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SynapCountJ a plugin for ImageJ



PLUGIN for IMAGEJ, that allows you to count the number an calculate the ensity

of synapses of a neuron

Gadea Mata Martínez ga ea!mata"gmail!com gmata!e#t"rio$asalu !es

bassoon (a pres%naptic scaffolding protein! and s%napsin ( a s%naptic $esicular protein!& )n% set of two s%naptic markers can work perfectl%& Image marked with bassoon Image marked with synapsin The program was designed to work with images from neurons in culture& /owe$er( in future $ersions we will deal with the problems of brain slices& INST !! TION: '%naptCount0 can work on Windows (1p( 2ista and 3! GNU/Linux and Mac To install the SynapCount" plugin %ou should proceed as follows4 1& "& nstall Neuron" plugin4 http455www&imagescience&org5meijering5software5neuronj5 nstall #io$%ormats plugin4 http455www&loci&wisc&edu5software5bio+formats φ Cop% the file 7acro87ake8Binar% into the macros where &mage" is installed4  95 mage05macros S !& 6& #nrar the file s%napcountj&rar 1 http://www.ues& n the e-ample( these images are obtained from the same neuron in culture using two antibod%‐investigacion/enfermedades‐neurodegenerativas?start=1 2 https://esus.cibir.SynapCountJ a plugin for ImageJ 2 SCOPE OF THE WORK: The plugin that will be presented below is a joint work between the team “ Structural Synaptic Plasticity”1 of The Biomedical Research Center or La Rioja (C B R! and “ Programming and Symbolic Computation Team”" of #ni$ersit% of La Rioja& DESCRIPTION: '%napses are the points of connection between neurons& The rele$ance of s%napses comes from the fact that the% are related to the computational capabilities of the brain& The possibilit% of changing the number of s%napses ma% be an important asset in the treatment of some neurological diseases( such as )l*heimer& This plugin pro$ides a semi+automatic method for counting s%napses& This tool needs two images which are obtained using immunostanning .unirioja.

ou can create a folder or put them into an e-isting one& φ /elp < =Refresh 7enu= or start &mage" (reboot if alread% open!& WORKING WITH NEURONJ P!"GIN: >irst( we are going to draw the dendritic neuronal morpholog% from one of those pictures( an immunoc%tochemistr% from a dendritic structural marker such M'P(# should work as well& >irst of all( we must draw the structure of the neuron& To this end we we ha$e to load the Neuron" plugin (:lugins < Neuron"!& )fterwards we can see this toolbar4 Then( we open one of the images e-plained at the beginning of this manual (bassoon or s%napsin! clicking on this button & ?ow( we ha$e to draw the structure4 'a$e coordinates structure @elete branch Create branch 'a$e image structure The tracing of dendrites is initiated b% mo$ing the mouse to the beginning of a dendrite of interest and clicking the (left! mouse button& The Neuron" plugin shows the path from the current mouse position in the image to the clicked point& 7o$e the mouse roughl% along the dendrite until the path suggested b% Neuron" starts to de$iate too much from what is considered the correct trace & Clicking the mouse button again makes the program fi.SynapCountJ a plugin for ImageJ 3 φ Cop% the files SynapCount"&jar into the :lugins where &mage" is installed4  95 mage05:lugins .the displa%ed path and start the computation of paths from the newl% clicked point& This procedure can be repeated till the end of the dendrite has been reached( which is indicated b% double‐ clicking the mouse button& .

SynapCountJ a plugin for ImageJ 4 Ahen the structure is draw( we should e-tract the trace from the image& Click on the button and choose the first option “Tab+delimited te-t file4 single file for all tracings”& The plugin sa$e a file with the e-tension =&t-t=& This file contains the coordinates of all the dendrites selected and all the traces under the name of tracing?1( tracing?" and so on& >inall%( we close the Neuron" plugin . WORKING WITH THE P!"GIN: 'tart the plugin SynapCount" (:lugins < SynapCount"!& The following dialog is open4 .

in our case is call green&t-t. The following dialog is open4 .lif (Leica Files)4 Aork for batch a set of pairs of images compressed in a file e-tension =&lif=& t takes the necessar% data from a file with =&-ml= created before& 6& Directory (Tif files)4 Aork for batch a set of pairs of images stored in directories& Ind#$#dua% ana%y&#& Bpen the two images with the bassoon and s%napsin markers (our e-amples! & )fterwards introduce the si*e of images and choose the file which contains the information of the structure of the neuron (it was sa$e before.SynapCountJ a plugin for ImageJ 5 There are three options4 1& Individual: Aork indi$iduall% with each pair of photos& This option allows to sa$e the information to work in batch& "& File .

