Practical Methods for In Vivo Cortical Physiology with 2-Photon Microscopy and Bulk Loading of Fluorescent Calcium Indicator Dyes

2014 ◽  
pp. 117-141
Author(s):  
Stephen D. Van Hooser ◽  
Elizabeth N. Johnson ◽  
Ye Li ◽  
Mark Mazurek ◽  
Julie H. Culp ◽  
...  
Keyword(s):  
Calcium Indicator ◽  
Bulk Loading ◽  
Cortical Physiology ◽  
Indicator Dyes

scholarly journals Light Dependence of Calcium and Membrane Potential Measured in Blowfly Photoreceptors In Vivo

1998 ◽  
Vol 112 (2) ◽  
pp. 113-124 ◽  
Author(s):  
Johannes Oberwinkler ◽  
Doekele G. Stavenga
Keyword(s):  
Membrane Potential ◽  
Light Intensity ◽  
Light Adaptation ◽  
Photoreceptor Cell ◽  
Dynamic Range ◽  
Bright Light ◽  
Molecular Probes ◽  
Calcium Indicator ◽  
Indicator Dyes

Light adaptation in insect photoreceptors is caused by an increase in the cytosolic Ca2+ concentration. To better understand this process, we measured the cytosolic Ca2+ concentration in vivo as a function of adapting light intensity in the white-eyed blowfly mutant chalky. We developed a technique to measure the cytosolic Ca2+ concentration under conditions as natural as possible. The calcium indicator dyes Oregon Green 1, 2, or 5N (Molecular Probes, Inc., Eugene, OR) were iontophoretically injected via an intracellular electrode into a photoreceptor cell in the intact eye; the same electrode was also used to measure the membrane potential. The blue-induced green fluorescence of these dyes could be monitored by making use of the optics of the facet lens and the rhabdomere waveguide. The use of the different Ca2+-sensitive dyes that possess different affinities for Ca2+ allowed the quantitative determination of the cytosolic Ca2+ concentration in the steady state. Determining the cytosolic Ca2+ concentration as a function of the adapting light intensity shows that the Ca2+ concentration is regulated in a graded fashion over the whole dynamic range where a photoreceptor cell can respond to light. When a photoreceptor is adapted to bright light, the cytosolic Ca2+ concentration reaches stable values higher than 10 μM. The data are consistent with the hypothesis that the logarithm of the increase in cytosolic Ca2+ concentration is linear with the logarithm of the light intensity. From the estimated values of the cytosolic Ca2+ concentration, we conclude that the Ca2+-buffering capacity is limited. The percentage of the Ca2+ influx that is buffered gradually decreases with increasing Ca2+ concentrations; at cytosolic Ca2+ concentration levels above 10 μM, buffering becomes minimal.

Evidence against electrophoresis as the principal mode of protein transport in vitellogenic ovarian follicles of Drosophila

Development ◽  
1987 ◽  
Vol 101 (2) ◽  
pp. 279-288
Author(s):  
J. Bohrmann ◽  
H. Gutzeit
Keyword(s):  
Nurse Cell ◽  
Protein Transport ◽  
Exchange Chromatography ◽  
Heterologous Proteins ◽  
Nurse Cells ◽  
Cell Cytoplasm ◽  
Two Dimensional Gel Electrophoresis ◽  
Electrophoretic Transport ◽  
Indicator Dyes

Charged cell constituents in polytrophic insect follicles are thought to be transported in the nurse cell-oocyte syncytium by way of electrophoresis. This concept, proposed by Woodruff & Telfer (1980) was based on electrophysiological data and microinjection of heterologous proteins using Hyalophora follicles. By microinjecting fluorescently labelled acidic and basic proteins into the nurse cells or oocyte of vitellogenic Drosophila follicles, we failed to obtain evidence for charge-dependent migration of these molecules. We have also analyzed the proteins of nurse cells and oocyte on isoelectric focusing gels, by means of two-dimensional gel electrophoresis, and by ion exchange chromatography to see if basic or acidic proteins accumulate in vivo in nurse cells and oocyte, respectively. For the bulk of the follicular proteins we found no accumulation. Further evidence against an electrophoretic transport system in Drosophila was obtained by estimating the intracellular pH from the colour of indicator dyes microinjected into the follicles; the results indicate that the pH in the nurse cell cytoplasm is lower than that in the ooplasm. According to the model developed for Hyalophora, electrophoretic transport would be favoured by high pH in the nurse cell cytoplasm.

Abstract 17071: Cell Communication Inference in Macrophages

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Nika Taghdiri ◽  
Kevin R King ◽  
David Calcagno ◽  
Zhenxing Fu ◽  
Kenneth Huang ◽  
...  
Keyword(s):  
Image Analysis ◽  
Analytical Methods ◽  
Cell Injury ◽  
Diffusion Mechanism ◽  
Cell Communication ◽  
In Vivo Studies ◽  
Cell Dynamics ◽  
Calcium Indicator

