APPLICATION NOTE

VISQUE™ InVivo Elite

Preclinical Optical Imaging System

ThisdocumentdescribeshowtomergeimagesbyusingCleVue™designedexclusivelyforVISQUE™InVivoElite.TheCleVuesoftwareprovidestwooptions:(a)Previewand(b)Color,formergingimages.

Thedocumentcoversthreedifferentscenarios:1)Mergingafluorescent/bioluminescentimagewithapreviewimage2)Mergingfluorescentimageswithoutblendinganoverlappedarea3)Mergingfluorescentimageswithblendinganoverlappedarea

Formoredetailedinformation,pleaserefertotheCleVueSoftwareUserManual.

How to Merge Images

Drop down list to selecta preview image

Drop down list to selecta fluorescent/luminescent image

A B

Figure 1. Merging a luminescent image with a preview image

APPLICATION NOTE

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1.Beforemergingafluorescent/luminescentimagewithapreviewimage, youneedtoimporttheimagesfirst.2.UsetheFILE > Import menu to import the images. Selectafluorescentorluminescentimagetomerge. Selectapreviewimagetomerge.

3.Ifnecessary,adjustthewindowlevelofthepreviewimagebyusingtheROI W/L iconorScreen Stretch pane.4.SelectthePROCESSING > Merge > Preview menu. Themergewindowappearsasshowninthefigureright.5.SelectthefluorescentorluminescentimagefromtheFluorescence & Luminescencedrop-downlist(A).6.SelectthepreviewimagefromthePreviewdrop-downlist(B).7.ClicktheMerge button to display the merged image in the Result pane.

• Aftermergingafluorescentorluminescentimagewitha previewimage,youcanadjustthepseudocolororwindow levelofthefluorescentorluminescentimage,butnotofthe previewimage.• Theresultimagewillnotbesavedautomatically. UsetheFile > Save or Save Asmenutosaveit.

Merging a fluorescent/bioluminescent image with a preview imageThisscenariodescribeshowtomergeafluorescentorluminescentimagewith apreviewimage(photographicimageofthetargetobject)inTIFFformat(*.tif). Step 3 Importaluminescentimage. Importapreviewimage.

Step 4-6 Selecttheluminescentandpreviewimagesinthemergewindow.

Step 7 ThemergedimageisdisplayedintheResultpane.

Luminescent.tiforLuminescent-Dark.tif

Preview.tif

A.Fluorescence&Luminescence drop-down list

B.Preview drop-down list

Example of merging a luminescent image with a preview image

Theluminescentimageisoverlaidonthepreviewimage.

How to Merge Images

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Assume that the followingproceduremergesa fluorescent imagemergedwithapreviewimageinCIFformatandanotherfluorescentimageinTIFFformat.1.BeforemergingafluorescentimageinCIFformatwithanotherfluorescentimage inTIFFformat,youneedtoconvertyourCIFimagetoaTIFFimagefirst. UsetheFILE > Import menu to import the CIF image. Ifnecessary,selectacolorbartoimplementintheimagebyusingthe Properties pane > Display > Pseudo Color. AdjustthewindowleveloftheimagebyusingtheROI W/LiconorScreen Stretch pane. ClicktheFILE > Export > Image,andthenselecttheSave Options > Displayed ImagecheckboxtosaveinTIFFformat.

2.NowyouhavetwodifferentfluorescentimagesinTIFFformat.Beforemerging them,youneedtoimporttheimagesfirst.3.UsetheFILE > Import menu to import the images.SelectthefluorescentimagewhichisconvertedtotheTIFFimageinstep1. SelectanotherfluorescentimagesavedinTIFFformat.

4.SelectthePROCESSING > Merge > Preview menu. Themergewindowappearsasshowninthefigureleft.5.Selectthefluorescentimage(step3-)fromFluorescence & Luminescencedrop-downlist(A).6.Selectthefluorescentimage(step3-)fromthePreviewdrop-downlist(B).7.ClicktheMerge button to display the merged image in the Result pane.

Merging fluorescent images without blending an overlapped

areaThisscenariodescribeshowtomergeafluorescentor luminescent imagewithanadditionalfluorescentorluminescentimage.Thisisusefulforcombiningfluorescentor luminescent imagesacquiredat twodifferentwavelengths, forexample,whenimaging two different tumors simultaneously in a target animal.

• Aftermergingfluorescentimages,youcanadjustthepseudo colororwindowlevelofthefluorescentimagedescribedinthe step5(3-),butnotofthefluorescentimagedescribedinthe step6(3-).• Iftwodifferentfluorescentorluminescentimageshavean overlappedarea,thefluorescentimagedescribedinstep5 (3-)willbeoverlaidontheotherfluorescentimagedescribed instep6(3-).• Toblendanoverlappedareaoftwoimages,referto Merging fluorescent images with blending an overlapped area.

Example of merging fluorescent images without blending an overlapped areaStep 3 Importafluorescentimage. Importanotherfluorescentimage.

Step 4-6 Selecttwodifferentfluorescentimagesinthemergewindow.

Step 7 TheResultpanedisplaystheresult. Merginganoverlappedarea

CIFimageconvertedtoTIFFimage

Fluorescentimagein TIFFformat

A.Fluorescence&Luminescence

drop-down listB.Preview drop-down list

Thefluorescentimage is overlaidonthe other fluorescentimage.

Youcanonlyadjustthisfluorescentimage.

