2+ release in autonomic neurones

1992 ◽  
Vol 70 (S1) ◽  
pp. S64-S72 ◽  
Author(s):  
Kenji Kuba ◽  
Mitsuo Nohmi ◽  
Shao-Ying Hua
Keyword(s):  
Sympathetic Ganglion ◽  
Laser Scanning ◽  
Ganglion Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Release Mechanism ◽  
Voltage Dependent ◽  
Intracellular Ca ◽  
Intracellular Organelles ◽  
Cultured Neurones

Spatial and temporal changes in the intracellular free Ca2+ concentration in response to Ca2+ influx at the cell membrane and to Ca2+ release from intracellular organelles were studied by recording fluorescence of Ca2+-sensitive probes, fura 2 or indo 1, with conventional epifluorescence or confocal laser-scanning microscopy combined with recordings of Ca2+-dependent membrane responses in bullfrog sympathetic ganglion cells. It was found that an increase in the intracellular Ca2+ induced by (an) action potential(s) in freshly isolated ganglion cells bathed in Ringer's solution was solely a result of Ca2+ influx, while a rise in the intracellular Ca2+ by Ca2+ current in voltage-clamped cultured neurones was caused by not only Ca2+ influx but also Ca2+ release. This Ca2+ release was suggested to occur by a voltage-dependent (and graded) mode of activation of a Ca2+-induced Ca2+ release mechanism, explaining the lack of Ca2+ release by action potentials (because of their short-lasting depolarization) in freshly isolated neurones. In both cases, there was an inward spread of an increase in intracellular Ca2+. On the other hand, all or nothing activation of Ca2+-induced Ca2+ release occurred in the presence of caffeine, leading to the oscillation of Ca2+ in the cells. Characteristics of this mode of Ca2+ release and unique properties of drugs to block Ca2+ release were described. Finally, the physiological significance of different types of Ca2+ release was discussed.Key words: Ca2+ current, Ca2+-induced Ca2+ release, Ca2+ transient, confocal microscope, bullfrog sympathetic ganglion cells.

scholarly journals Physiological controls on seawater uptake and calcification in the benthic foraminifer <i>Ammonia tepida</i>

2009 ◽  
Vol 6 (11) ◽  
pp. 2669-2675 ◽  
Author(s):  
L. J. de Nooijer ◽  
G. Langer ◽  
G. Nehrke ◽  
J. Bijma
Keyword(s):  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Free Calcium ◽  
Benthic Foraminifer ◽  
Amorphous Precursor ◽  
Intracellular Ca ◽  
Ca Ions ◽  
First Time ◽  
Cellular Ca

Abstract. To analyze the relation between seawater uptake and calcification, we incubated juveniles of the benthic foraminifer Ammonia tepida with various fluorescent probes and visualised them afterwards with confocal laser scanning microscopy. Vesicle membranes, Ca ions and vacuole fluids were followed with various tracers and showed for the first time that endocytosis of seawater is part of the calcification process in Ammonia tepida. Data on the intracellular Ca ion cycling allowed for calculating a preliminary cellular Ca budget during foraminiferal calcification. This showed that the free calcium involved in the production of a new chamber cannot be sufficient and suggests that foraminifera may precipitate their calcite from an amorphous precursor.

scholarly journals Calcium Dynamics in Thorny Excrescences of CA3 Pyramidal Neurons

1997 ◽  
Vol 78 (1) ◽  
pp. 10-18 ◽  
Author(s):  
David B. Jaffe ◽  
Thomas H. Brown
Keyword(s):  
Laser Scanning ◽  
Pyramidal Neurons ◽  
Mossy Fiber ◽  
Hippocampal Slices ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
High Temporal Resolution ◽  
Calcium Dynamics ◽  
Intracellular Ca ◽  
Ca3 Pyramidal Neurons

