Impaired Intracellular Ca2+Dynamics in Live Cardiomyocytes Revealed by Rapid Line Scan Confocal Microscopy

2005 ◽  
Vol 11 (3) ◽  
pp. 235-243 ◽  
Author(s):  
David M. Plank ◽  
Mark A. Sussman
Keyword(s):  
Confocal Microscopy ◽  
Fluorescence Intensity ◽  
Linear Regression Analysis ◽  
Data Interpretation ◽  
Live Cells ◽  
Correction Factors ◽  
Pixel Intensity ◽  
Scanning Confocal Microscopy ◽  
Intracellular Ca ◽  
Average Pixel Intensity

Altered intracellular Ca2+dynamics are characteristically observed in cardiomyocytes from failing hearts. Studies of Ca2+handling in myocytes predominantly use Fluo-3 AM, a visible light excitable Ca2+chelating fluorescent dye in conjunction with rapid line-scanning confocal microscopy. However, Fluo-3 AM does not allow for traditional ratiometric determination of intracellular Ca2+concentration and has required the use of mathematic correction factors with values obtained from separate procedures to convert Fluo-3 AM fluorescence to appropriate Ca2+concentrations. This study describes methodology to directly measure intracellular Ca2+levels using inactivated, Fluo-3-AM-loaded cardiomyocytes equilibrated with Ca2+concentration standards. Titration of Ca2+concentration exhibits a linear relationship to increasing Fluo-3 AM fluorescence intensity. Images obtained from individual myocyte confocal scans were recorded, average pixel intensity values were calculated, and a plot is generated relating the average pixel intensity to known Ca2+concentrations. These standard plots can be used to convert transient Ca2+fluorescence obtained with experimental cells to Ca2+concentrations by linear regression analysis. Standards are determined on the same microscope used for acquisition of unknown Ca2+concentrations, simplifying data interpretation and assuring accuracy of conversion values. This procedure eliminates additional equipment, ratiometric imaging, and mathematic correction factors and should be useful to investigators requiring a straightforward method for measuring Ca2+concentrations in live cells using Ca2+-chelating dyes exhibiting variable fluorescence intensity.

scholarly journals TIRF Microscope Image Sequences of Fluorescent IgE-FcεRI Receptor Complexes inside a FcεRI-Centric Synapse in RBL-2H3 Cells

Data ◽  
2019 ◽  
Vol 4 (3) ◽  
pp. 111
Author(s):  
Rachel Drawbond ◽  
Kathrin Spendier
Keyword(s):  
Immunological Synapse ◽  
Immunoglobulin E ◽  
Image Sequences ◽  
Live Cells ◽  
Background Intensity ◽  
Pixel Intensity ◽  
Microscope Image ◽  
Signaling Complex ◽  
Receptor Complexes ◽  
Average Pixel Intensity

Total internal reflection fluorescence (TIRF) microscope image sequences are commonly used to study receptors in live cells. The dataset presented herein facilitates the study of the IgE-FcεRI receptor signaling complex (IgE-RC) in rat basophilic leukemia (RBL-2H3) cells coming into contact with a supported lipid bilayer with 25 mol% N-dinitrophenyl-aminocaproyl phosphatidylethanolamine, modeling an immunological synapse. TIRF microscopy was used to image IgE-RCs within this FcεRI-centric synapse by loading RBL-2H3 cells with fluorescent anti-dinitrophenyl (anti-DNP) immunoglobulin E (IgE) in suspension for 24 h. Fluorescent anti-DNP IgE (IgE488) concentrations of this suspension increased from 10% to 100% and corresponding non-fluorescent anti-DNP IgE concentrations decreased from 90% to 0%. After the removal of unbound anti-DNP IgE, multiple image sequences were taken for each of these ten conditions. Prior to imaging, anti-DNP IgE-primed RBL-2H3 cells were either kept for a few minutes, for about 30 min, or for about one hour in Hanks buffer. The dataset contains 482 RBL-2H3 model synapse image stacks, dark images to correct for background intensity, and TIRF illumination profile images to correct for non-uniform TIRF illumination. After background subtraction, non-uniform illumination correction, and conversion of pixel units from analog-to-digital units to photo electrons, the average pixel intensity was calculated. The average pixel intensity within FcεRI-centric synapses for all three Hanks buffer conditions increased linearly at a rate of 0.42 ± 0.02 photo electrons per pixel per % IgE488 in suspension. RBL-2H3 cell degranulation was tested by detecting β-hexosaminidase activity. Prolonged RBL-2H3 cell exposure to Hanks buffer inhibited exocytosis in RBL-2H3 cells.

