Measurement of [Ca] in single cells and pH in single organelles by fluorescence microscopy

1988 ◽  
Vol 46 ◽  
pp. 46-47
Author(s):  
F.R. Maxfield ◽  
M.L. Shelanski
Keyword(s):  
Single Cells ◽  
Living Cells ◽  
Free Calcium ◽  
Ion Concentrations ◽  
Calcium Indicator ◽  
Controlled Illumination ◽  
Computer Controlled ◽  
Temporal And Spatial ◽  
Intracellular Sorting ◽  
Indicator Dyes

Microscope spectrofluorometry and digital image processing provide the ability to study changes in ion concentrations in living cells with high temporal and spatial resolution. We have used fluorescein labeled macromolecules to measure the pH of specific endosomal compartments (1-3). The ratio of fluorescence intensities at 450 nm and 490 nm excitation provides a measure of the pH (4). The acidification of endosomes detected by this technique provide an explanation for endosome functions including intracellular sorting of ligands and receptors, release of iron from transferrin, and penetration of viruses and toxins into the cytosol (3).Using the tetracarboxylate calcium indicator dyes synthesized by R. Tsien and his colleagues (5), the same instruments can be used for measuring intracellular free calcium, [Ca2+]i, in single cells. We have used the system to measure [Ca2+]i changes associated with cell motility (6-9).Cells are examined using a Leitz Diavert microscope with a computer-controlled illumination and photometry system.

scholarly journals Calcium waves along the cleavage furrows in cleavage-stage Xenopus embryos and its inhibition by heparin.

1996 ◽  
Vol 135 (1) ◽  
pp. 181-190 ◽  
Author(s):  
A Muto ◽  
S Kume ◽  
T Inoue ◽  
H Okano ◽  
K Mikoshiba
Keyword(s):  
Calcium Transients ◽  
Free Calcium ◽  
Calcium Waves ◽  
Cleavage Furrow ◽  
Cleavage Stage ◽  
Xenopus Embryos ◽  
Calcium Indicator ◽  
Spatio Temporal ◽  
Indicator Dyes ◽  
Insp3 Receptor

Calcium signaling is known to be associated with cytokinesis; however, the detailed spatio-temporal pattern of calcium dynamics has remained unclear. We have studied changes of intracellular free calcium in cleavage-stage Xenopus embryos using fluorescent calcium indicator dyes, mainly Calcium Green-1. Cleavage formation was followed by calcium transients that localized to cleavage furrows and propagated along the furrows as calcium waves. The calcium transients at the cleavage furrows were observed at each cleavage furrow at least until blastula stage. The velocity of the calcium waves at the first cleavage furrow was approximately 3 microns/s, which was much slower than that associated with fertilization/egg activation. These calcium waves traveled only along the cleavage furrows and not in the direction orthogonal to the furrows. These observations imply that there exists an intracellular calcium-releasing activity specifically associated with cleavage furrows. The calcium waves occurred in the absence of extracellular calcium and were inhibited in embryos injected with heparin an inositol 1,4,5-trisphosphate (InsP3) receptor antagonist. These results suggest that InsP3 receptor-mediated calcium mobilization plays an essential role in calcium wave formation at the cleavage furrows.

IMAGING CALCIUM WAVES IN EGGS AND EMBRYOS

1993 ◽  
Vol 184 (1) ◽  
pp. 213-219 ◽  
Author(s):  
I Gillot ◽  
M Whitaker
Keyword(s):  
Latent Period ◽  
Calcium Concentration ◽  
Calcium Influx ◽  
Embryonic Cell ◽  
Calcium Wave ◽  
Free Calcium ◽  
Calcium Indicator ◽  
Free Calcium Concentration ◽  
Indicator Dyes ◽  
Indicator Dye

Sea urchin eggs and those of most other deuterostomes are activated at fertilization by an increase in cytoplasmic free calcium concentration ([Ca2+]i) that triggers the onset of the embryonic cell division cycles. We can image the calcium wave using fluorescent calcium indicator dyes and confocal microscopy. There are two components to the [Ca2+]i increase at fertilization. The first is due to a rapid calcium influx caused by a calcium action potential; this leads to a small increase in [Ca2+]i just beneath the plasma membrane with spherical symmetry. After a latent period of some 15 s, there is a second large and rapid increase in [Ca2+]i localized to the region of sperm-egg contact: during the latent period [Ca2+]i does not change but within 1 s of the end of the latent period [Ca2+]i reaches 2 micromolar. The calcium wave then spreads across the egg with a velocity of 5 micrometre s-1. Behind the advancing wavefront, the calcium concentration is uniformly high, even within the egg nucleus, though there are no indications that intranuclear calcium concentration differs from [Ca2+]i. [Ca2+]i falls uniformly towards resting levels over the next 500 s. In cases where there is an apparent inhomogeneity in [Ca2+]i in either the cortex or the nucleus, we find that the calcium indicator dye is inhomogeneously distributed. This appears to be due to uptake of the indicator dye (Fluo-3), probably into mitochondria. The artefact can be avoided by using a dextran-conjugated dye.