Jaffe, David B. and Thomas H. Brown. Calcium dynamics in thorny excrescences of CA3 pyramidal neurons. J. Neurophysiol. 78: 10–18, 1997. Confocal laser scanning microscopy was used to visualize Ca2+ transients in a particular type of dendritic spine, known as a thorny excrescence, in hippocampal CA3 pyramidal neurons. These large excrescences or thorns, which serve as the postsynaptic target for the mossy-fiber synaptic inputs, were identified on the basis of their location, frequency, and size. Whole cell recordings were made from superficial CA3 pyramidal neurons in thick hippocampal slices with the use of infrared video microscopy; cells with proximal apical dendrites close to the surface of the slice were selected. Changes in intracellular Ca2+ levels were monitored by imaging changes in fluorescence of the dyes Calcium Green-1 and Fluo-3. Dual-emission fluorescence imaging was also employed with the use of a combination of Fluo-3 and the Ca2+insensitive dye seminaphthorhodafluor-1. This method was used todecrease the potential influence of background fluorescence on the calculated changes in intracellular Ca2+ concentration ([Ca2+]i). Somatic depolarization produced increases in [Ca2+]i in both the thorn and the immediately adjacent dendrite. Changes in [Ca2+]i were time locked with the onset of depolarization and the decay began immediately after the termination of depolarization. The peak increase in the Ca2+ signal was significantly greater in the thorns than in the adjacent dendritic shafts. With the use of high-temporal-resolution methods (line scans), differences were also seen in the time course of Ca2+ signals in these two regions. The decay time constants of the Ca2+ signal were faster in thorns than in the adjacent dendritic shafts. These observations suggest that voltage-gated Ca2+ channels are localized directly on the dendritic spines receiving mossy-fiber input. Furthermore, Ca2+ homeostasis within thorny excrescences is distinct from Ca2+ regulation in the dendritic shaft, at least over brief time periods, a finding that could have important implications for synaptic plasticity and signaling.

Nucleoplasmic calcium regulation in rabbit aortic vascular smooth muscle cells

2003 ◽  
Vol 81 (3) ◽  
pp. 301-310 ◽  
Author(s):  
Bernard Abrenica ◽  
Grant N Pierce ◽  
James S.C Gilchrist
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Signal Intensity ◽  
Laser Scanning ◽  
Muscle Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Intracellular Ca

In this study, we investigated whether nucleoplasmic free Ca2+ in aortic vascular smooth muscle cells (VSMCs) might be independently regulated from cytosolic free Ca2+. Understanding mechanisms and pathways responsible for this regulation is especially relevant given the role of a numerous intranuclear Ca2+-sensitive proteins in transcriptional regulation, apoptosis and cell division. The question of an independent regulatory mechanism remains largely unsettled because the previous use of intensitometric fluorophores (e.g., Fluo-3) has been criticized on technical grounds. To circumvent the potential problem of fluorescence artifact, we utilized confocal laser scanning microscopy to image intracellular Ca2+ movements with the ratiometric fluorophore Indo-1. In cultured rabbit VSMCs, we found sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA) pumps and ryanodine receptor (RyR) Ca2+ channel proteins to be discretely arranged within a perinuclear locus, as determined by fluorescent staining patterns of BODIPY® FL thapsi gargin and BODIPY® FL-X Ry. When intracellular Ca2+ stores were mobilized by addition of thapsigargin (5 μM) and activatory concentrations of ryanodine (1 μM), Indo-1 ratiometric signals were largely restricted to the nucleoplasm. Cytosolic signals, by comparison, were relatively small and even then its spatial distribution was largely perinuclear rather homogeneous. These observations indicate perinuclear RyR and SERCA proteins are intimately involved in regulating VSMC nucleoplasmic Ca2+ concentrations. We also observed a similar pattern of largely nucleoplasmic Ca2+ mobilization upon exposure of cells to the immunosuppressant drug FK506 (tacrolimus), which binds to the RyR-associated immunophillin-binding proteins FKBP12 and FKBP12.6. However, initial FK506-induced nucleoplasmic Ca2+ mobilization was followed by marked reduction of Indo-1 signal intensity close to pretreatment levels. This suggested FK506 exerts both activatory and inhibitory effects upon RyR channels. The latter was reinforced by observed effects of FK506 to only reduce nucleoplasmic Indo-1 signal intensity when added following pretreatment with both activatory and inhibitory concentrations of ryanodine. These latter observations raise the possibility that VSMC nuclei represent an important sink of intracellular Ca2+ and may help explain vasodilatory actions of FK506 observed by others.Key words: Ca2+, RyR, SERCA, cell nucleus, FK506, thapsigargin, ryanodine.

