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LEAFGUI:UserManual(LeafExtractionandAnalysisFrameworkGraphicalUserInterface)
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Index
Howto
cite
the
LEAF
GUI..Page
3
Introduction..Page3
Installation..Page 4OverviewoftheLEAFGUIlayout.Page5
LEAFGUIOperation........Page 6SettingtheScale.Page6ImageCropping..Page6Thresholding.Page 7ImageStorageandAreaMask.Page9CleaningtheBinaryImage.Page10ImageSegmentation..Page 13SummaryStatistics.Page14UsingtheQueuePage17ClearingtheCurrentImages.Page18UndoingthePreviousStepPage18InterruptingtheGUI..Page18DropDownMenus..Page19
Caveats...Page25Contacts.Page27Table1:HotKeys.Page28
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4
MatlabandmanyofthefunctionalaspectsoftheLEAFGUIrelyonspecificfunctionsfromthe
Matlabimageprocessinglibrary.Weprovidebriefdescriptionsofsuchfunctionshereandrefer
theusertotheMatlabdocumentationforsomeofthemorecomplicatedoperations.
InstallationThecodefortheLEAFGUIsoftwareiswritteninMatlab.However,thankstoMatlabsGUI
developmentenvironment,theGUIcanbecompiledasastandaloneexecutablethatcanrun
onmostplatforms.Sourcecode,downloadinstructionsandversionscompiledforPCs,Macs
andLinuxoperatingsystemsareavailableatwww.leafgui.org.
http://www.leafgui.org/http://www.leafgui.org/8/7/2019 Leaf GUI User Manual
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SetthescaleCroptheimage
Adaptive
Thresholding
Global
Thresholding
AreaMask
Dropdownmenus OriginalImage
Window
Modified
Image
Window
OriginalZoom
OutputPreference
(xlsortxt)SummaryStatistics
Options
Image
Segmentation
Options
CleanBinaryImageOptions
Zoom
Rectangle
(movable,resizable)
SendtoQueueUndo!
ClearImagesInterrupt!
ImageStorage
Combine
Images
LEAFGUIcontainssevenoperationalpanels,SetScaleandCrop,Filtering,Thresholding,ImageStorage,CleanBinaryImage,
Statistics.Inthefollowingsectionswedescribethefunctionalitiesofthepanels.LEAFGUIalsohasfourdisplaypanels,OriginalImage
displayingtheirrespectivezoomedregions.ThereisalsoaQueueontherighthandsideoftheGUIwhereuserscanstorethumbnailso
OverviewoftheLEAFGUIlayout
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LEAFGUIOperationWenowdescribeoperationsonleafimageswhichcanbeperformedusingleftsidepanelsof
themainwindow(seeOverviewabove).Weassumethatauserhasalreadyloadedanimageof
aleaf(seeFile>OpenImageinDropDownMenussectionforinstructionsonhowtoloadan
image).SettingtheScaleThispanelallowstheusertosetthescale,inpixelspermillimeterfortheimage. Ifthescaleis
knownforexample,50pixelspermm,theusercanenterthis
valueandproceed(Figure1).Alternatively,iftheimage
containsascalebar,theusercanenterthelengthofthe
scalebarinmm,thenclickonthesetscalebutton(Demo
Video2).Whentheuserplacesthecursorovertheraw
image,crosshairswillappear.Theuserthenclicksateither
endofthescalebar,thendoubleclicksonthebaritself.Thenewlycalculatedscale,in
pixels/mmwillappearinthescalewindow.Notethatthecalculationofsummarystatisticssuc
asareoleorveindimensionsrequiresthattheuserenterorsetthescale,andifthescaleisnot
enteredwhenselectingtheseoptions,anerrormessagewillappear.Becausemanyusers
beworkingwithcollectionsofimagesthatallhavethesamescale,thescaleisnotresetwhen
userclearstheimages(seeClearImagesbelow).
h
will
a
Figure1Settingthescaleandcroppingpanel.
