Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca<sup>2+</sup> Signaling In Situ

Bernard T. Drumm; Grant W. Hennig; Salah A. Baker; Kenton M. Sanders

doi:10.3791/58989

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellula...

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Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca²⁺ Signaling In Situ

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

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09:34 min

January 7, 2019

DOI: 10.3791/58989-v

Bernard T. Drumm¹, Grant W. Hennig², Salah A. Baker¹, Kenton M. Sanders¹

¹Department of Physiology and Cell Biology,University of Nevada Reno School of Medicine, ²Department of Pharmacology, The Robert Larner, M.D. College of Medicine,University of Vermont

Overview

This study focuses on improving the analysis of calcium imaging data in tissue experiments, specifically using genetically encoded Ca²⁺ indicators (GECIs). It provides detailed protocols that facilitate the quantification of calcium signals through advanced analytical techniques.

Key Study Components

Research Area

Calcium imaging
Signal quantification
Tissue analysis

Background

Improved data acquisition from calcium imaging experiments
Importance of accurate analysis techniques in capturing calcium signaling
Use of genetically-encoded indicators for in situ imaging

Methods Used

Spatiotemporal mapping
Particle-based analysis
Microscopy techniques (confocal microscope)

Main Results

Protocols yield spatial, temporal, and intensity data critical to understanding calcium signaling
Enhanced accuracy in measuring calcium events and their properties
Established a framework for analyzing calcium transient clusters in tissue samples

Conclusions

The study demonstrates advanced methods for quantifying calcium signals in tissues
Highlights the importance of refined analytical approaches for improved biological insights in calcium signaling

Frequently Asked Questions

What are genetically encoded calcium indicators?

Genetically encoded calcium indicators (GECIs) are proteins that fluoresce in response to calcium ion concentrations, allowing for real-time calcium imaging in living cells.

Why is proper analysis important in calcium imaging?

Proper analysis ensures that important data related to spatial and temporal calcium signals are accurately captured and interpreted, which is crucial for understanding signaling pathways in biology.

What techniques are used to analyze calcium signals?

Techniques used include spatiotemporal mapping and particle-based analysis, which help quantify the amplitude and frequency of calcium events.

What is the role of the small intestine in this study?

The small intestine is the model system used to examine interstitial cells of Cajal (ICC) that express GECIs, providing a platform for calcium imaging studies.

How is data saved and analyzed in this study?

Data from calcium imaging is saved as TIFF image stacks and analyzed using software like ImageJ and Volumetry to create detailed maps and quantify calcium signaling.

What is a spatiotemporal map?

A spatiotemporal map visualizes calcium activity over time and space within a biological sample, helping researchers understand the dynamics of calcium signals.

How does improved analysis affect biological research?

Improved analysis enhances the clarity and accuracy of data interpretation in calcium signaling studies, leading to better insights into cellular processes and disease mechanisms.

Genetically encoded Ca²⁺ indicators (GECIs) have radically changed how in situ Ca²⁺ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca²⁺ signals is required. The protocols in this paper facilitate the quantification of Ca²⁺ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

The capacity to acquire large amounts of data from calcium imaging experiments has drastically improved, especially with tissue imaging experiments. Our protocols describe appropriate analysis techniques to quantify and describe these data sets. Our protocols generate a range of spatial, temporal, and intensity values that are intrinsic to calcium signaling in entire tissues and this can be lost in more rudimentary-based analysis.

One hour before the imaging transfer a small intestine harvested from a mouse with interstitial cells of Cajal or ICC that specifically express a genetically-encoded calcium indicator to the stage of a spinning disk confocal microscope. And continuously perfuse the tissue with 37 degrees Celsius Krebs-Ringer bicarbonate or KRB solution. Using a 60 to 100 X magnification locate the stellate-shaped myenteric plexus ICC or ICC-MY between the circular and longitudinal smooth muscle layers of the small intestine.

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