Three-Dimensional Imaging of Sulfides in Silicate Rocks at Submicron Resolution with Multiphoton Microscopy

2011 ◽  
Vol 17 (6) ◽  
pp. 937-943 ◽  
Author(s):  
Antoine Bénard ◽  
Sabine Palle ◽  
Luc Serge Doucet ◽  
Dmitri A. Ionov
Keyword(s):  
Laser Scanning ◽  
Second Harmonic ◽  
Multiphoton Microscopy ◽  
3D Structure ◽  
Three Dimensional ◽  
Harmonic Signal ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Silicate Rocks ◽  
Submicron Resolution

AbstractWe report the first application of multiphoton microscopy (MPM) to generate three-dimensional (3D) images of natural minerals (micron-sized sulfides) in thick (∼120 μm) rock sections. First, reflection mode (RM) using confocal laser scanning microscopy (CLSM), combined with differential interference contrast (DIC), was tested on polished sections. Second, two-photon fluorescence (TPF) and second harmonic signal (SHG) images were generated using a femtosecond-laser on the same rock section without impregnation by a fluorescent dye. CSLM results show that the silicate matrix is revealed with DIC and RM, while sulfides can be imaged in 3D at low resolution by RM. Sulfides yield strong autofluorescence from 392 to 715 nm with TPF, while SHG is only produced by the embedding medium. Simultaneous recording of TPF and SHG images enables efficient discrimination between different components of silicate rocks. Image stacks obtained with MPM enable complete reconstruction of the 3D structure of a rock slice and of sulfide morphology at submicron resolution, which has not been previously reported for 3D imaging of minerals. Our work suggests that MPM is a highly efficient tool for 3D studies of microstructures and morphologies of minerals in silicate rocks, which may find other applications in geosciences.

Human round spermatids from azoospermic men exhibit oocyte-activation and Ca2+ oscillation-inducing activities

Zygote ◽  
2007 ◽  
Vol 15 (4) ◽  
pp. 337-346 ◽  
Author(s):  
H. Yazawa ◽  
K. Yanagida ◽  
A. Sato
Keyword(s):  
Laser Scanning ◽  
Round Spermatid ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Obstructive Azoospermia ◽  
Oocyte Activation ◽  
Cleavage Rate ◽  
Mouse Oocytes ◽  
Intracellular Ca ◽  
The Mean

SummaryDuring mammalian fertilization, intracellular Ca2+ oscillations are important for both oocyte activation and embryonic development. As the ability of round spermatids (ROS) to induce Ca2+ oscillations and oocyte activation is different between species, we examined Ca2+ oscillation- and oocyte activation-inducing abilities of human ROS originating from patients with non-obstructive azoospermia. Human ROS from 11 non-obstructive azoospermic patients were collected during their TESE–ICSI cycles. Following injection into mature unfertilized mouse oocytes, we examined the oocyte-activating and Ca2+ oscillation-inducing activities of ROS by using Ca2+ imaging and confocal laser scanning microscopy (mouse test). In these 11 cases, clinical TESE–ICSI using mature testicular spermatozoa was successful, with the exception of one case in which only one sperm-injected oocyte was not fertilized. The mean fertilization rate was 70.1% (40–100%); the mean cleavage rate was 97.9% (46/47). Two pregnancies were established from 10 transfer cycles (PR; 20%). When the ROS from these patients were injected into mouse oocytes, the ROS from all patients induced at least some intracellular Ca2+ oscillations (25–100%). In all patients, 40 out of 82 oocytes injected with ROS exhibited normal oscillation patterns of [Ca2+]i.Human spermatogenetic cells acquired oocyte-activating and Ca2+ oscillation-inducing abilities at the round spermatid stage, an earlier stage than found for rodent cells. These data indicate that human ROS might be useful for clinical treatments of non-obstructive azoospermic patients exhibiting mature spermatozoa in biopsied specimens.