ImageCropping
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Therearetwoimagecroppingoptions
available,rectangularandpolygonalcropping
(Figure2).Rectangularcropping,whichwill
likelybethepreferredchoiceofmostusers
duetoitseaseofuse,isavailableasabutton
onthemainguipanel(Figure1),while
polygonalcroppingcanbefoundonthe
Imagedropdownmenu.Bothoptionsallow
theusertocropawayunwantednoisethat
occursaroundtheleafimage,orfocusona
regionofinterest. Polygonalcroppingallows
theusertoplacepointsaroundtheregiontheywanttocropbyclickingamouse.Whenthe
regionofinterestisclosed,anewrectangularimagewillbeformedthatjustenclosesthe
polygon.Blackpixelswillpadtheimagetoformarectangle.Alternatively,theusercanplacea
rectanglearoundtheleaforregionofinterest.Thisapproachtypicallyyieldsthebestresultsas
itdoesnotcreateblackregionsaroundthedesiredregionthatmustbedealtwithlater(see
DemoVideo3).
Figure2.Polygonandrectangularcroppingoptions.
ThresholdingThethresholdingoptionswithintheLEAFGUIallowtheusertoconvertRGBorgrayscaleimages
intobinaryimages(1sand0s).Thisisacriticalstepasalmostallsubsequentcleaningand
segmentationfunctionswithintheLEAFGUIactonbinaryimages.Therearethreeoptions
withinthethresholdingbuttongroup: adaptivethresholding,globalthresholding,ora
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8
combinationofthetwo(Figure3). Adaptivethresholdingisan
approachthattakesintoaccounttheunevenilluminationofmany
leafimages.Tocorrectforthis,adaptivethresholdingusesalocal
window,thesizeofwhichinpixelsissetbytheuser.Thiswindow
isthenmovedacrosstheimageandpixelsareseparatedinto
foregroundandbackgrounddependingonwhethertheirvaluesare
greaterthanthethresholdlevelsetbytheuser.Inaddition,we
provideaglobalthresholdingoption.Globalthresholdingfirst
convertstheimagetograyscale,thenseparatesforegroundfrom
backgroundbasedonwhetherthepixelintensitiesaregreateror
lessthanthethresholdlevel.Bothadaptiveandglobalthresholding
utilizesliderstosetthethresholdlevelswhichcanspeeduptheprocesswhentryingmultiple
levelsouttoseehowwelltheysegmenttheimage.
Wehavefoundthatacombinationofbothadaptiveandglobalthresholdingworksbest
onimagesofclearedleaves:adaptivethresholdingtendstocapturesmallveinsbetterwhile
globalcaptureslargeveinsbetter.Tocombineimages,firstutilizetheadaptiveapproachtoget
theModifiedImagewhereyouwantit.BycheckingtheAdaptiveImagecheckboxatthe
bottomoftheThresholdingpanel,theuserstoresacopyofthethresholdedimageandan
additional
copy
is
sent
to
the
Queue
(see
below).
This
applies
to
global
thresholding
as
well,
and
oncebothboxesarechecked,theuserpressestheCombinebutton,andthetwothresholded
imagesarecombinedintoasingleimagewhichisnowdisplayedintheModifiedImagepanel
(seeDemoVideo4).
Figure3ThresholdingPanelwithCombiningoption.
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9
ImageStorageandAreaMaskMany
of
the
cleared
leaf
images
we
have
processed
have
significantnoisearoundtheleafimageitselfthatremainsin
theimagesevenafterthresholdingsteps.Therearemultip
approachesbywhichtheusercouldremovethatnoise
includingcroppingorcombinationsofoptionswithintheClean
BinaryImagepanel(seebelow),butwehavefoundthatsimply
creatingabinarymaskoftheleafimageisbyfarthefastestapproach.Todothistheuserfirst
setstheglobalthresholdinglevelhighenough(orlowenoughdependingonimagedetails)so
thatallthepixelswithintheinterioroftheleafareblackandthebackgroundiswhite.Often
therearestillregionsthatneedtoberemovedandthiscanbeaccomplishedthroughthe
RemoveRegionsoptionintheCleanBinaryImagepanel(seebelow).Oncetheuserissatisfied
withthesegmentationandcleaning,theusercreatesacomplementoftheimagewhichisthen
storedbycheckingtheAreaImagecheckboxwithintheAreaMaskpanel,whichalsosendsa
copytotheQueue(Figure4).Theimageisnowstored,andcanbeusedinsubsequentstepto
removebackgroundnoisebypressingtheMaskbutton(DemoVideo4).Thismultiplies
whateverimageisintheModifiedImagewindowbythestoredimagemask.Theresulting
imagecontainsonlythepartoftheOriginalImagethatwouldbecontainedwithinthewhite
Maskregion.
le
CheckingtheAreaImage,VeinImageorSkeletonImagetabscausesacopyofthese
imagestobesenttotheQueueforfutureuseifneededandasverificationtotheuserthatthe
Figure4.AreaMaskPanelwithArea,VeinandSkeletonimagestorageoptions.