Fluorescence ratio imaging of dynamic intracellular ionic signals

1988 ◽  
Vol 46 ◽  
pp. 42-43
Author(s):  
R. Y. Tsien ◽  
A. Minta ◽  
M. Poenie ◽  
J.P.Y. Kao ◽  
A. Harootunian
Keyword(s):  
Binding Sites ◽  
Living Cells ◽  
Molecular Engineering ◽  
Ph Indicators ◽  
Highly Sensitive ◽  
Fluorescence Ratio Imaging ◽  
Ratio Imaging ◽  
Technical Advances ◽  
Indicator Dyes ◽  
Fluorescein Dyes

Recent technical advances now enable the continuous imaging of important ionic signals inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+, or Mg2+. The Ca2+ indicators, exemplified by fura-2 and indo-1, derive their high affinity (Kd near 200 nM) and selectivity for Ca2+ to a versatile tetracarboxylate binding site3 modeled on and isosteric with the well known chelator EGTA. The most commonly used pH indicators are fluorescein dyes (such as BCECF) modified to adjust their pKa's and improve their retention inside cells. Na+ indicators are crown ethers with cavity sizes chosen to select Na+ over K+: Mg2+ indicators use tricarboxylate binding sites truncated from those of the Ca2+ chelators, resulting in a more compact arrangement of carboxylates to suit the smaller ion.

scholarly journals Timing of Ca2+ Release from Intracellular Stores and the Electrical Response of Limulus Ventral Photoreceptors to Dim Flashes

2000 ◽  
Vol 115 (6) ◽  
pp. 735-748 ◽  
Author(s):  
Richard Payne ◽  
James Demas
Keyword(s):  
Latent Period ◽  
Electrical Response ◽  
Cyclopiazonic Acid ◽  
Receptor Potential ◽  
Ion Concentration ◽  
Free Calcium ◽  
Flash Intensity ◽  
Stochastic Variation ◽  
Highly Correlated ◽  
Indicator Dyes

Light-induced release of Ca2+ from stores in Limulus ventral photoreceptors was studied using confocal fluorescence microscopy and the Ca2+ indicator dyes, Oregon green-5N and fluo-4. Fluorescence was collected from a spot within 4 μm of the microvillar membrane. A dual-flash protocol was used to reconstruct transient elevations of intracellular free calcium ion concentration (Cai) after flashes delivering between 10 and 5 × 105 effective photons. Peak Cai increased with flash intensity to 138 ± 76 μM after flashes delivering ∼104 effective photons, while the latent period of the elevation of Cai fell from ∼140 to 21 ms. The onset of the light-induced elevation of Cai was always highly correlated with that of the receptor potential. The time for Cai to exceed 2 μM was approximately equal to that for the receptor potential to exceed 8 mV (mean difference; 2.2 ± 6.4 ms). Cai was also measured during steps of light delivering ∼105 effective photons/s to photoreceptors that had been bleached with hydroxylamine so as to reduce their quantum efficiency. Elevations of Cai were detected at the earliest times of the electrical response to the steps of light, when a significant receptor potential had yet to develop. Successive responses exhibited stochastic variation in their latency of up to 20 ms, but the elevation of Cai and the receptor potential still rose at approximately the same time, indicating a shared process generating the latent period. Light-induced elevations of Cai resulted from Ca2+ release from intracellular stores, being abolished by cyclopiazonic acid (CPA), an inhibitor of endoplasmic reticulum Ca2+ pumps, but not by removal of extracellular Ca2+ ions. CPA also greatly diminished and slowed the receptor potential elicited by dim flashes. The results demonstrate a rapid release of Ca2+ ions that appears necessary for a highly amplified electrical response to dim flashes.

Regulation of proneural gene expression and cell fate during neuroblast segregation in the Drosophila embryo

Development ◽  
1992 ◽  
Vol 114 (4) ◽  
pp. 939-946 ◽  
Author(s):  
J.B. Skeath ◽  
S.B. Carroll
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
Single Cells ◽  
Regulatory Protein ◽  
Drosophila Embryo ◽  
Proneural Gene ◽  
Developmental Pathway ◽  
Neurogenic Genes ◽  
Temporal And Spatial ◽  
Epidermal Development

