Name: clock scan protocol for image analysis: imagej plugins
Uploaded: 2017-06-19T13:00:00
Description: Watch this Scientific Journal Video about Clock Scan Protocol for Image Analysis: ImageJ Plugins at JoVE.com

We thank Dr. Tanja Maritzen and Dr. Fabian Feutlinske (Leibniz Institute of Molecular Pharmacology, Berlin, Germany) for sharing with us their version of the Fuji ImageJ Clock Scan plugin and inspiring us to develop this version of the program. We are also grateful to Dr. Fritz Melchers (Department of Lymphocyte Development, Max Planck Institute for Infection Biology) for his kind permission to use the images from the database of his department for the purpose of testing and improving the plugin. Support: Center for Translational Neurosciences; NIH grant: P30-GM110702-03.

1. Software Installation
<ol>
	<li>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 &#34;ImageJ&#34;.</li>
	<li>Copy &#34;Clock_Scan-1.0.1. jar&#34; and &#34;Multi_Clock_Scan-1.0.1.jar&#34; plugin files using the link provided in materials table and paste them into the ImageJ plugin directory. Alternatively, use &#34;Plugins | Install pl...

<ol>
	<li>Dobretsov, M., Romanovsky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Clock-scan"+protocol+for+image+analysis.">"Clock-scan" protocol for image analysis.</a> Am J Physiol Cell Physiol. 291, 869-879 (2006).</li><li>Feutlinske, F., Browarski, M., Ku, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stonin1+mediates+endocytosis+of+the+proteoglycan+NG2+and+regulates+focal+adhesion+dynamics+and+cell+motility.">Stonin1 mediates endocytosis of the proteoglycan NG2 and regulates focal adhesion dynamics and cell motility.</a> Nat Commun. 6, 8535 (2015).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9, 671-675 (2012).</li><li>Schindelin, J., Arganda-Carreras, I., Frise, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9, 676-682 (2012).</li><li>Dobretsov, M., Hastings, S. L., Stimers, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-uniform+expression+of+alpha+subunit+isoforms+of+the+Na+/K++pump+in+rat+dorsal+root+ganglia+neurons.">Non-uniform expression of alpha subunit isoforms of the Na+/K+ pump in rat dorsal root ganglia neurons.</a> Brain Res. 821, 212-217 (1999).</li><li>Hayar, A., Gu, C., Al-Chaer, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+patch+clamp+recording+and+calcium+imaging+of+neurons+in+the+intact+dorsal+root+ganglion+in+rats.">An improved method for patch clamp recording and calcium imaging of neurons in the intact dorsal root ganglion in rats.</a> J Neurosci Methods. 173, 74-82 (2008).</li><li>Dobretsov, M., Pierce, D., Light, K. E., Kockara, N. T., Kozhemyakin, M., Wight, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mouse+model+to+selectively+identify+alpha3+Na,K-ATPase+expressing+cells+in+the+nervous+system.">Transgenic mouse model to selectively identify alpha3 Na,K-ATPase expressing cells in the nervous system.</a> Society for Neuroscience. , 1 (2015).</li><li>Lein, E. S., Hawrylycz, M. J., Ao, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+atlas+of+gene+expression+in+the+adult+mouse+brain.">Genome-wide atlas of gene expression in the adult mouse brain.</a> Nature. 445, 168-176 (2007).</li><li>Romanovsky, D., Mrak, R. E., Dobretsov, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+decline+in+density+of+human+nerve+and+spinal+ganglia+neurons+expressing+the+alpha3+isoform+of+Na/K-ATPase.">Age-dependent decline in density of human nerve and spinal ganglia neurons expressing the alpha3 isoform of Na/K-ATPase.</a> Neuroscience. 310, 342-353 (2015).</li><li>Campbell, J., Singh, D., Hollett, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+selective+photoconductive+stimulation+of+live+neurons.">Spatially selective photoconductive stimulation of live neurons.</a> Front Cell Neurosci. 8, 142 (2014).</li><li>Yuryev, M., Pellegrino, C., Jokinen, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Calcium+Imaging+of+Evoked+Calcium+Waves+in+the+Embryonic+Cortex.">In vivo Calcium Imaging of Evoked Calcium Waves in the Embryonic Cortex.</a> Front Cell Neurosci. 9, 500 (2015).</li><li>Qiao, M., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Method+for+Labeling+Electrically+Coupled+Cells:+Application+to+Retina.">Genetic Method for Labeling Electrically Coupled Cells: Application to Retina.</a> Front Mol Neurosci. 8, 81 (2015).</li></ol>

