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V.SimultaneousAnalysisofMultipleImagesUsingImageJ

SallySoria-Dengg,GEOMARHelmholtzCentreforOceanResearchKiel

Düsternbrookerweg20,24105Kiel,GermanyAnalysingmultipleimagessimultaneouslyisanotherfeatureofImageJwhichmakesdiscanalysiseasierandfaster.InthecontextofVIRTUE-s,thisissuitablefordeterminingtheareacoverageofthediscsatastagewhenfoulingisstillsparse.Inthisexample,severalphotomicrographsofasinglediscareusedtoextrapolatetothetotalareaofthedisccoveredbydiatoms.Asaprerequisite,areferencescale(stagemicrometre)needstobephotographed(cf.tutorialMicroscopyandImageJ).Inaddition,itisimportantthatthemagnificationofthereferencescaleandtheimagestobeanalysedaswellas,ifpossible,thefocus/adjustmentsarethesame.Also,inyourimagetheareasofinterestshouldhaveaverygoodcontrasttothebackground.1. DownloadtheversionofFijiImageJwhichiscompatiblewiththeoperatingsystem

ofyourcomputerfromthiswebsite:https://imagej.net/Fiji/Downloads(ThefollowingdescriptionreferstoVersion2.0.0ofImageJasof2018-12-04(checkinMenuFiji-AboutImageJ).Ifadifferentversionisused,somemodificationsmayhavetobemade,butinprincipletheprocedureshouldstillbeapplicable.)

2. OpentheprogramFijiandyouwillseethetoolselectionbarunderthemenubar.

(FiguresinthistutorialarefromtheMacversion.UnderWindowsorLinux,detailsmaylookdifferent).

(IfyouareworkingwithafreshlyinstalledversionofFiji,noadjustmentsneedtobemade.IfyouhaveusedthisversionofFijibeforeandchangedsomesettingsinEdit-Options,youshouldgotoEdit-Options-Reset,confirmtheresetandrestartFijitomakesureyouareusingthedefaultsettingsrequiredinthistutorial.CheckunderProcess-Binary-Optionsandensurethatthebox“Blackbackground”isactivated.)

3. Setthescale.Loadthepictureofyourreferencescalebydraggingthefileand

droppingitonthetoolselectionbar.Inourexample(file8X@20180112_174424.jpeginthecollectionofpracticeimages)wehaveadotwithaknowndiameterof0.6mmfromaMotic4-dotcalibrationslide.

Choosethe“Straight”tool(blackarrow)fromthetoolselectionbaranddrawastraightlineacrossthediameterofthedot(yellowarrow).

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ClickonAnalyze–SetScaleinthemenubarandfillintheinformationon“Knowndistance”(0.6)and“Unitoflength”(mm).SelectGlobaltousethisscaleforalltheimagesyouaregoingtoanalyse.ConfirmwithOK.Closetheimageofthereferencescale.

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4. Nowopentheimagesyouwanttoanalyse.Youcanselectalltheimagestobeanalysedatthesametimeanddraganddropthemonthetoolselectionbar.Alltheimageswillappearonyourscreen.ChecktomakesurethatallimagesareopeninImageJ.Ifthisisnotthecase,youmayhavetodraganddropthemissingimagesindividuallyontothetoolselectionbar.

Forthistutorialwehaveagroupof6imagesofrandomlychosenareasonadiscdeployedforfourweeksinDecember.Noticethatgrowthisstillsparseandnoindividualorganismscanbecountedunderthismagnificationunderastereomicroscope.Thedarkareasarecoloniesofdiatoms(microscopicalgae)thatwereidentifiedunderalightmicroscopebeforetakingthephotographs.Wenowwanttoestimatethetotalareaoccupiedbythediatomsonthediscfromthese6samples.

5. CreateaStack.SelectImage–Stacks–ImagestoStack.Giveanametoyourstack.

Inthisexamplewewillnamethestack“December”inreferencetothemonthofdeployment.Activate“UseTitlesasLabels”and“KeepSourceImages”.Thelatterwillretaintheoriginalimages.Youcanusetheseasreferenceimagesafterthepictureshavebeenconverted.Click“OK”.

