Intracellular Ca2+ measurements in live cells by rapid line scan confocal microscopy: simplified calibration methodology

2004 ◽  
Vol 25 (3-4) ◽  
pp. 123-133 ◽  
Author(s):  
David M. Plank ◽  
Mark A. Sussman
Keyword(s):  
Confocal Microscopy ◽  
Live Cells ◽  
Line Scan

Intracellular Ca2+ Measurements in Live Cells by Rapid Line Scan Confocal Microscopy: Simplified Calibration Methodology

2004 ◽  
Vol 10 (S02) ◽  
pp. 1390-1391
Author(s):  
David M. Plank ◽  
Mark A. Sussman
Keyword(s):  
Confocal Microscopy ◽  
Live Cells ◽  
Line Scan

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

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 AIE-Driven Fluorescent Polysaccharide Polymersome as Enzyme-responsive FRET Nanoprobe to Study the Real-time Delivery Aspects in Live-Cells

2021 ◽  
Author(s):  
Nilesh Umakant Deshpande ◽  
Mishika Virmani ◽  
Manickam Jayakannan
Keyword(s):  
Energy Transfer ◽  
Confocal Microscopy ◽  
Real Time ◽  
Fluorescence Resonance Energy Transfer ◽  
Imaging Techniques ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Intracellular Enzyme ◽  
Bio Imaging

We report aggregation induced emission (AIE) driven polysaccharide polymersome as fluorescence resonance energy transfer (FRET) nanoprobes to study their intracellular enzyme-responsive delivery by real-time live-cell confocal microscopy bio-imaging techniques. AIE...

Comparison study of live cells by atomic force microscopy, confocal microscopy, and scanning electrochemical microscopy

2007 ◽  
Vol 85 (3) ◽  
pp. 175-183 ◽  
Author(s):  
Xiaocui Zhao ◽  
Nils O Petersen ◽  
Zhifeng Ding
Keyword(s):  
Atomic Force Microscopy ◽  
Confocal Microscopy ◽  
Scanning Electrochemical Microscopy ◽  
Time Lapse ◽  
Live Cells ◽  
Tapping Mode ◽  
Force Microscopy ◽  
Atomic Force ◽  
Tapping Mode Afm ◽  
Electrochemical Microscopy

In this report, three kinds of scanning probe microscopy techniques, atomic force microscopy (AFM), confocal microscopy (CM), and scanning electrochemical microscopy (SECM), were used to study live cells in the physiological environment. Two model cell lines, CV-1 and COS-7, were studied. Time-lapse images were obtained with both contact and tapping mode AFM techniques. Cells were more easily scratched or moved by contact mode AFM than by tapping mode AFM. Detailed surface structures such as filamentous structures on the cell membrane can be obtained and easily discerned with tapping mode AFM. The toxicity of ferrocenemethanol (Fc) on live cells was studied by CM in reflection mode by recording the time-lapse images of controlled live cells and live cells with different Fc concentrations. No significant change in the morphology of cells was caused by Fc. Cells were imaged by SECM with Fc as the mediator at a biased potential of 0.35 V (vs. Ag/AgCl with a saturated KCl solution). Cells did not change visibly within 1 h, which indicated that SECM was a noninvasive technique and thus has a unique advantage for the study of soft cells, since the electrode scanned above the cells instead of in contact with them. Reactive oxygen species (ROS) generated by the cells were detected and images based on these chemical species were obtained. It is demonstrated that SECM can provide not only the topographical images but also the images related to the chemical or biochemical species released by the live cells.Key words: live cells, atomic force microscopy, confocal microscopy, scanning electrochemical microscopy.

scholarly journals Confocal Microscopy in Conjunction With Haemocytometry to Evaluate the Imperative Physical Characteristics of Multicellular Tumor Spheroids