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imageshaveindeedbeenstored.Inaddition,theoptiontostoreacopyoftheVeinImageand
SkeletonImagegivestheusertheoptiontomanuallyinspectthesetwosteps,modifyingthe
imagesifdesiredbeforecallingtheVeinStatsalgorithm.Inaddition,ifcopiesoftheseimages
arestored,theyareusedintheBinaryw/Edges,NodesandTipsvisualizationoptiondescribed
below.
CleaningtheBinaryImageTherearemultipleoptionsintheCleanBinaryImagepanelthatallowtheusertoimprovethe
qualityoftheleafveinimagebeforeitissaved,orpassedtosubsequentstatisticsalgorithms
(Figure5). Asindicatedbythepaneltitle,allofthealgorithmsinthesectionworkonbinary
images,thus,theimageintheModifiedImagewindowmustbebinaryinorderforthese
algorithmstowork.
RemoveRegions(Ctrl+R)thisstepremovescontiguouswhitepixel
regionswhosenumberofpixelsisequaltoorlessthanthevalue
setbytheuser(1000inFigure5).Theactualvaluethattheuser
utilizesheredependsentirelyontheimage,desiredoutcome,and
thecomfortleveloftheuserinthewholesaleremovalof
contiguousregions:usewithcaution(DemoVideo6).
Figure5CleanBinaryImagepanel.
RemovePerimeter(Ctrl+P)removesasinglepixelwideperimeter
oftheleaf(whitepixels).Thisstepcanberepeatedasmanytimes
asneeded(DemoVideo6).
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RemoveLabeled(Ctrl+L)thisbuttonopensupanewpopupwindow.Withinthewindow,any
contiguousblackpixelregionsareidentified,labeled(theyareassignedaspecificsequential
numericalidentifier)andrandomlyassignedacolortoallowtheusertobetteridentifyseparate
regions.Thecursorarrowisreplacebycrosshairs,andtheusercannowclickonanyregions
whichheorshewantstoremovefromtheModifiedImage.Aftertheuserisfinished,pressing
theEnterbuttononthekeyboardbringsupanewimagewithanewcolorschemeinwhichall
regionstheuserselectedhavebeenremoved.Anadditionalpopupwindowopensaskingifthe
userwouldliketoremoveanyadditionalregions.Selectingyesreturnstothepreviousstep:
selectingnoreturnstheimagelabeledimagetotheprimaryGUIwindow(DemoVideo6).
RemoveSpurs(Ctrl+S)removesanywhitepixelsthatareconnectedtoonlyasingleother
whitepixel(either4connectedor8connected).Thisstepcanberepeatedasmanytimesasis
necessary,andcanbeperformedbothonthestandardbinaryimage(whiteveins,black
background),orthecomplementofthatimage(whitebackground,blackveins)toimprovethe
performanceoftheEdgeStatsalgorithm,byremovingspuriousedges:usewithcaution(Demo
Video6).
FillRegions(Ctrl+Y)thisbuttonopensupanentirelyseparatepopupwindow.Withinthis
window,theuserinteractivelyplacesthecursor(crosshairs)byclickingwithintheimageto
formapolygon.Whenthepolygonregionisclosed(acirclewillformwhenthecursorisplaced
over
the
first
vertex),
the
popup
window
closes,
and
the
region
that
the
user
created
is
now
filledwithwhitepixelsintheModifiedImage(DemoVideo6).Thisstepcanbeusedtoremove
noiseortofillinveinregionsthattheuserthinksshouldbefilled.
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ClearBorder(Ctrl+B)thisstepremovesanycontiguouswhitepixelregions,suchasunwanted
noise,thatoverlaptheimageborder(DemoVideo4).
Skeletonthisbuttonreducestheveinimagetoitsskeleton,whichisasinglepixelwide
representationofthenetwork.Thiscanbedoneiterativelybyclickingthebuttonrepeatedlyor
entirelybyinvokingtheSkeletonizeFully(Ctrl+F)optionintheCleanBinarydropdownmenu.
TheskeletonisintegralintheEdgeStatsalgorithm,sovisualizingtheskeletonwillbeimportant
forverifyingthatthereisgeneralagreementbetweentheskeletonimageandtheoriginal
image.ThiscanfurtherbevisualizedusingsomeoftheoptionsintheCompositedropdown
menu:eitherOriginalw/SkeletonorBinaryw/edges,nodesandtips(seebelow).