Introduction: Tissue macrophages play diverse roles in the cardiovascular system during health and disease. They have diverse functions within tissues, but our understanding of their dynamics is limited because most macrophage characterization assays are destructive and have low temporal resolution. We asked whether these cells are dynamic and interconnected. Methods: Here, we describe experimental and analytical methods for measuring cell dynamics and inferring communication between cells in vitro and in vivo. We created a mouse (Csf1r-Cre x GCaMP5) expressing the Cre-inducible genetically encoded calcium indicator GCaMP5 under the regulation of the innate immune promoter, Csf1r, to non-destructively quantify high-frequency cell dynamics and differentiated them in culture using m-CSF. We developed custom image analysis routines and parameterization strategies for classifying calcium responses. Results: Our studies revealed that calcium reporter BMDMs display minimal fluctuations at baseline but exhibit a dynamic response to immunogenic DNA sensing. DNA-induced isolated cell injury and death, which precipitated cell communication that spread with a velocity of [9μm/s], consistent with an extracellular diffusion mechanism. We developed quantitative image analysis methods that corrected for random calcium fluctuations and identified statistically significant areas of correlated calcium changes suggestive of communication. An analytical pipeline enabled quantification of calcium spike dynamics and correlations of dynamic calcium profiles of single cell sharing a local microenvironment. This resulted in an “improbable synchrony” metric that allowed localization of communication in time and space. We adapted the pipeline for in vivo studies and tested them in a dorsal window chamber model using intravital microscopy. At 2Hz sampling frequency, we identified 27 potential communication events as they responded to complex microenvironmental cues in vivo. Conclusion: The experimental and analytical methods for inferring cell communication provide a new quantitative toolkit for investigating known as-yet undiscovered cell communication pathways.

scholarly journals Thy1 transgenic mice expressing the red fluorescent calcium indicator jRGECO1a for neuronal population imaging in vivo

PLoS ONE ◽  
2018 ◽  
Vol 13 (10) ◽  
pp. e0205444 ◽  
Author(s):  
Hod Dana ◽  
Ondrej Novak ◽  
Michael Guardado-Montesino ◽  
James W. Fransen ◽  
Amy Hu ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Neuronal Population ◽  
Calcium Indicator ◽  
Population Imaging

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.

Capillary perfusion pattern and microvascular geometry in heterogeneous hypoxic areas of hypoperfused rat myocardium

1995 ◽  
Vol 268 (6) ◽  
pp. H2183-H2194 ◽  
Author(s):  
F. Vetterlein ◽  
M. Prange ◽  
D. Lubrich ◽  
J. Pedina ◽  
M. Neckel ◽  
...  
Keyword(s):  
Capillary Flow ◽  
Flow Distribution ◽  
Histochemical Staining ◽  
Low Flow ◽  
Lower Percentage ◽  
Capillary Perfusion ◽  
Indicator Dyes ◽  
Myocardial Biopsies

The origin of heterogeneities in tissue oxygenation due to low-flow ischemia was studied in hypoperfused myocardium of anesthetized rats. In frozen sections of myocardial biopsies the localization of increases in NADH fluorescence, an indicator of tissue hypoxia, was compared with microvascular flow distribution and capillary geometry. The latter parameters were accomplished through capillary labeling with indicator dyes in vivo and enzyme-histochemical staining in vitro, respectively. Most NADH-fluorescent areas were found to have developed despite sustained capillary flow. When the fractions of arterial, venous, and intermediate capillary segments were analyzed within circumscribed hypoxic fields (< 200 microns diam), frequencies of 30.7 +/- 6.1, 35.3 +/- 5.3, and 30.8 +/- 5.0%, respectively, were found. In contrast, a significantly higher fraction of arterial segments (63.2 +/- 3.3%) and a lower percentage of venous segments (16.4 +/- 2.5%) were determined in nonhypoxic islands enclosed by hypoxic tissue. These results support the view that the latter zones are located near the arterial portion of the capillary bed where their oxygenation is favored during low-flow states. This effect appears to contribute to the supply heterogeneities in hypoperfused myocardium.

Encapsulation of Insulin-Secreting Cells Expressing a Genetically Encoded Fluorescent Calcium Indicator for Cell-Based Sensing In Vivo

2016 ◽  
Vol 6 (4) ◽  
pp. 1600869 ◽  
Author(s):  
Christophe Boss ◽  
Umberto De Marchi ◽  
Aurélie Hermant ◽  
Mouna Conrad ◽  
Federico Sizzano ◽  
...  
Keyword(s):  
Calcium Indicator ◽  
Insulin Secreting Cells

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.

scholarly journals In vivo multiphoton microscopy of cardiomyocyte calcium dynamics in the beating mouse heart

2018 ◽  
Author(s):  
Jason S. Jones ◽  
David M. Small ◽  
Nozomi Nishimura
Keyword(s):  
Action Potentials ◽  
Multiphoton Microscopy ◽  
Three Dimensional ◽  
Heart Beat ◽  
Beating Heart ◽  
Mouse Heart ◽  
Calcium Dynamics ◽  
Intact Mouse ◽  
Calcium Indicator

AbstractWe demonstrated intravital multiphoton microscopy in the beating heart in an intact mouse and optically measured action potentials with GCaMP6f, a genetically-encoded calcium indicator. Images were acquired at 30 fps with spontaneous heart beat and continuously running ventilated breathing. The data were reconstructed into three-dimensional volumes showing tissue structure, displacement, and GCaMP activity in cardiomyocytes as a function of both the cardiac and respiratory cycle.

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