Laser Scanning Confocal Microscopy and Three-Dimesnsional Reconstruction of the Mitotic Spindles of Unequally Dividing Embryonic Cells

1990 ◽  
Vol 48 (3) ◽  
pp. 166-167
Author(s):  
J. Holy ◽  
G. Schatten
Keyword(s):  
Confocal Microscopy ◽  
Mitotic Spindle ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Confocal Microscopy ◽  
Two Dimensions ◽  
Embryonic Cells ◽  
Mitotic Spindles ◽  
Cleavage Division ◽  
Scanning Confocal Microscopy

One of the classic limitations of light microscopy has been the fact that three dimensional biological events could only be visualized in two dimensions. Recently, this shortcoming has been overcome by combining the technologies of laser scanning confocal microscopy (LSCM) and computer processing of microscopical data by volume rendering methods. We have employed these techniques to examine morphogenetic events characterizing early development of sea urchin embryos. Specifically, the fourth cleavage division was examined because it is at this point that the first morphological signs of cell differentiation appear, manifested in the production of macromeres and micromeres by unequally dividing vegetal blastomeres.The mitotic spindle within vegetal blastomeres undergoing unequal cleavage are highly polarized and develop specialized, flattened asters toward the micromere pole. In order to reconstruct the three-dimensional features of these spindles, both isolated spindles and intact, extracted embryos were fluorescently labeled with antibodies directed against either centrosomes or tubulin.

Do We have a Satisfactory Cell Viability Assay? Review of the Currently Commercially-Available Assays

2020 ◽  
Vol 17 (1) ◽  
pp. 2-22 ◽  
Author(s):  
Abdel-Baset Halim
Keyword(s):  
Cell Viability ◽  
Data Interpretation ◽  
Positive Control ◽  
Live Cells ◽  
Proof Of Concept ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
Current Cell ◽  
Dying Cells ◽  
Differential Permeability

:Cell-based assays are an important part of the drug discovery process and clinical research. One of the main hurdles is to design sufficiently robust assays with adequate signal to noise parameters while maintaining the inherent physiology of the cells and not interfering with the pharmacology of target being investigated.:A plethora of assays that assess cell viability (or cell heath in general) are commercially available and can be classified under different categories according to their concepts and principle of reactions. The assays are valuable tools, however, suffer from a large number of limitations. Some of these limitations can be procedural or operational, but others can be critical as those related to a poor concept or the lack of proof of concept of an assay, e.g. those relying on differential permeability of dyes in-and-out of viable versus compromised cell membranes. While the assays can differentiate between dead and live cells, most, if not all, of them can just assess the relative performance of cells rather than providing a clear distinction between healthy and dying cells. The possible impact of relatively high molecular weight dyes, used in most of the assay, on cell viability has not been addressed. More innovative assays are needed, and until better alternatives are developed, setup of current cell-based studies and data interpretation should be made with the limitations in mind. Negative and positive control should be considered whenever feasible. Also, researchers should use more than one orthogonal method for better assessment of cell health.

Observation of Fine Structure in Bicontinuous Phase-Separated Domains of a Polymer Blend by Laser Scanning Confocal Microscopy

2001 ◽  
Vol 34 (15) ◽  
pp. 5186-5191 ◽  
Author(s):  
Hiroshi Jinnai ◽  
Hiroshi Yoshida ◽  
Kohtaro Kimishima ◽  
Yoshinori Funaki ◽  
Yoshitsugu Hirokawa ◽  
...  
Keyword(s):  
Fine Structure ◽  
Confocal Microscopy ◽  
Polymer Blend ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Scanning Confocal Microscopy

scholarly journals The Contraction of Smooth Muscle Cells of Intrapulmonary Arterioles Is Determined by the Frequency of Ca2+ Oscillations Induced by 5-HT and KCl

2005 ◽  
Vol 125 (6) ◽  
pp. 555-567 ◽  
Author(s):  
Jose F. Perez ◽  
Michael J. Sanderson
Keyword(s):  
Pulmonary Hypertension ◽  
Smooth Muscle ◽  
Confocal Microscopy ◽  
Smooth Muscle Cells ◽  
Phase Contrast ◽  
Muscle Cells ◽  
Low Frequencies ◽  
Scanning Confocal Microscopy ◽  
Agonist Concentration ◽  
Mouse Lungs