ImageComplement(Ctrl+K)thissimplycreatesacomplementoftheimage (0sbecome1s,
1sbecome0s)intheintheModifiedImagewindow,whetherbinaryorRGB(DemoVideo6).
CreativeuseoftheImageComplementoptionexpandsthemethodsbywhichtheusercan
cleanthebinaryimagebeforepassingittothestatisticalgorithms.
FullorZoomRadioButtonsthesemutuallyexclusivebuttonsallowtheusertospecify
whethertheywanttheoperationswithintheCleanBinaryImagepaneltobeperformedonthe
entireimage(Fulloption)oronlywiththeregiondelineatedbythezoomwindow(Zoom
option)(DemoVideo6).Thiscanbeahelpfulstepifnoiseisunequallydistributedwithinthe
ModifiedImage.
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ImageSegmentationTheImageSegmentationpanel(Figure6)allowstheuserto
transform
the
leaf
vein
image
using
several
methods
that
highlight
aspectsoftheimagethatareusedinsubsequentstatistical
algorithms.TheseimagescanbesavedintheQueue(seebelow)
tobeusedfordemonstrationpurposes,orforpublication.
LabelLeafVeinsthisstepidentifiescontiguouswhitepixel
regions(leafveins)andassignseachaspecificsequentialnumericalidentifier.Eachregionis
thenrandomlycolorcodedtoallowtheusertovisualizehowwelltheveinnetworkis
connected.Anentirelyconnectednetworkwouldbeasinglecolor;whileadisconnected
networkwouldhaveseveralcolors(seeDemoVideo7).
Figure6ImageSegmentationPanel
LabelAreolesthisstepisidenticaltotheLabelLeafVeinsstepexceptthatareolesinsteadof
veinsarelabeledandrandomlycolorcoded. Theregionsidentifiedherearethesamethose
thatwillbeprocessedbytheAreoleStatsalgorithminthefollowingsection(seeDemoVideo
7).
BinarizeRGBthisbuttonconvertsanRGBimageasmightbecreatedinthepreviousstep
(LabelAreoles)intoabinaryimage(DemoVideo7).
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SummaryStatisticsTheSummaryStatisticspanelcontainsthebuttonsthatinvokethealgorithmsthatwilllikelybe
of
greatest
interest
to
most
users.
Note,
while
we
have
made
every
effort
to
optimize
these
algorithmsforspeed,forlargeleavesorlargeleafimages,theycantakesometimeto
complete,sobepatient.
AreaStatsthisbuttoncalculatestheareaandperimeteroftheentireleaf.Theareaisdefined
asthetotalnumberofpixelsintheimagetimesthescalefactorsquared(mm/pixel)2.The
perimeteristhelengthinpixelsoftheboundaryaroundthecontiguousbinaryleafimagetimes
thescalefactor(mm/pixel).Forreference,thescalefactorisalsoincludedintheoutputofthis
algorithm.
Figure7SummaryStatisticPanel
AreoleStatsthisbuttoninvokesanalgorithm thatcalculates
anumberofdifferentstatisticsforeachindividual areoleinthe
image.ThesearereturnedasaTextfileorasa tableinan
Excelspreadsheetdependingonuseroutput preferences
(Figure7).Whenclicked,adialogboxwillopen askingifthe
imagetobeprocessedhasbackgroundornot.If theimageis
forexampleanentireleafsurroundedbyblackbackground,theanswerisyes.Iftheimageisa
subsamplewithintheleaf,theanswerisno.Theunderlyingalgorithmcalculatesstatisticsforall
contiguousblackregionsintheimage,ofwhichthebackgroundisone,soinansweringthis
questiontheuserisessentiallyremovingthebackgroundfromthestatisticsreturned.Notethat
ifprocessingasubsampleofanimage,theuserwillprobablywanttoremoveareoles
overlappingtheborderastheyarenotcomplete.Thiscanbedonebycreatingacomplementof
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theimageandusingtheClearBorderbuttonintroducedpreviouslyandcreatinganimage
complementagain(seeDemoVideo8).
Thestatisticsreturnedinclude:
1)AreoleNumber,theuniquenumericalIDassignedtothatareole,from1tothenumberof
areoles.
2)AreoleArea(mm2),thenumberofpixelsineachareoletimestheareaofasinglepixel.
