Clock Scan Protocol for Image Analysis: ImageJ Plugins

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.

Optogenetic Tools for Control of Public Goods in Saccharomyces cerevisiae

“…On the seventh day, the pictures were imaged with ChemiDoc imaging system (catalog no. 12003154; Bio-Rad) at an exposure of 0.06 s in the bright-field setting and analyzed using an ImageJ plug-in Clockscan ( 90 ).…”

Ecm29-Dependent Proteasome Localization Regulates Cytoskeleton Remodeling at the Immune Synapse

“…Quantification of BCR clustering at the IS center was performed by using an adaptation of the clock scan analysis plugin for Fiji ( Dobretsov et al, 2017 ) implemented in a personalized macro with machine learning correction with the advanced Weka segmentation tool ( Arganda-Carreras et al, 2017 ). Data obtained from the images was then curated and filtered using Rstudio.…”

Optogenetic tools for public goods control in Saccharomyces cerevisiae

“…The patterned plates were analyzed using a published ImageJ plug-in, Clock Scan 90 which outputs averaged radial intensity values for the image.…”

“…We quantify the growth of yeast on a plate from images using a custom MATLAB script that examines intensity versus radius along angular slices through the center of the plate and identifies the bounds of features such as valleys and rings. Because it’s hard to accurately identify these features from individual angular slices or the single, composite intensity profile given by a clockscan 90 , we use a bootstrap-based approach to repeatedly identify potential features from randomly selected sets of angular slices and select the most frequently identified potential features as true features.…”