2021 ◽  
Author(s):  
Aziz UR RAHMAN
Keyword(s):  
Confocal Microscopy ◽  
Drug Uptake ◽  
Live Cells ◽  
Multicellular Tumor Spheroids ◽  
Tumor Spheroids ◽  
Oriented Growth ◽  
Trypan Blue Exclusion ◽  
Trypan Blue Exclusion Method

Abstract Background: Tumor tissues resist penetration of therapeutic molecules. Multicellular tumor spheroids (MCTSs) were used as an in vitro tumor model. The aim of this study was to determine the growth of MCTSs with the age of spheroids, which could be applied and compared with in vivo drug uptake and penetration. Method: Spheroids were generated by liquid overlay techniques, and their diameter was measured by confocal microscopy for up to two weeks. The trypan blue exclusion method was used to count dead and live cells separately via a hemocytometer. Results: The pentaphysical characteristics of spheroids, including diameter, cell number, volume per cell, viability status, and estimated shell of viable and core of dead cells, were determined. The growth of spheroids was linear over the first week but declined in the 2nd week, which may be due to an overconcentration of dead cells and degraded products inside the spheroids, hence lowering the ratio of live cells in spheroids. Compaction of spheroids occurs from day 3 to day 7, with the mature spheroids having a low amount of extracellular space compared to intracellular volume. Conclusion: Age-oriented growth of MCTSs provides a rationale to predict less rapid penetration as spheroids get older and could be correlated with in vivo tumors to predict pharmaceutical and therapeutic intervention.

scholarly journals Multi-Dimensional Laser Confocal Microscopy on Live Cells in Submicroliter Volumes Using Glass Capillaries

2006 ◽  
Vol 39 (4) ◽  
pp. 103-106 ◽  
Author(s):  
Emilie Flaberg ◽  
György Stuber ◽  
Laszlo Szekely
Keyword(s):  
Confocal Microscopy ◽  
Live Cells ◽  
Laser Confocal Microscopy ◽  
Glass Capillaries

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 Fast volumetric imaging with line-scan confocal microscopy by an electro-tunable lens

2021 ◽  
Author(s):  
Khuong Duy Mac ◽  
Muhammad Mohsin Qureshi ◽  
Myeongsu Na ◽  
Sunghoe Chang ◽  
Hyuk-Sang Kwon ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
High Performance ◽  
Imaging System ◽  
Ex Vivo ◽  
Resonant Mode ◽  
Biological Tissues ◽  
Line Scan ◽  
Rapid Acquisition ◽  
Tunable Lens

AbstractIn microscopic imaging of biological tissues, particularly real-time visualization of neuronal activities, rapid acquisition of volumetric images poses a prominent challenge. Typically, two-dimensional (2D) microscopy can be devised into an imaging system with 3D capability using any varifocal lens. Despite the conceptual simplicity, such an upgrade yet requires additional, complicated device components and suffers a reduced acquisition rate, which is critical to document neuronal dynamics properly. In this study, we implemented an electro-tunable lens (ETL) in the line-scan confocal microscopy, enabling the volumetric acquisition at the rate of 20 frames per second with the maximum volume of interest of 315 × 315 × 80 μm3. The axial extent of point-spread-function (PSF) was 17.6 ± 1.6 μm and 90.4 ± 2.1 μm with the ETL operating in either stationary or resonant mode, respectively, revealing significant depth elongation by the resonant mode ETL microscopy. We further demonstrated the utilities of the ETL system by volume imaging of cleared mouse brain ex vivo samples and in vivo brains. The current study foregrounds the successful application of resonant ETL for constructing a basis for a high-performance 3D line-scan confocal microscopy system, which will enhance our understanding of various dynamic biological processes.

scholarly journals New Lipophilic Fluorescent Dyes for Exosome Labeling: Monitoring of Cellular Uptake of Exosomes

2020 ◽  
Author(s):  
Takashi Shimomura ◽  
Ryo Seino ◽  
Kaori Umezaki ◽  
Asako Shimoda ◽  
Takatoshi Ezoe ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Zeta Potential ◽  
Cellular Uptake ◽  
Fluorescent Probes ◽  
Fluorescent Dyes ◽  
Live Cells ◽  
Nanoparticle Tracking Analysis ◽  
Excellent Performance ◽  
Potential Measurement ◽  
Highly Sensitive