3)ConvexArea(mm2)theareaoftheconvexhullthatjustenclosestheareole.
4)Eccentricity(dimensionless,rangebetweenzeroandone)thisistheratioofthedistance
betweenthefociofanellipseanditsmajoraxis.Theellipsehasthesamenormalizedsecond
momentofareaastheregion.
5)Solidity(dimensionless,rangebetweenzeroandone)thisistheratiooftheAreoleAreatoits
ConvexArea.
6)Perimeter(mm)thedistancearoundtheboundaryofareole.
7)EquivalentDiameter(mm)thisisthediameterofacirclethathasthesamenormalized
secondcentralmomentsastheregion.
8)MajorAxisandMinorAxis(mm)oftheellipsethathasthesamenormalizedsecondcentral
momentsastheregion.
9)Centroid(coordinates,x,y)thecenterofdensityoftheregion.
10)
Mean
Distance
(mm)
the
average
Euclidean
distance
between
each
areole
pixel
and
the
nearestveinpixel.
11)VarianceDistance(mm)thevarianceintheMeanDistance.
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AnexampleofatablewithstatisticsfromtheAreoleStatsoutputcanbeseenontheLEAFGUI
website.ForfurtherinstructionseeDemoVideo8.
VeinStatsthisinvokesanalgorithmthatreturnsthefollowingstatistics:
1)EdgeNumbertheuniquenumberassignedtoidentifythatedge,from1tothenumberof
edges.
2)Length(mm)thelengthoftheedge.Thisiscalculatedasthedistancealongtheskeleton,
withthedistancebetweenpixelssharingaboundaryas1andthedistancebetweenpixels
connectedbytheircornersasthesquarerootof2.Thesedistancesarethenconvertedtomm
usingthescalepreviouslysetbytheuser.
3)Width(mm)a.k.a.diameter,iscalculatedusingthedistancetransformtofirstidentifythe
distanceofallveinpixelstothenearestareolepixel.Thedistanceofeveryskeletonpixel
(radius)isthendoubledandmultipliedbythescalefactortoequal(approximately)the
diameterofeachedgeatthatpoint.Eachskeletonpixelthereforehasacorresponding
diameter,andtheaverageacrossallofthediameterswithinagivenedgeisthentheWidthof
thatedge.
4)Area(mm2)iscalculatedasthesumoftheWidthateachskeletonpixelasintheprevious
steptimesthelengthofasinglepixelacrossalloftheskeletonpixelscontainedwithinagiven
edge(seemainmanuscript).Thisisthe2Dareaofeachedge.
5)
Surface
Area
(mm
2
)
is
calculated
using
the
same
approach
is
the
previous
step,
except
that
eachskeletonpixelistreatedasthecenterofacylinderinsteadofarectangle.Thediameter(d)
ofthecylinderisascalculatedabove.Thelength(lorheight)ofthecylinderislengthofasingle
pixel,andthesurfaceareaisthecalculatedusingthefamiliarformulaforthesurfacearea(SA)
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ofacylinder(notincludingends),SA=2p(d/2)*l.Thetotalsurfaceareaofanedgeisthenthe
sumofalloftheindividualcylinderscorrespondingtoeachskeletonpixel.
6)Volume(mm3)iscalculatedinthesamewayasSurfaceArea,excepttheformulaforcylinder
Volume(V)isusedinstead,V=p(d/2)2*l.
7)Centroid1isthemeanofallxcoordinatesfortheskeletonpixelsinagivenedge.
8)Centroid2isthemeanofallycoordinatesfortheskeletonpixelsinagivenedge.
UsingtheQueueTheQueue(Ctrl+Q)buttonstoresacopyofwhateverimageiscurrentlyintheModifiedImage
window,andsendsathumbnailcopyoftheimagetoaseriesofsmallpanelsrunningalongthe
righthandedgeoftheLEAFGUI.Uptotenimagescanbestoredinthisway.Atanypointthe
usercanreturnoneoftheimagestotheModifiedImagewindowbyselectingtheposition
(numberedtoptobottom)occupiedbythatimagefromtheQueuedropdownmenu.For
example,selectingImage4fromthedropdownmenuplacesthefourthimagefromthetopof
theQueueintheModifiedImagewindow.Inaddition,alloftheimagesintheQueuecanbe
savedsimultaneouslytothefoldercontainingtheLEAFGUIsoftwarebyselectingtheSaveAllIn
Queue(Ctrl+H)optionsfromthedropdownmenu.Moreover,byselectingClearQueue(Ctrl+P),
theusercancleartheQueuetoallowformoreimagetobestored.
