manual synapcountj v1 en

8/13/2019 Manual Synapcountj v1 En

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SynapCountJ a plugin for ImageJ 1

SynapCountJ

PLUGIN for IMAGEJ,

that allows you to count

the number an calculate the ensity

of synapses of a neuron

Gadea Mata Martnez

gaea!mata"gmail!com

gmata!e#t"rio$asalu!es
mailto:[email protected]:[email protected]:[email protected]:[email protected]


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SCOPE OF THE WORK:

The plugin that will be presented below is a joint work between the team Structural Synaptic Plasticity1

of The Biomedical Research Center or La Rioja (CBR! 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 techni,ues& n the e-ample

these images are obtained from the same neuron in culture using two antibod% markers. bassoon (a

pres%naptic scaffolding protein! and s%napsin ( a s%naptic $esicular protein!& )n% set of two s%naptic markers

can work perfectl%&

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(p 2ista and 3! GNU/Linuxand Mac S !&

To install the SynapCount"plugin %ou should proceed as follows4

1& nstall Neuron"plugin4http455www&imagescience&org5meijering5software5neuronj5

"& nstall #io$%ormatsplugin4 http455www&loci&wisc&edu5software5bio+formats

6& #nrar the file s%napcountj&rar

Cop% the file 7acro87ake8Binar% into the macros where &mage"is installed4

95mage05macros

1 http://www.cibir.es/cibirinvestigacion/enfermedadesneurodegenerativas?start=12 https://esus.unirioja.es/psycotrip/

Image marked with bassoon Image marked with synapsin
http://www.imagescience.org/meijering/software/neuronj/http://www.imagescience.org/meijering/software/neuronj/http://www.loci.wisc.edu/software/bio-formatshttp://www.imagescience.org/meijering/software/neuronj/http://www.loci.wisc.edu/software/bio-formats


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Cop% the files SynapCount"&jar into the :lugins where &mage"is installed4

95mage05:lugins

;ou can create a folder or put them into an e-isting one&

/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

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- 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&

Create branch

@elete branch'a$e coordinates structure

'a$e image structure


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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 dendritesselected 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


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There are three options4

1& Individual:Aork indi$iduall% witheach pair of photos& This option allows to sa$e the information

to work in batch&

"& File .lif (LeicaFiles)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= createdbefore&

6& Directory (Tif files)4 Aork for batch a set of pairs of images stored in directories&

Ind#$#dua% ana%y&

pen 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 ofthe structure of the neuron (it was

sa$e before. in our case is call green&t-t.

The following dialog is open4


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@istance in pi-el4which represents the unit in pi-els which is going to be taken&

nown 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&

Working individually

To stud% one dendrite4 for doing that it is necessar% to choose one from trace from the

(Choose Tracing combobo-! which depicts all dendrites& 'elect the option 7anual

Threshold&

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&

n all this cases whenclicking on the button it will show this follow dialog4


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The plugin show the images with the two markers and the one with the structure o$erlapped thus4

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 structureof the neuron&

To find the s%napses weshould 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, allow you to :

riginal4 Return to the original image&

Count4 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

)elp4 @ispla% help about this dialog&

*4 @oes the final ,uantification after that the program return the followings results4

The image named +G# ,e-initi.a shows the s%napses which are marked with blue points&


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the results table shows the number of s%napses dendritic length anal%*ed and s%naptic densit% b%

the units of length introduced before&

n the last image name %inal +G# the three channels appears o$erlapped&


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Working semiautomatically

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 k it will show the same images seen in the pre$ious case

(indi$idual kind!&

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 &


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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 k&

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

samethan theimages 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


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Then press 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# ,e-initi.a! for each image from

e-periment&


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Wor'#n( )#t* d#re0tor#e&

SynapCount"can also work directl% with tiff files & To do that %ou ha$e

'elect thedirector% 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"&


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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 togadea&mataIgmail&comor 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&
mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]

manual synapcountj v1 en

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