Leaf GUI User Manual

8/7/2019 Leaf GUI User Manual

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LEAFGUI:UserManual(LeafExtractionandAnalysisFrameworkGraphicalUserInterface)


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LEAFGUIUserManual Priceetal.

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/


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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

[email protected]

ContactinformationasofAugust2010:

AssistantProfessor

SchoolofPlantBiology

UniversityofWesternAustralia

35StirlingHighway

Crawley,Perth

WesternAustralia6009

DrJoshuaWeitz

AssistantProfessor

SchoolofBiology


Atlanta,GA,30332

[email protected]

DrOlgaSymonova

PostdoctoralFellow,WeitzLab

SchoolofBiology


Atlanta,GA,30332

[email protected]

DrYuriyMileyko

DepartmentofMathematics

DukeUniversity,Box90320

Durham,NC277080320

[email protected]

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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.

Leaf GUI User Manual

Documents

Transcript of Leaf GUI User Manual