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6. Astackisnowcreatedwithyourimages.(Alloriginalimageswillalsoberetainedasindividualpictures.)Onyourscreenyouwillseeoneimagewithaslidebar(blackarrow)atthebottomedgeofthewindow.Bymovingtheslideryoucanswitchbetweentheindividualimagesinthestack.Afilenametotheupperleft(yellowarrow,here‘8X@20180112_180158’)showswhichimageinthestackiscurrentlydisplayed.Theredarrowgivesthesizerepresentedbythecompleteframe.Inthisexample,youarelookingataportionofthediscwithanareaof8.16mmx6.12mm=49.94mm2.

7. Processtheimages.Youonlyneedtoprocessoneimagebecauseyouradjustments

willbeappliedtoalltheotherimagesinthestack.SelectImage–Type–8bit.Allimageswillbetransformedtogreyscaleimages.

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8. Improvethequalityoftheimagesforeasierthresholding.SelectImage–Adjust–Brightness/Contrast.Adjusttheslidebarstogetabrighterimagewithhighcontrast.Checktheotherimagesandtoseeifthequalityoftheseimageshasalsoimproved.(Atthispointyoudonotneedtopress“Apply”.Pressingitwillmainlychangethelookofthe“B&C”displayinthenextsteps.)

9. Thresholdtheimages.(Thisisnecessarybecausethenextoperationswillonlywork

onbinaryimages)SelectImage–Adjust–AutoThreshold.Awindowwillappear.Activate“Stack”anddeactivate“Whiteobjectsonblackbackground”(sincecurrentlywehavedarkobjectsonabrightbackground).SelecttheMethod“Default”.Click“OK”.Theimagesarenowleftinabinarymodewithblackandwhiteasonlyremainingcolours.Inaddition,thesetwocolourshavebeeninverted.(BlackwasdefinedasthebackgroundcolourinProcess-Binary-Optionsinstep2,andsotheprogramexpectswhiteareasastheobjectstoanalyse.)

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Comparethethresholdedimageswiththeoriginalpicturesandcheckifalltheregionsyouwanttoanalysearenowwhiteagainstablackbackground.(Ifyouarerunningoutofspaceonthescreen,minimizethe6individualpicturesandrecallthemindividuallyfromtheWindowmenu.)

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10. SelectAnalyze–SetMeasurements.Choose“Area”,“Limittothreshold”and“StackPosition”.Point“Redirectto:”tothenameofyourstack(inourexampleto“December”).Click“OK”.

11. Makemeasurements.SelectAnalyze–AnalyzeParticles.Adjustthesettingsto:

Size:0–Infinity.Circularity:0.00–1.00.(Thesesettingswilltakeallshapesandsizesintoaccount.)ChooseShow:Maskstogiveyouvisualfeedbackontheareasanalyzed.Activate:DisplayresultsandSummarize.Click“OK”.

12. Anewwindowwillaskifallimagesshouldbeprocessed.Select“Yes”.

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13. SeveralwindowswiththeresultswillappearbutonlytheSummaryisofinterestforus.Intheexamplebelowthenumberof“particles”(i.e.whiteareas)analyzedineachofthe6imagesisgiven(redbox),aswellasthetotalareaofalltheparticlesineachimage(bluebox).

14. Calculatethetotalareacoveredbydiatomsonthedisc:

• Totalareaanalysed(TAA)=areaofoneimage×numberofimages=

=49.94mm2×6(seeStep6)=299.64mm2=2.996cm2

• Totalareacoveredbydiatomsintheimages(TAD)

=6.736+6.286+2.483+4.518+3.773+4.518=28.314mm2 =0.283cm2

• PercentageCoverinanalysis=(TAD÷TAA)×100=(0.283cm2÷2.996cm2)×100=9.45%

• Totalareaofthedisc(AD).

Wehaveadiscwithadiameterof8.5cm.Areaofthedisc=πr2,wherer=radius=diameter÷2=4.25cm

Areaofthedisc=3.1416×(4.25)2=56.75cm2• Extrapolatetheareacoveredbydiatomstothetotalareaofthedisc(TADD)

=(TAD÷TAA)xAD

=(0.283cm2÷2.996cm2)×56.75cm2=0.0945×56.75cm2=5.36cm2

v.012020 Contact:sdengg@geomar.de