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 publications1,9. Briefly, this protocol allows the generation of integral radial pixel-intensity profiles by quantifying the intensity of...

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 &#34;background&#34; pixel intensity. The protocol scales these profiles according to the cell radius, measured in the direction of the scan. Thus, the distance from the center to the ROI border of each individual radial scan is always 100% of the X scale. Finally, the program averages these individual profiles into one integral radial pixel-intensity profile. Because of scaling, the mean pixel-intensity profile, produced by the &#34;Clock Scan&#34; protocol, depends neither on the ROI size nor, within reasonable limits, on the ROI shape. This method allows direct comparison or, if required, averaging or subtraction of profiles of different ROIs. The protocol also allows correction of the integral pixel intensity profiles, of any object for background noise, by a simple subtraction of the average intensity of pixels located outside the object. Although it has only been tested in biological samples, our protocol provides a valuable addition to other existing image analysis tools used in studies of images of physical or chemical processes that are arranged around a point of origin (such as diffusion of substances from a point source)1.
However, the major limitation of the original image analysis method was that the protocol was developed as a Visual Basic 6 (VB6) (code, and therefore, it was platform-dependent and difficult to distribute (requiring VB6). To address this problem and to join similar recent efforts by other investigators2, we converted the VB6 Clock Scan program code into two Java-based plugins, compatible with the NIH-sponsored and freely-available open-source and platform-independent image analysis programs, ImageJ3 and Fiji ImageJ4. Furthermore, these plugins have now several new functions that expand the capability of the original protocol to process multiple ROIs and image stacks. Many image analysis applications are not user-friendly, with regards to performing statistical analysis of multiple objects, and thus, often only representative data are shown. With the multi Clock Scan ImageJ plugin, it is possible to facilitate the analysis of multiple objects simultaneously. A robust statistical evaluation of microscopy data, with regards to signal intensity distribution in single cells/objects, is now possible with this plugin extension. Here, we describe the Clock Scan plugins and show examples of their applications in image analysis.

<table border="0" cellpadding="0" cellspacing="0" width="896">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Computer</td>
			<td style="width:71px;">Any</td>
			<td style="width:289px;"></td>
			<td style="width:320px;">compatible with software listed below</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ImageJ or Fiji ImageJ</td>
			<td style="width:71px;">NIH</td>
			<td>https://imagej.nih.gov/ij/ or https://fiji.sc/</td>
			<td>bundled with Java 1.8 or higher</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Clock-scan plugins</td>
			<td style="width:71px;">freeware</td>
			<td>https://sourceforge.net/projects/clockscan/</td>
			<td>Clock_Scan-1.0.1 jar and Multi_Clock_Scan-1.0.1/ jar</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Origin 9.0</td>
			<td>OriginLab</td>
			<td style="width:289px;">Northampton, MA, USA</td>
			<td style="width:320px;">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</td>
		</tr>
	</tbody>
</table>

clock scan protocol for image analysis: imagej plugins

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.

The images that are used here for illustration purpose, are taken from databases created during our previous cell and tissue biological studies5,6,7 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.
<p ...

Watch this Scientific Journal Video about Clock Scan Protocol for Image Analysis: ImageJ Plugins at JoVE.com

Clock Scan Protocol for Image Analysis: ImageJ Plugins

This paper describes two novel ImageJ plugins for &#39;Clock Scan&#39; 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.

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 ...

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