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

Kendisinin geçici eşleme ve parçacık analiz teknikleri intrasellüler Ca sinyal2 + in S...

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

Kendisinin geçici eşleme ve parçacık analiz teknikleri intrasellüler Ca sinyal2 + in Situ ölçmek için uygulamaları

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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.

Genetik olarak kodlanmış Ca^{2 +} göstergeleri (turk) nasıl yerinde Ca^{2 +} görüntüleme gerçekleştirilir kökten değişti. Veri kurtarma gibi kayıtları en üst düzeye çıkarmak için Ca^{2 +} sinyal uygun analiz gereklidir. Bu kağıt Protokoller'de Ca sinyalleri^{2 +} yerinde kronolojik zamanmekansal haritalama ve analiz parçacık tabanlı kullanarak kaydedilen miktar kolaylaştırmak.

Kalsiyum görüntüleme deneylerinden büyük miktarda veri elde etme kapasitesi, özellikle doku görüntüleme deneyleriyle büyük ölçüde artmıştır. Protokollerimiz, bu veri kümelerini ölçmek ve tanımlamak için uygun analiz tekniklerini açıklar. Protokollerimiz tüm dokularda kalsiyum sinyaline özgü bir dizi mekansal, zamansal ve yoğunluk değerleri üretir ve bu daha ilkel tabanlı analizlerde kaybedilebilir.

Görüntülemeden bir saat önce, cajal veya ICC'nin interstisyel hücreleri olan ve genetik olarak kodlanmış kalsiyum indikatını dönen disk konfokal mikroskobu aşamasına ifade eden bir fareden alınan ince bağırsağı aktarır. Ve sürekli 37 derece Santigrat Krebs-Ringer bikarbonat veya KRB çözeltisi ile doku perfüzyon. 60-100 X büyütme kullanarak ince bağırsağın dairesel ve uzunlamasına düz kas katmanları arasında stellat şekilli mienterik pleksus ICC veya ICC-MY bulun.

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