ABSTRACTWe have developed three types of exosomal membrane binding fluorescent probes, Mem Dye-Green, Mem Dye-Red and Mem Dye-Deep Red, to monitor exosome uptake into cells. The dyes contain a cyanine group as a fluorescent scaffold, which allows for highly sensitive fluorescence imaging of the exosome. These dyes can also be used to observe the dynamics of exosomes in live cells. The use of PKH dyes (Figure 1), which are currently the most widely-used fluorescent probes for exosome labeling, has some limitations. For example, PKH dyes tend to aggregate to form exosome-like nanoparticles, and these nanoparticles are uptaken by cells. Moreover, Mehdi suggested that the use of PKH dyes triggers an enlargement of the exosome size owing to membrane fusion or intercalation. To overcome the limitations of PKH dyes, we introduce amphiphilic moieties to the cyanine. To investigate the characteristics of the Mem Dyes as exosome labeling probes, we perform nanoparticle tracking analysis (NTA), zeta potential measurement and confocal microscopy. The Mem Dyes show excellent performance for exosome labeling (no aggregation and less size shift).

Abstract 599: Lighting Up P2Y12 Oligomers: Insights into the Role of Oligomerization in P2Y12 Function

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Aasma Khan ◽  
Jeffrey L Caplan ◽  
Donna Woulfe
Keyword(s):  
Flow Cytometry ◽  
Confocal Microscopy ◽  
G Protein ◽  
Time Lapse ◽  
Bimolecular Fluorescence Complementation ◽  
P2y12 Receptor ◽  
Live Cells ◽  
Bleeding Disorders ◽  
Mutant Forms

Introduction: Little is known about the role of P2Y12 oligomerization in receptor function and whether P2Y12 receptor mutations associated with human bleeding disorders may be explained by alterations in oligomerization. Objectives: 1) To determine whether P2Y12 homo- and hetero-oligomers are constitutive or dynamically regulated. 2) To explore whether P2Y12 mutants R256Q and R265W (previously detected in patients with abnormal bleeding, but with unaltered ADP binding) have different oligomerization affinities or kinetics and determine whether differences in P2Y12 oligomerization explain the functional defects. Methods: We employed a Venus-based Bimolecular Fluorescence Complementation (BiFC) approach in HEK293T cells transiently co-expressing P2Y12 or its mutant forms (R256Q or R265W) tagged with either the N-terminal (P2Y12-VN) or C terminal fragment (P2Y12-VC) of Venus, to characterize their homomeric interactions, in live cells using confocal microscopy and quantitative flow cytometry assays. Results: Agonist-independent formation of P2Y12 receptor homo-oligomers were detected on cell membranes. Time lapse imaging showed movement of P2Y12 receptor pairs from the endoplamic reticulum and Golgi network to the plasma membrane, suggesting that they are constitutive and required for export. Co-expression of P2Y12-VN with increasing amounts of P2Y12-VC demonstrated a dose-dependent increase in the fluorescence intensity of Venus, and reached saturation at a ratio of 1:3. Interestingly, the fluorescence intensities of homomeric P2Y12-R256Q-VN and R256Q-VC and, separately, P2Y12-R265W-VN and P2Y12R265W-VC were almost 4 times stronger than that of the wild type receptor as quantified in flow cytometry-based BiFC. Similar results were obtained in confocal microscopy. This suggests that these P2Y12 mutants form an increased number of dimers or oligomers with increased self-affinities. Conclusion: We demonstrate that P2Y12 forms constitutive homo-oligomers. Two mutations associated with bleeding disorders in patients have altered receptor-receptor interactions. Future investigation will explore the effect of mutations and receptor oligomers on G protein coupling and receptor: G protein stoichiometry.

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