Clock Scan Protocol for Image Analysis: ImageJ Plugins

Summary

This paper describes two novel ImageJ plugins for 'Clock Scan' image analysis. These plugins expand the functionality of the original visual basic 6 program and, most importantly, make the program available to a large research community by bundling it with the ImageJ free image analysis software package.

Abstract

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a closed or segmented convex-shaped region of interest, leading to the generation of an averaged integral radial pixel-intensity profile. This protocol was originally developed in 2006, as a visual basic 6 script, but as such, it had limited distribution. To address this problem and to join similar recent efforts by others, we converted the original clock scan protocol code into two Java-based plugins compatible with NIH-sponsored and freely available image analysis programs like ImageJ or Fiji ImageJ. Furthermore, these plugins have several new functions, further expanding the range of capabilities of the original protocol, such as analysis of multiple regions of interest and image stacks. The latter feature of the program is especially useful in applications in which it is important to determine changes related to time and location. Thus, the clock scan analysis of stacks of biological images may potentially be applied to spreading of Na⁺ or Ca⁺⁺ within a single cell, as well as to the analysis of spreading activity (e.g., Ca⁺⁺ waves) in populations of synaptically-connected or gap junction-coupled cells. Here, we describe these new clock scan plugins and show some examples of their applications in image analysis.

Introduction

The goal of this work is to present a Clock Scan protocol that is platform-free and freely available to any researcher interested in this type of image analysis. The Clock Scan protocol was originally developed in 2006¹, with the goal of improving existing methods of pixel intensity quantification within convex-shaped regions of interest (ROI), a method which has better integrative capacity and improved spatial resolution. During the acquisition, the protocol sequentially collects multiple radial pixel-intensity profiles, scanned from the ROI center to its border, or to a predetermined distance outside the ROI for the purpose of measuring the &....

Protocol

1. Software Installation

1. Install the latest versions of bundled Java and either ImageJ or Fiji ImageJ as recommended on the respective websites (see materials table for links to the corresponding websites). In the text below, both programs are referred to as "ImageJ".
2. Copy "Clock_Scan-1.0.1. jar" and "Multi_Clock_Scan-1.0.1.jar" plugin files using the link provided in materials table and paste them into the ImageJ plugin directory. Alternatively, use "Plugins | Install plugin" menu option to install these files after they have been saved on the computer hard drive.

2. Clock Scan ana....

Results

The images that are used here for illustration purpose, are taken from databases created during our previous cell and tissue biological studies^5,6,7 and from the Allen Mouse Brain Atlas⁸. Both plugins were successfully tested using ImageJ 1.50i/Java 1.8.0_77, ImageJ 2.0.0-rc-44/1.50e/ Java 1.8.9_66 and Fiji ImageJ 2.0.0-rc54/1.51g/Java 1.8.0_66 program environment.

Discussion

Clock Scan Protocol: The Clock Scan protocol is a fast and simple tool of image analysis. The advantages of this protocol, compared to existing common approaches of image analysis (such as linear pixel intensity scans or calculation of mean pixel intensity of the ROI), have been described in details in previous publications^1,9. Briefly, this protocol allows the generation of integral radial pixel-intensity profiles by quantifying the intensity of.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Computer	Any		compatible with software listed below
ImageJ or Fiji ImageJ	NIH	https://imagej.nih.gov/ij/ or https://fiji.sc/	bundled with Java 1.8 or higher
Clock-scan plugins	freeware	https://sourceforge.net/projects/clockscan/	Clock_Scan-1.0.1 jar and Multi_Clock_Scan-1.0.1/ jar
Origin 9.0	OriginLab	Northampton, MA, USA	This program was used to generate some graphs of the original Clock Scan data. Any other graphic software can be used to perform this function