E-C coupling failure in mouse EDL muscle after in vivo eccentric contractions

1998 ◽  
Vol 85 (1) ◽  
pp. 58-67 ◽  
Author(s):  
Christopher P. Ingalls ◽  
Gordon L. Warren ◽  
Jay H. Williams ◽  
Christopher W. Ward ◽  
R. B. Armstrong
Keyword(s):  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Eccentric Contractions ◽  
Confocal Laser ◽  
Defect Site ◽  
Uptake Rates ◽  
Intracellular Ca ◽  
Edl Muscle

The objectives of this research were to determine the contribution of excitation-contraction (E-C) coupling failure to the decrement in maximal isometric tetanic force (Po) in mouse extensor digitorum longus (EDL) muscles after eccentric contractions and to elucidate possible mechanisms. The left anterior crural muscles of female ICR mice ( n = 164) were injured in vivo with 150 eccentric contractions. Po, caffeine-, 4-chloro- m-cresol-, and K+-induced contracture forces, sarcoplasmic reticulum (SR) Ca2+release and uptake rates, and intracellular Ca2+ concentration ([Ca2+]i) were then measured in vitro in injured and contralateral control EDL muscles at various times after injury up to 14 days. On the basis of the disproportional reduction in Po (∼51%) compared with caffeine-induced force (∼11–21%), we estimate that E-C coupling failure can explain 57–75% of the Po decrement from 0 to 5 days postinjury. Comparable reductions in Po and K+-induced force (51%), and minor reductions (0–6%) in the maximal SR Ca2+ release rate, suggest that the E-C coupling defect site is located at the t tubule-SR interface immediately after injury. Confocal laser scanning microscopy indicated that resting [Ca2+]iwas elevated and peak tetanic [Ca2+]iwas reduced, whereas peak 4-chloro- m-cresol-induced [Ca2+]iwas unchanged immediately after injury. By 3 days postinjury, 4-chloro- m-cresol-induced [Ca2+]ibecame depressed, probably because of decreased SR Ca2+ release and uptake rates (17–31%). These data indicate that the decrease in Po during the first several days after injury primarily stems from a failure in the E-C coupling process.

scholarly journals Murine Salmonellosis Studied by Confocal Microscopy: Salmonella typhimurium Resides Intracellularly Inside Macrophages and Exerts a Cytotoxic Effect on Phagocytes In Vivo

1997 ◽  
Vol 186 (4) ◽  
pp. 569-580 ◽  
Author(s):  
Agneta Richter-Dahlfors ◽  
Alison M.J. Buchan ◽  
B. Brett Finlay
Keyword(s):  
Confocal Microscopy ◽  
Salmonella Typhimurium ◽  
Mouse Liver ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Phagocytic Cells ◽  
Intracellular Location ◽  
Subcellular Level

Salmonella typhimurium is considered a facultative intracellular pathogen, but its intracellular location in vivo has not been demonstrated conclusively. Here we describe the development of a new method to study the course of the histopathological processes associated with murine salmonellosis using confocal laser scanning microscopy of immunostained sections of mouse liver. Confocal microscopy of 30-μm-thick sections was used to detect bacteria after injection of ∼100 CFU of S. typhimurium SL1344 intravenously into BALB/c mice, allowing salmonellosis to be studied in the murine model using more realistic small infectious doses. The appearance of bacteria in the mouse liver coincided in time and location with the infiltration of neutrophils in inflammatory foci. At later stages of disease the bacteria colocalized with macrophages and resided intracellularly inside these macrophages. Bacteria were cytotoxic for phagocytic cells, and apoptotic nuclei were detected immunofluorescently, whether phagocytes harbored intracellular bacteria or not. These data argue that Salmonella resides intracellularly inside macrophages in the liver and triggers cell death of phagocytes, processes which are involved in disease. This method is also applicable to other virulence models to examine infections at a cellular and subcellular level in vivo.

3-D imaging of cells using a confocal laser scanning microscope and digital image processing

1988 ◽  
Vol 46 ◽  
pp. 96-97
Author(s):  
Thomas M. Jovin ◽  
Michel Robert-Nicoud ◽  
Donna J. Arndt-Jovin ◽  
Thorsten Schormann
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Scanning Microscope ◽  
Laser Scanning Microscope ◽  
Biochemical Processes ◽  
Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

Sign in / Sign up

Export Citation Format

Share Document