ClearingCurrentImages(Ctrl+D)
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ClearsallimagesfromtheGUIwindowandQueue:Dothisstepbeforestartingtoworkwitha
newimage.Onclickingthisbuttonadialogboxwillopenaskingiftheuserreallywantstoclear
alltheimages.Thisistopreventaccidentalclearingofallimages.
Undo(Ctrl+U)Undoesthelaststep:Astheuserisworking,copiesofthelast20imagesthatwereprocessed
arestored,andcanbereturnedtotheModifiedImagewindowbyclickingUndo.However,
thereisnoredooptionmeaningthatonceausergoesbacktoapreviousimage,theimages
thathadbeensubsequenttoitarenolongerstored.
InterruptTheinterruptbuttonallowstheusertointerruptanyalgorithmthatistakingalongtime.Some
ofthestatisticsalgorithms,particularlyVeinStats,cantakeawhileonalargeleaf.Thisfunction
givestheusertheoptiontohaltanyprocessescurrentlyrunning.
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DropDownMenusManyfunctionsareavailableindropdownmenusthatarenotontheLEAFGUImainwindow.
Inaddition,mostofthefunctionsavailableonthemainwindowarealsolistedindropdown
menus.Thisisprimarilysotheusercanseethehotkeysavailable(seealsoTable1onpage
28).
FileOpenImage(Ctrl+O)Opensastandardwindowinwhichtheusercanlocateandclickonthe
imagetheywouldliketoopeninLEAFGUI.
SaveModifiedImage(Ctrl+M)Opensupafigurewindowwithwhichtheusercansavethe
ModifiedImageintheformatoftheirchoice.
SaveOriginalImage(Ctrl+I)Opensupafigurewindowwithwhichtheusercansavethe
OriginalImageintheformatoftheirchoice.
QuitLEAFGUI(Ctrl+W)ClosestheentireLEAFGUIprogram,afteraskingifthatsreallywhat
youwanttodo.
ImageMeasureScale SeeSettingtheScalepage6.
MeasureDistance(inpixels)placesamovable,resizablelineontheimagethatdisplaysits
lengthinpixels.Thisfeaturecanbeusefulformeasuringleaffeaturessuchastotalleafwidthor
length(followinguserconversionfrompixelstomm).
Undo(Ctrl+U)SeeUndopage18.
ClearImages(Ctrl+D)SeeClearingCurrentImagespage18.
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OriginaltoModifiedsendstheimagethatiscurrentlyintheOriginalImagepaneltothe
ModifiedImagepanel.
ModifiedtoOriginalsendstheimagethatiscurrentlyintheModifiedImagepaneltothe
OriginalImagepanel.
CropwithRectangle SeeImageCroppingpage6.
CropwithPolygonSeeImageCroppingpage6.
CompositeFunctionsOriginalw/skeletonthiscreatesacompositeimageusingthebinaryimageintheModified
Imagewindow.Theresultisarepresentationoftheleafveinnetworkwithblueveins,
superimposedredSkeleton,andablackbackground.Thisallowstheusertoinvestigate
whetherthealgorithmcorrectlyidentifiesnodesandedges(seealsoBinaryplusedges,nodes
andtips).
Binaryplusedge,nodesandtipsthiscreatesacompositeimageusingthebinaryimageinthe
ModifiedImagewindow.Theresultisarepresentationoftheleafveinnetworkwithblack
background,blueveins,andsuperimposedlightblueSkeleton,rednodes,andyellowtips.This
allowstheusertoinvestigatewhetherthealgorithmiscorrectlyidentifyingnodes,edgesand
tips(seealsoOriginalw/skeleton).
Connected
Nodes
this
algorithm
creates
a
skeletonized
image
of
the
binary
network
overlain
withnodesencircledinblueandrededgesshowingwhichnodesareconnected.Thisoption
allowstheusertoassesstheextenttowhichthealgorithmsarecorrectlyidentifyingtheactual
nodesandedges.
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SuperimposePerimeterthissuperimposesaredcopyoftheperimeterofthebinaryveinimage
thatisdisplayedintheModifiedImageWindowontopofagrayscaleversionoftheimagein
theOriginalImageWindow.Thisstepallowstheusertoinvestigatehowwelltheresultsfrom
previousthethresholdingandcleaningalgorithmsworked.