SynapCountJ a plugin for ImageJ 6 δ δ δ δ δ δ δ @istance in pi-el4 which represents the unit in pi-els which is going to be taken& Cnown distance4 shows the measure of the pi-el unit taken& :i-el )spect Ratio #nit of length4 name of the unit of measure that represents the information before& Red4 select the image for the red channel& Dreen4 select the image for the green channel& @iameter in pi-els4 'et the diameter of the structure of the dendrite& ?otice that this $alue determined the area within the anal%sis will take place& ) $alue around "E pi-els works fine for us& φ Working individually γ To stud% one dendrite4 for doing that( it is necessar% to choose one from trace from the (“Choose Tracing combobo-”! which depicts all dendrites& Threshold”& γ γ To stud% all dendrites one b% one4 for doing that( it is necessar% to select the option “Traces one b% one” and “7anual Threshold”& To stud% all the whole neuron4 for doing that( it is necessar% to select “Ahole structure”& 'elect the option “7anual n all this cases( when clicking on the button “BC”( it will show this follow dialog4 .

allow you to : riginal4 Return to the original image& Count 4 show the number of s%napses calculated for the selected range of $alues& The counting will appear at the right of “?umber of '%napses” label )elp 4 @ispla% help about this dialog& * 4 @oes the final .SynapCountJ a plugin for ImageJ 7 The plugin show the images with the two markers and the one with the structure o$erlapped( thus4 φ φ φ The red channel is for the image with s%napsin & The green channel is for the image with bassoon& The blue channel is for the image with the structure of the neuron& To find the s%napses( we should look for the points where the three channels match up( these points are white& /owe$er( it is worth noting that the s%napses are not onl% the full% white points( but also the ones whose color is close enough to white& 'o( we select a range of white $alues in which we estimate there are s%napses& ?otice that onl% the s%napses located on the dendritic tree are selected( in such a wa% we reduce the false positi$es and confine the dendrites to anal%*e& Ahen we choose a range of white $alues( the areas in that range are highlighted to pro$ide a $isual estimation of the s%napses& The buttons which appear in the dialog box.a” shows the s%napses which are marked with blue points& .uantification( after that the program return the followings results4 φ The image named “+G# .e-initi.

SynapCountJ a plugin for ImageJ 8 φ the results table shows the number of s%napses( dendritic length anal%*ed and s%naptic densit% b% the units of length introduced before& φ n the last image( name “%inal +G#”( the three channels appears o$erlapped& .

SynapCountJ a plugin for ImageJ 9 φ Working semiautomatically γ γ To stud% one dendrite. for doing that( it is necessar% to choose one trace (“Choose Tracing drop+ down menu”! ( and then to introduce manuall% which are the $alues of red and green threshold& To stud% all dendrites one b% one4 for doing that( it is necessar% to select “Traces one b% one” ( after that and to introduce which are the $alues of red and green threshold& This mode works with indi$idual traces( indicating s%naptic densit% from dendrites and whole neuron& n all cases( after clicking on the button “Bk”( it will show the same images seen in the pre$ious case (indi$idual kind!& φ Save information To further process se$eral images in batches( we can now sa$e the preselected setting such threshold ( scale and dendrite diameter & )ll this information is sa$e in a “&-ml” file& The settings can be reuse with pictures from the same e-periment& ?otice( that antibodies concentrations( e-position time( etc&( must be similar in order to use a batch processing & .

-%#+.SynapCountJ a plugin for ImageJ 10 Wor'#n( )#t* +#%e& . /!e#0a +#%e&1 Choose the file “&lif” which stores all images of a batch on “:ath file &Lif”& Choose the file “&-ml” which stores the information about the batch job & Click “Bk”& Important note: t is necessar% that the structure of each neuron must be pre$iousl% outlined b% ?euron0& The structural files ( “&t-t”! must be inside a folder called FtracingsF that needs to be in the same director% where the file e-tension “&lif” is located& n addition( it is necessar% that the names of the files which store the information about structure( are the same than the images obtained from file “&lif”& G-ample4 f the image generated b% Lieca is4 batch "6E"11&lif H 'eriesEE"&tif Then( the file which sa$es its structure has to be named 4 batch "6E"11&lif H 'eriesEE"&t-t .

e-initi.SynapCountJ a plugin for ImageJ 11 Then press BC and the program will continuous to process all the pictures included in the file&lif& The results table will show the densit% b% traces and whole neurons of all cells included& )part from this table( in the same director% where is the file “ /li-0( the plug+in sa$es the images contained in the file “/li-”& This images are separated b% two color channels (red and green!& n addition( it stores the dates( about e-periment includes in the file “/li-”& This dates are sa$ed in “/txt” and “/xls” format& )lso result( the plug+in sa$es all images results (“ %inal +G#” and “+G# .a”! for each image from e-periment& .

SynapCountJ a plugin for ImageJ 12 Wor'#n( )#t* d#re0tor#e& SynapCount" can also work directl% with tiff files & To do that %ou ha$e γ 'elect the director% where to find&&& 9 a folder named “red” which store the images of red channel with %our fa$orite antibod% 9 a folder named “green” which store the images of green channel with %our second fa$orite antibod%& 9 a folder named “tracings” which store the structure of each neuron from batch& Important Note4 all files from the same neuron must ha$e the same name& >or instance in our e-periment the files are call series11! ( the one in the folder green contains the bassoon immunostainnig( the one in the folder red the s%napsin staining and the one sa$ed on tracing folder the structure generated b% Neuron"& .

SynapCountJ a plugin for ImageJ 13 Important Note4 all files which are of a same neuron( are named in the same wa%& 2T3at4s all -ol*s0/ This is a plugin in working process( would reall% appreciate %our comments to impro$e it& :lease write me to gadea&mataIgmail&com or gmata&e-tIriojasalud&es n the near future we like to de$elop an automatic structure recognition plug+in and a dendritic spine recognition algorithm& 'ta% tuned& .