Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Summary

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca²⁺ signals with minimal user input and bias.

Abstract

Intracellular Ca²⁺ signals are commonly studied with fluorescent Ca²⁺ indicator dyes and microscopy techniques. However, quantitative analysis of Ca²⁺ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca²⁺ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca²⁺ signaling.

Introduction

Ca²⁺ is a ubiquitous second messenger signaling molecule and cytosolic Ca²⁺ levels are highly regulated. Intracellular Ca²⁺ signals are complex and include isolated transients, oscillations, and propagating waves^1-4. Spatial and temporal control of Ca²⁺ is thought to underlie physiological signal specificity, and therefore the analysis of Ca²⁺ signal patterns is of considerable interest to investigators in multiple fields⁵.

Ca²⁺ indicator dyes such as Fluo-4 and Fura-2 are commonly employed to measure intracellular Ca²⁺ signals with....

Protocol

1. Vessel Dissection and Imaging

1. Harvest tissue from domestic juvenile pigs as described in Martens et al²⁷. Place harvested swine right ventricles into a polydimethylsiloxane (PDMS) bottom dissection dish containing HEPES buffered physiological saline solution (PSS).
   1. With the aid of a stereomicroscope, dissect and remove a segment of the left anterior descending coronary artery (~8 mm length, 0.5 mm diameter) from surrounding tissue using forceps and spring scissors by removing the vessel segment from the surrounding cardiac tissue layers. NOTE: Take care to not puncture the vessel wall.
   2. Place a PDMS block (1 x 0....

Results

A custom algorithm, LC_Pro, was developed and implemented in order to perform automated analysis of Ca²⁺ dynamics on confocal image sequences. As depicted in Figure 1, the algorithm utilizes sequential processing modules that A) detect and track sites of dynamic Ca²⁺ change above statistical (p < 0.01) noise, B) define regions of interest (ROI) automatically at active site centers, and C) calculate average fluorescence intensities at ROIs to determine specific event parameters. A.......

Discussion

Decoding complex Ca²⁺ signals at the cellular and multicellular level will require rigorous experimental and analytical approaches. Here, an approach is described in which time resolved confocal image sequences of Ca²⁺ dependent fluorescence are subjected to an automated analysis that identifies and quantifies statistically relevant Ca²⁺ signals within intact cellular fields In the specific case presented, an artery segment was isolated from pig heart, pinned opened to expose the endothel.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dissection dish	Fisher Sci	#08-772-70
Polydimethylsiloxane (PDMS)	Fisher Sci	#NC9644388	elastomer kit, must be molded into dishes
HEPES-buffered PSS	Sigma	#H3375-250G	HEPES acid
Stereomicroscope	Nikon Inst.	#MNA42000
Forceps	Fine Science Tools	#11223-20
Spring scissors	Fine Science Tools	15003-08
Tungsten wire	Scientific Inst Svcs	#406
Fluo-4 AM	Life Tech.	#F-14201
Pluronic F-127	Life Tech.	#P3000MP
Metal pins	Fine Science Tools	#26002-10
Cover-glass bottom chamber			Custom designed
Spinning disc confocal microscope	Perkin Elmer	RS-3
ImageJ software			download at: http://rsbweb.nih.gov/ij/download.html
LC_Pro plugin for imageJ			download at: http://rsbweb.nih.gov/ij/plugins/lc-pro/index.html
R software			download at: http://www.r-project.org/
R traceplot script			download at: https://docs.google.com/file/d/0B-PSp1D9e2fjV3NIcGppUzkxdEk/edit?usp=sharing