APPLICATION NOTE

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1.Beforemergingfluorescentimagesandapreviewimage,youneedtoimport, adjustthewindowlevelorpseudocolor,andthenexportthemasTIFFimages. UsetheFILE > Importmenutoimportthefluorescentimagesandapreviewin TIFFformat. Selectacolorbartoimplementineachfluorescentimagebyusingthe Properties pane > Display > Pseudo Color. Adjustthewindowlevelofeachfluorescentandpreviewimagebyusingthe ROI W/LiconorScreen Stretch pane. ClicktheFILE > Export > Image,andthenselecttheSave Options > Displayed ImagecheckboxtosaveinTIFFformat.

2. Close all image windows, and then import all images exported in step 1.3.SelectthePROCESSING > Merge > Colormenu.Themergewindowappearsas showninthefigureright.4.SelectonefluorescentimagefromtheImage Adrop-downlistandselecttheother fluorescentimagefromtheImage B drop-down list.5.ClicktheMerge button to display the merged image in the Result pane.6.SelectthePROCESSING > Merge > Color menu again.7.Selectthethirdimage(importedinstep2butnotusedinstep4)fromtheImage A drop-downlistandselecttheresultimagemergedinstep5fromtheImage B drop-down list.8.ClicktheMerge button to display the merged image in the Result pane.9.Repeatsteps6through8untilyoumergealltheimages.

Merging fluorescent images with blending an overlapped areaThisscenariodescribeshowtomergefluorescentorluminescentimageswithblendingintensitiesofeachimage. Thisisusefulforcombiningfluorescentorluminescentimagesacquiredatdifferentwavelengthsbyaddingintensitiesoftheimages.

Assumethatthefollowingproceduremergesfourfluorescentimagesandapreviewimage.ItisrecommendedthatyouadjustthewindowlevelorpseudocolorofeachimageandexportthemasTIFFimages,sinceyoucannotadjustthewindowlevelorpseudocoloraftermerging. Drop down list to select

another fluorescent/luminescent imageDrop down list to selecta fluorescent/luminescent image

A B

Figure 2. Merging fluorescent images with blending intensities

Step 1 Importfluorescentimagesandapreviewimage.

Step 3-4 / 6-7 Selecttwodifferentfluorescentimagesinthemergewindow.

Step 5 / 8 ThemergedimageisdisplayedintheResultpane.

Example of merging fluorescent images with blending an overlapped area

PE Cy5.5 GFP ICG Preview image

PE + Cy5.5 PE + Cy5.5 + GFP PE + Cy5.5 + GFP + ICG+ Preview imagePE + Cy5.5 + GFP + ICG

Intensities within overlapped areas are blended.

A. Image A drop-down list B. Image B drop-down list

How to Merge Images

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Setting the Reverse Mapping to True for a preview imageWith the Reverse Mapping parameter set to True,youcanapplythereversemappingtoapreviewimagebeforemergingafluorescentimagewiththepreviewimage.Insomecases,thismaybeusefulinordertoimprovevisibilityoffluorescencein the merged image.

1. UsetheFILE > Import menu to importapreviewimageinTIFFformat.2. In the Properties pane, set the Display > Reverse Mapping parameter to True.3.AdjustthewindowlevelofthepreviewimagebyusingtheROI W/LiconorScreen Stretch pane.4.Followtheproceduresdescribed in theMerging fluorescent images with blending an overlapped areascenariotomergeimages.

Reverse Mapping = False (default) Reverse Mapping = True

Example of setting the Reverse Mapping parameter

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APPLICATION NOTE

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Hints and Cautions for merging imagesWhen you merge images, please keep the following information in mind.

How to Merge Images

Binning

• Setting Path - Setting pane > Imaging > Binning > 2 � 2 or 4 � 4

• WhenyoumergeafluorescentorluminescentimageacquiredwiththeBinning parameter set to 2 � 2 or 4 � 4andaphotographicimageofthe targetobject(previewimage),theresolutionofthefluorescentorluminescentimagewillbeincreasedbythebinningfactor.Thisistruebecause;

- ApreviewimageisalwaysacquiredwiththeBinning parameter set to 1 � 1, - CleVuemergestwoimageswhentheimagesarebothinsameaspectratioregardlessoftheirsize.

• Forexample,ifyouacquireafluorescentimagewiththeBinning parameter set to 2 � 2,youwillacquireapreviewimagewitharesolutionof 1600 �1200andafluorescentimagewitharesolutionof800�600.Andifyoumergethefluorescentimagewiththepreviewimage,theresolution ofthefluorescentimagewillbeincreasedto1600�1200andthenthefluorescentimagewillbemergedintothepreviewimage.

Raw

• Setting Path - Properties pane > Image Processing > Raw

• IfyoumergefluorescentorluminescentimagesaftersettingtheImage ProcessingparametertoavalueotherthanRaw,youcannotverifytheraw fluorescentorluminescentimage.• ItisrecommendedthatyousettheImage Processing parameter to Rawtoverifyarawfluorescentorluminescentimageaftermerging.

Setting the window level for a photographic image of the target object

Itisrecommendedthatyouadjustthewindowlevelofaphotographicimageofthetargetobjectbeforemerging. Thisistruebecauseyoucannotadjustthewindowlevelofthepreviewimageaftermergingafluorescentorluminescentimagewithapreviewimage.

Displaying an image in Intensity or Radiance units

• Setting Path - Image in CIF format > Properties pane > Display > Units

• WhenyouacquireafluorescentorluminescentimagewithInVivoElite,aCleVueimagefile(*.cif)willbegenerated.Inthisfile,youcandetermine whethertodisplaytheimageinintensityorradianceunits.However,ifyousaveamergedimageasCIFformat,youcannotchangetheUnits setting oftheimage.ThisistruebecausetheUnitsparameterisnotavailableontheimagefilewhichismergedandthensavedasCIFformat.