CleanBinaryRemoveRegions (Ctrl+R)SeeRemoveRegionspage10.
RemovePerimeter(Ctrl+P)SeeRemovePerimeterpage10.
FillSingles(Ctrl+E)fillsanysingleblackpixelsthatareentirelysurroundedbywhitepixels
(DemoVideo6).Thisisahelpful,andtypicallyanecessarystepincleaningtheimage.Dueto
imagenoisethereareoftensingleblackpixelssurroundedbywhitepixelsthataredifficultto
seeinboththeModifiedimagewindowandthecorrespondingzoomwindow.Fillingthepixels
eliminatesthisproblemandimprovestheperformancesofsubsequentstatisticsalgorithms,
particularlytheEdgeStatsandAreoleStatsalgorithms.Toremoveregionslargerthanasingle
pixel,theRemoveRegionsoptioninthecomplementofthebinaryimagecouldbeused.
FillHolesthisoptionfillsanyregionswithintheimagethatareentirelysurroundedbywhite
pixels.ThisstepcouldbeusefulingeneratingaMask(seeabove).
FillRegions(Ctrl+Y)SeeFillRegionspage11.
RemoveSpurs(Ctrl+N)SeeRemoveSpurspage11.
Clear
Border
(Ctrl+E)
See
Clear
Border
page
12.
RemoveLabeled(Ctrl+G)SeeRemoveLabeledpage11.
ImageComplement(Ctrl+Q)SeeImageComplementpage12.
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Thin(Ctrl+X)Removestheexteriormostpixelsfromcontiguousregions.Thismightbehelpfulif
theuserthinkstheveinsonthebinaryimagearemuchthickerthantheactualveins.However,
keepinmindthatforveinsthreepixelswideorless,thisstepwillthintheveintoasinglepixel,
thusveinsthatarethreepixelswideandveinsthataretwopixelswidewillbecomeasingle
pixelwide:usewithcaution.
DiagonalFillthisoptioneliminateseightconnectedpixelsinthebackground(seeMatlab
documentation).
DilateImage performsimagedilationwitha3x3structuringelement.Formoreinformationon
dilationseeMatlabdocumentation.
ErodeImage performsimageerosionwitha3x3structuringelement.Formoreinformationon
erosionseeMatlabdocumentation.
CloseImageperformsamorphologicalcloseoperation(dilationfollowedbyerosion)witha
3x3discasastructuringelement.FormoreinformationseeMatlabdocumentation.
SkeletonizeIteratively(Ctrl+F)sameresultasSkeletonabove,butdoessoiteratively,i.e.pixels
areremovedfromtheboundariesofthebinaryimageonelayeratatime.
ZoomRefreshZoom(Ctrl+Z)usethisstepafterresizingormovingtheZoomWindowintheOriginal
Image.
This
step
will
also
update
the
red
reference
window
in
the
Modified
Image
window
whichsimplyallowstheusertoseeeasilytheregionontheModifiedImagethattheZoom
WindowontheOriginalImagecorrespondsto.
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ImageSegmentationLabelLeafVeinsseeLabelLeafVeinspage13.
LabelAreolesseeLabelAreolespage13.
BinarizeRGBseeBinarizeRGBpage13.
DistanceTransformthisbuttonmeasuresthedistancefromeveryblackpixelintheimageto
everywhitepixel,thenconvertsthosedistancestoaheatmapsotheusercanvisualizehowfar
awayareoleinteriorsarefromveins,orhowfartheveininteriorsarefromareoles.Thedata
correspondingtothisfigureisreturnedfromtheEdgeStatsalgorithm.
StatisticsAreaStatsseeAreaStatspage14.
AreoleStatsseeAreoleStatspage14.
VeinStatsseeVeinStatspage16.
ConnectivityMatrixthiscommandgeneratesamatrixshowingwhichNodesareconnectedto
whichEdges.TodeterminetheConnectivityMatrix,the3x3neighborhoodaroundeachnodeis
searchedtoseewhatedgesitisconnectedto.TheConnectivityMatrixisfirstrepresentedasan
NxN (N=numberofnodes)matrixwhererowsandcolumnindicesrepresentthelistofnodes,
andmatrixentriesrepresenttheedgesthatconnectnodes.Tosavespace,theconnectivity
matrixisthencondensedtoanEx3(E=numberofedges)matrixshowingwhichlabelednodes
(columns1and2)areconnectedbywhichlabelededges(column3).
