Introductiontoworking
withImageJ
Downloadandinstall
•ImageJ isopensourcesoftware
•BasedonJava
• Hint:UsewithlatestavailableJavaversion
• forMacuseMacJavaversionifOracle’sJavafails
•ThereareseveralforksofIJ
• ImageJ
• ImageJ2
• Fiji- http://fiji.sc/Fiji
• "It can be described as a "batteries-included" distribution of ImageJ (and ImageJ2), bundling Java, Java3D and a lot of
plugins organized into a coherent menu structure. Fiji compares to ImageJ as Ubuntu compares to Linux.”
• http://fiji.sc/Downloads
Mainwindow
workflow direction
Addnew
optionsto
toolbar
Redarrowother
choiceson
RMB
Drag&
Drop to
open file
Alloperationsare
performedonlast
activewindow
Double
clickopens
options
Ifyouknowthenameof acommand (including
plugins) butforgetwhereitishidden amidst
themenus, type Ctrl +L tobring up
the CommandFinder
Menu- File
Closes thecurrentimageandopens thenextimage(ifany)initsdirectory.HoldingAlt opens
theprevious image(ifany)initsdirectory.
Capturesfullscreenasvideo
ROI
•ROI(RegionOfInterest)areselectedareasofanimagewhichcanbeusedforlocalised
measurementsoralterationsofanimage.
•TheAnalyze→Measure commandwillonlymeasurethepixelswithintheROI.
•Mostofthecommandsinthe'Process'menuwilloperatewithinaselectedROI.
•HoldingShift whiledrawinganROIwilladd(merge)ittotheexistingROI.HoldingAlt (Ctrl+Alt)
whiledrawingwillsubtractfromthecurrentROI.
•ImageJnormallyhasasingleROIwhichispresentoneverysliceofastack.TheROIManager
(Analyze→Tools→RoiManager)allowsyoutodealwithmultipleROIs.AddinganROItothe
managerwillstoretheROI,andwillkeeptrackofwhichsliceitcamefrom.
•TheROIManagercanstoremultipleROIs,loadandsavethem,addandsubtractshapes.
Menu- Edit
Limited
Copy selectedareato
internalclipboard
Negation
areaselection andline
selection
Scenario- getequivalentdiameterof
object
Equivalentdiameter- diameterofacirclewiththesameareaastheregion.Computedas
sqrt(4*Area/pi),oneofmanymeasuresofregionproperties.
◦
◦
◦
◦
Openimage(Ctrl+O)
SelectPolygonSelection toolandoutline object
Add toROImanagerEdit->Selection->AddtoROImanager (Ctrl+T)
Displaymeasuresofselectedregion Analyze->Measure (Ctrl+M) ordirectlyformROImanager, read
areaanduseittocalculatediameter
◦
◦
◦
◦
Youcan fitequivalentcircleEdit->Selection->Fitcircle(andtakealookforothertools modyfying selections)
Addcircleselection toROImanagerEdit->Selection->AddtoROImanager (Ctrl+T)
Nowonecanswitchbetweenthose two
Display measuresofselectedregionAnalyze->Measure (Ctrl+M) andread areas
◦ Turnonother measurementsAnalyze->SetmeasurementsorResults/Setmeasurements
◦ Backtooriginal selectionandgeneratemaskEdit->Selection->CreateMask
◦ Generateflatimagee.g.fordocumentation - selectROIyouwantandclickFlatten(orDraw – see the
difference)
Menu- Image
adjustements - no
UNDO!!
stacks
selection
geometrical
Usespacetopaninzoommode(eqw
ofhandfromtoolbar
Autosettingscan'tbereseted
getmore
control
donowant
anychanges
apply
adjustments modify image
Scenario- addtexttoimage
•Selectimagewindowyouwanttomodify
•ClickTexttool buttonontoolbar
• Double clickforconfiguration
•Drawboxinimageandstarttyping
• Thisisoverlaythatisnot mergedwithimageyet
• ItcanbeaddedtoROImanager
•Pressctrl+d tofinishandmergewithimage
Stacks,hyperstacks
http://fiji.sc/Stack_Manipulation
"Hyperstacks are multidimensional images, extending image stacks to four (4D) or five (5D)
dimensions:
x (width), y (height), z (slices), c (channels or wavelengths) and t (time frames).
Hyperstacks are displayed in a window with three labelled scrollbars (see Stacks and
Hyperstacks↑). Similarly to the scrollbar in Stacks, the frame slider (t) has a play/pause icon."
Scenario- extractchannelfromstack
•OpenstackdataCtrl+O
•SelectImage->Stacks->Tools->MakeSubstack
•Selectchannelyouwanttoextractandframerange(bydefaultallframesareproposed)
Scenario- getoneframefromstack
•Openstackdatactrl+O
•Pickframeandchannel
•SelectallEdit->Selection-All (ctrl+a)
•Copytointernalclipboard(ctrl+c)
•PasteasnewimageFile->New->Internalclipboard
Scenario- cutstack
•Openstackdata ctrl+O
•SelectImage->Stacks->Tools->SliceKeeperorSliceRemover
Scenario- cutonecellfromstack
•Openstackdata ctrl+O
•DrawROI
•SelectImage->Duplicate orImage->Crop
Scenario- usefluoroscopydatafor
automaticsegmentation
•Openstackdatactrl+O
•Selectfluoroscopychannel
•Selectwandtoolfromtoolbarandclickonobject
•DoubleclickonWandtoolandadjusttolerance
• Tip:ifyoudonotseeanychangesonselection,closeWandoptionsandclickonimageagainandopenagain
options
• Tip:Pressctrl+shift+a todeleteselection(usefulifthewholeimageisselected)
• Tip:Selectionprocesscanbeimprovedbysmoothingfluoroscopydata(Process->Smooth)butbecarefulto
notsmootheverything.
•Addselection toROImanager(ctrl+t)
•SelectsecondcellandaddittoROImanageraswell
•CombinetwoROIsintooneusingORoperationfromROImanager
•Afterswitchingchannelsselected regioncanbeprocessed
Menu- Process
Mathoperationson
image
Mathoperationson
images
Menu- Analyze
Basicstatisticson
Resultswindow
Histogram
Intensityprofile
(needs selection)
Scenario- synchronizewindows
Loadtwo(ormore)datasetstobesynchronized
SelectAnalyze->Tools->SynchronizeWindows
◦ Selectwindowstobesynchronized
◦ ClickSynchronize
Menu- Plugin
Macrorelated
options
Scenario- createcommandhistory
•SelectPlugins->Macro-Record
•Noweveryactionisrecordedinmacroscript
•Macrocanbesavedandthenre-played(Plugins->Macro->Run)
•Goodmethodfordocumentingwork
Scenario- installplugin
PluginsdownloadedfromInternetcanbeinstalledby:
◦ Selecting Plugin->InstallPlugin
◦ RestartingFiji
AfterinstallationpluginshouldbeavailableinPluginsmenu
Scenario- writesimplemacro
Howtoprocessfolderfullofimages?
•ClickPlugins->New->Macro
• Build-inmacroeditor opens
•SelectTemplates->IJ1Macro->Processfolder
•YoucanmodifyprocessFile functioninsertingthererecordedcommands
•RunmacrobyclickingRunbuttonorsaveitandrunfromPlugins->Macros->Run
Literature
1.
P.Bankhead,“AnalyzingfluorescencemicroscopyimageswithImageJ,”
2.
T.Collins,“ImageJformicroscopy,”Biotechniques,vol.43,no.S1,pp.S25–S30,2007.
3.
ImageJmanual