Increased resistance of the small blood vessels within the lungs is associated with pulmonary hypertension and results from a decrease in size induced by the contraction of their smooth muscle cells (SMCs). To study the mechanisms that regulate the contraction of intrapulmonary arteriole SMCs, the contractile and Ca2+ responses of the arteriole SMCs to 5-hydroxytrypamine (5-HT) and KCl were observed with phase-contrast and scanning confocal microscopy in thin lung slices cut from mouse lungs stiffened with agarose and gelatin. 5-HT induced a concentration-dependent contraction of the arterioles. Increasing concentrations of extracellular KCl induced transient contractions in the SMCs and a reduction in the arteriole luminal size. 5-HT induced oscillations in [Ca2+]i within the SMCs, and the frequency of these Ca2+ oscillations was dependent on the agonist concentration and correlated with the extent of sustained arteriole contraction. By contrast, KCl induced Ca2+ oscillations that occurred with low frequencies and were preceded by small, localized transient Ca2+ events. The 5-HT–induced Ca2+ oscillations and contractions occurred in the absence of extracellular Ca2+ and were resistant to Ni2+ and nifedipine but were abolished by caffeine. KCl-induced Ca2+ oscillations and contractions were abolished by the absence of extracellular Ca2+ and the presence of Ni2+, nifedipine, and caffeine. Arteriole contraction was induced or abolished by a 5-HT2–specific agonist or antagonist, respectively. These results indicate that 5-HT, acting via 5-HT2 receptors, induces arteriole contraction by initiating Ca2+ oscillations and that KCl induces contraction via Ca2+ transients resulting from the overfilling of internal Ca2+ stores. We hypothesize that the magnitude of the sustained intrapulmonary SMC contraction is determined by the frequency of Ca2+ oscillations and also by the relaxation rate of the SMC.

scholarly journals Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy

2007 ◽  
Vol 2 (9) ◽  
pp. 2295-2301 ◽  
Author(s):  
Partha Mukhopadhyay ◽  
Mohanraj Rajesh ◽  
György Haskó ◽  
Brian J Hawkins ◽  
Muniswamy Madesh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Confocal Microscopy ◽  
Simultaneous Detection ◽  
Superoxide Production ◽  
Live Cells ◽  
Mitochondrial Superoxide

scholarly journals The DNA content of trophozoites and cysts of Giardia lamblia by microdensitometric quantitation of Feulgen staining and examination by laser scanning confocal microscopy.

1994 ◽  
Vol 42 (11) ◽  
pp. 1413-1416 ◽  
Author(s):  
S L Erlandsen ◽  
E M Rasch
Keyword(s):  
Confocal Microscopy ◽  
Genome Size ◽  
Dna Content ◽  
Giardia Lamblia ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Evolutionary Development ◽  
Scanning Confocal Microscopy ◽  
Dividing Cells ◽  
C Value

We investigated direct measurement of the DNA content of the parasitic intestinal flagellate Giardia lamblia through quantitation by Feulgen microspectrophotometry and also by visualization of Feulgen-stained DNA chromosomes within dividing cells by laser scanning confocal microscopy. Individual trophozoites of Giardia (binucleate) contained 0.144 +/- 0.018 pg of DNA/cell or 0.072 pg DNA/nucleus. Giardia lamblia cysts (quadranucleate) contained 0.313 +/- 0.003 pg DNA or 0.078 pg DNA/nucleus. The genome size (C) value per nucleus ranged between 6.5-7.1 x 10(7) BP for trophozoites and cysts, respectively. Confocal microscopic examination of Giardia trophozoites undergoing binary fission revealed five chromosome-like bodies within each nucleus. Further information about genome size and DNA content within different Giardia species may help to clarify the pivotal role of these primitive eukaryotic cells in evolutionary development.

The Effect of Chloroquine on the Apoptosis Induced by Cisplatin in Human Gastric Cancer BGC823 Cells

2014 ◽  
Vol 926-930 ◽  
pp. 1124-1127
Author(s):  
Zhen Xun Jin ◽  
Li Li Zhang ◽  
Yan Wang ◽  
Lin Chuan Zeng ◽  
Yang Yu ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Confocal Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Combination Group ◽  
Human Gastric Cancer ◽  
Apoptotic Cells ◽  
Toxic Dose ◽  
Scanning Confocal Microscopy ◽  
Mtt Tests

The aim of this study is to investigate the effects and mechanism of chloroquine (CQ) on the apoptosis induced by cisplatin in human gastric cancer BGC823 cells. MTT assay was used to detect the state of cell growth. The appearances of cellular apoptosis were detected by laser scanning confocal microscopy and light microscopy. The expressions of LC3 and p62 were detected by laser scanning confocal microscopy. MTT tests showed that the non-toxic dose of CQ could increase the inhibition rate of BGC823 cells induced by cisplatin. Under the light microscope, the ratio of apoptotic cells in the group treated with non-toxic dose of CQ combined with cisplatin was higher than that in the group treated with cisplatin alone. Hoechst33342 staining showed that the ratio of apoptotic cells in the combination group was higher than that in the cisplatin group. The expression and colocalization of LC3 and p62 proteins were significantly increased in the combination group. These results indicate that CQ can enhance the cell apoptosis induced by cisplatin in BGC823 cells, which is through the inhibition of autophagy.

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