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QueueImage1Image10 thesefunctionssimplymoveacopyofwhateverimagehasbeenstoredin
thecorrespondingQueueposition,numberedtoptobottom.Forexample,iftheuserhas
previouslystoredacopyofabinaryleafveinimageinthesecondQueueposition,byclickingon
Image2intheQueuedropdownmenuthatimagewillbemovedtotheModifiedImage
window(seeDemoVideo5).
SendtoQueueSeeUsingtheQueuepage17.SaveAllinQueueSeeUsingtheQueuepage17.ClearQueue SeeUsingtheQueuepage17.
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CaveatsWhenandwhennottouseLEAFGUI
1)LEAFGUIworksbestonimageswherethereisgoodcontrastbetweenveinsandimage
background.Ifthegrayscaleintensityofthebackgroundistoosimilartotheveinsthemselves,
thresholdingstepswillnotbeabletosegmenttheimageswell,andtherewillbesignificant
noiseintheresultingbinaryimages.
2)Ifimagesareveryhighresolution,theskeletonizationalgorithmsuponwhichtheVeinStats
functionreliesmaycreatespuriousEdges,particularlyinthelargestveins.Useeitherthe
Originalw/SkeletonortheBinarywithedges,nodesandtipsoptionsintheCompositedrop
downmenutoinvestigatethis.ThereareseveralmethodsbuiltintotheLEAFGUItoreducethis
phenomenonincludingusingFillRegions,RemoveSpurs,DiagonalFill,DilateImage,Erode
Image,CloseImage,Bridge,ThinandFillandtheabilitytoperformmanyofthesefunctionson
subsectionsofimageswiththeFullorZoomoption;howeverallofthesemethodsshouldbe
usedwithcaution.
3)Theresultsfromthesoftwareareheavilydependentonthequalityoftheinitialimages.
Takingalittleextratimebeforehandtoimproveimagequalitywillgoalongwaytoward
improvingthesoftwaresperformance.
4)Someofthealgorithms,inparticularthestatisticsalgorithmscantakeasignificantamount
oftimetocomplete.Theamountoftimeincreasesexponentiallywithleafsize.Wehave
processedleaveswithover100kindividualnodes,butdoingsoonacomputerwitharelatively
slowprocessorisnotagoodidea,andmayproduceanoutofmemoryerror.Wehopeto
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developanonlineversionoftheGUIlinkedtoadedicatedserver,orevenperhapsa
supercomputer,butuntilthattime,wesuggestrunningLEAFGUIonamorepowerfulmachine.
5)AlmostallofthefunctionsinLEAFGUIareinterruptiblemeaningthatifanewfunctionis
invokedtoosoon,thepreviousfunctionmaynothavefinished.Thustoavoidconfusingresults
fromclickhappyusers,patienceisadvised.
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ContactsDrChuckPrice
PostdoctoralFellow,WeitzLab
SchoolofBiology
GeorgiaInstituteofTechnology
Atlanta,GA,30332
ContactinformationasofAugust2010:
AssistantProfessor
SchoolofPlantBiology
UniversityofWesternAustralia
35StirlingHighway
Crawley,Perth
WesternAustralia6009
DrJoshuaWeitz
AssistantProfessor
SchoolofBiology
GeorgiaInstituteofTechnology
Atlanta,GA,30332
DrOlgaSymonova
PostdoctoralFellow,WeitzLab
SchoolofBiology
GeorgiaInstituteofTechnology
Atlanta,GA,30332
DrYuriyMileyko
DepartmentofMathematics
DukeUniversity,Box90320
Durham,NC277080320
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Function HotKeyOpenImage O
SaveModifiedImage M
SaveOriginalImage I
QuitLeafGUI W
Undo U
ClearImages D
RemoveRegions R
RemovePerimeter P
FillSingles E
FillRegions Y
RemoveSpurs S
ClearBorder B
RemoveLabeled L
ImageComplement K
Skeleton F
RefreshZoom Z
LabelLeafVeins V
LabelAreoles A
BinarizeRGB G
SaveAllInQueue H
ClearQueue N
SendtoQueue Q
Table1.HotkeysavailableintheLEAFGUI.Thesearealsonotedinthedropdownmenus.Whereverpossible,wehavechosentheHotKeystomakethemmnemonicasindicatedbythe
grayshading.