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

JoVE Journal
Biology

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JoVE Journal Biology

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

时空映射和粒子分析技术在细胞内 ca2 +原位信令定量中的应用

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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

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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.

基因编码^{的 ca 2 +}指标 (geci) 从根本上改变了原位 ca^{2 +}成像的执行方式。为了最大限度地从此类记录中恢复数据, 需要对 ca^{2 +}信号进行适当分析。本文的规程利用时空映射和粒子分析, 便于对现场记录的 ca^{2 +}信号进行量化。

从钙成像实验中获取大量数据的能力已大大提高，尤其是组织成像实验。我们的协议描述了用于量化和描述这些数据集的适当分析技术。我们的协议生成一系列空间、时间和强度值，这些值是整个组织中钙信号所固有的，在更基本的分析中可能会丢失这些值。

在成像前一小时，从具有Cajal或ICC间细胞的小鼠中采集的小肠，这些小肠专门将基因编码的钙指示器表达到旋转盘共合显微镜的阶段。并持续用37摄氏度的克雷布斯-林格碳酸氢盐或KRB溶液对组织进行持续吸附。使用 60 到 100 X 放大镜定位小肠的圆形和纵向平滑肌肉层之间的硬质状肠状肠状丛ICC或ICC-MY。

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