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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
2
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
3
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
4
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
5
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
D-17-459
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.