The Drosophila embryonic central nervous system develops from sets of progenitor neuroblasts which segregate from the neuroectoderm during early embryogenesis. Cells within this region can follow either the neural or epidermal developmental pathway, a decision guided by two opposing classes of genes. The proneural genes, including the members of the achaete-scute complex (AS-C), promote neurogenesis, while the neurogenic genes prevent neurogenesis and facilitate epidermal development. To understand the role that proneural gene expression and regulation play in the choice between neurogenesis and epidermogenesis, we examined the temporal and spatial expression pattern of the achaete (ac) regulatory protein in normal and neurogenic mutant embryos. The ac protein is first expressed in a repeating pattern of four ectodermal cell clusters per hemisegment. Even though 5–7 cells initially express ac in each cluster, only one, the neuroblast, continues to express ac. The repression of ac in the remaining cells of the cluster requires zygotic neurogenic gene function. In embryos lacking any one of five genes, the restriction of ac expression to single cells does not occur; instead, all cells of each cluster continue to express ac, enlarge, delaminate and become neuroblasts. It appears that one key function of the neurogenic genes is to silence proneural gene expression within the nonsegregating cells of the initial ectodermal clusters, thereby permitting epidermal development.

scholarly journals Cytosolic free calcium changes induced by chemotactic peptide in neutrophils from patients with chronic granulomatous disease

Blood ◽  
1984 ◽  
Vol 63 (1) ◽  
pp. 231-233 ◽  
Author(s):  
PD Lew ◽  
C Wollheim ◽  
RA Seger ◽  
T Pozzan
Keyword(s):  
Chronic Granulomatous Disease ◽  
Calcium Concentration ◽  
Cytosolic Calcium ◽  
Human Neutrophils ◽  
Free Calcium ◽  
Granulomatous Disease ◽  
Chemotactic Peptide ◽  
Intact Cells ◽  
Calcium Indicator ◽  
Free Calcium Concentration

Abstract Cytoplasmic free calcium concentration (Ca2+)i was measured in neutrophils from patients with the classical X-linked form of chronic granulomatous disease (CGD) by trapping the fluorescent calcium indicator Quin 2 in intact cells. CGD neutrophils do not produce superoxide and are only slightly depolarized upon stimulation by the chemotactic peptide. N-formyl-methionyl-leucyl-phenylalanine (FMLP). The resting levels, as well as (Ca2+)i changes induced by FMLP in CGD cells, were quantitatively and kinetically similar to those observed in normal cells. We conclude that the defect in CGD cells is distal to, or independent of, the changes in (Ca2+)i induced by FMLP stimulation and that normal membrane depolarization does not seem to be necessary for receptor-mediated rise in free cytosolic calcium in human neutrophils.

Measurement and Interpretation of Cytoplasmic [Ca2+] Signals From Calcium-Indicator Dyes

Physiology ◽  
2000 ◽  
Vol 15 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Stephen M. Baylor ◽  
Stephen Hollingworth
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Skeletal Muscle Fibers ◽  
Calcium Indicator ◽  
Indicator Dyes

Ca2+-indicator dyes are widely used in biology yet difficult to characterize inside cells. Studies in skeletal muscle fibers provide important information about indicator behavior and about Ca2+ signaling within the cytoplasm.

scholarly journals Simultaneous quantification of multiple endogenous biothiols in single living cells by plasmonic Raman probes

2017 ◽  
Vol 8 (11) ◽  
pp. 7582-7587 ◽  
Author(s):  
Shan-Shan Li ◽  
Qi-Yuan Guan ◽  
Mengmeng Zheng ◽  
Yu-Qi Wang ◽  
Deju Ye ◽  
...  
Keyword(s):  
Principal Component Analysis ◽  
Single Cells ◽  
Principal Component ◽  
Component Analysis ◽  
Living Cells ◽  
Simultaneous Quantification ◽  
Single Living Cells ◽  
Single Living

Three endogenous biothiols in single cells were simultaneously quantified by plasmonic Raman probes and quantitative principal component analysis (qPCA).

Template-Free Fabrication of Aligned Nanoarray for Quantifying the Nanosurface-Single Cells Interaction

2018 ◽  
Author(s):  
Biran Wang ◽  
Liming Wang ◽  
Shiren Wang
Keyword(s):  
Standard Deviation ◽  
High Reliability ◽  
Low Cost ◽  
Single Cells ◽  
Living Cells ◽  
Nanowire Arrays ◽  
Template Free ◽  
Sense And Respond ◽  
Single Living Cells ◽  
Single Living

In this paper, we for the first-time synthesized vertically aligned polyaniline (PANI) nanowire arrays on flat-end AFM tips via template-free solution methods. 4-Aminothiophenol was used for tailoring the nucleation size, chain propagation and orientation of the PANI nanowires. The microscopy characterization indicated that diameter was centered at a mean of 33.7 nm with a standard deviation of 6.5 nm, and length was centered at a mean of 50.3 nm with a standard deviation of 7.6 nm. PANI nanowire arrays are non-toxic, low-cost, and tunable, and thus PANI nanowire-grown tips could perfectly simulate different nanosurfaces. Via the force spectroscopy, we demonstrate the feasibility in quantifying the nanostructure-cell interactions at the single cell level in real time with high reliability and accuracy. This work will enable a new tool in precisely quantifying the interactions of single living cells and nanosurface, and thus opens a new door to understand how single living cells sense and respond to the specific nanostructures.

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