ManipulatingimageswithMatlab

MonochromeimagesinMatlab arerepresentedbyandNxM matrix Nistheheightoftheimage,Misthewidth LoadanimageintovariableJ:J=imread(spine.tif); Letsgetsomeinformationabouttheimage:whos J

ManipulatingimageswithMatlab
MonochromeimagesinMatlab arerepresentedbyandNxM matrix Nistheheightoftheimage,Misthewidth LoadanimageintovariableJ:J=imread(spine.tif); Letsgetsomeinformationabouttheimage:whos J Rawdataistypicallyuint butimagesshouldbedoublesbetween0and1 usefunctionmat2gray toconverttheimage.

ManipulatingimageswithMatlab
MonochromeimagesinMatlab arerepresentedbyandNxM matrix Nistheheightoftheimage,Misthewidth LoadanimageintovariableJ:J=imread(spine.tif); Letsgetsomeinformationabouttheimage:whos J Rawdataistypicallyuint butimagesshouldbedoublesbetween0and1 usefunctionmat2gray toconverttheimage. Toviewtheimageuse:imtool(J)(alternatively,imshow(J))

ManipulatingimageswithMatlab
MonochromeimagesinMatlab arerepresentedbyandNxM matrix Nistheheightoftheimage,Misthewidth LoadanimageintovariableJ:J=imread(spine.tif); Letsgetsomeinformationabouttheimage:whos J Rawdataistypicallyuint butimagesshouldbedoublesbetween0and1 usefunctionmat2gray toconverttheimage. Toviewtheimageuse:imtool(J)(alternatively,imshow(J)) Trymanuallysetcontrast:imtool(J,[lower,upper]) Pixelslower areblack;Pixelsupper arewhite automaticallysetlowertotheminimumpixel andupper tothemaximumimtool(J,[]) Youcanalsoadjustthegammaandpixelmapping withimadjust(J,[],[],gamma);

FilteringimagesinMatlab
Aspatialfilterrequiresafiltermask.Exampleanaveragingfilter:

FilteringimagesinMatlab
Aspatialfilterrequiresafiltermask.Exampleanaveragingfilter: filt=ones(3,3); filt=filt*1/9; Eachpixelintheresultingimagehasavalueequaltothesumofthepixelvaluesofthe originalimagexfiltermaskwhenthemaskiscenteredonthatpixel:imfilter q=OriginalImage Filtermask

FilteringimagesinMatlab
Aspatialfilterrequiresafiltermask.Exampleanaveragingfilter: filt=ones(3,3); filt=filt*1/9; Eachpixelintheresultingimagehasavalueequaltothesumofthepixelvaluesofthe originalimagexfiltermaskwhenthemaskiscenteredonthatpixel:imfilter q=OriginalImage Filtermask

imfilt(q,filt)

TryapplyingaGaussianfiltertoyourimage(hint:Gaussiansarereadilymadeusingfspecial)

FilteringimagesinMatlab
Aspatialfilterrequiresafiltermask.Exampleanaveragingfilter: filt=ones(3,3); filt=filt*1/9; Eachpixelintheresultingimagehasavalueequaltothesumofthepixelvaluesofthe originalimagexfiltermaskwhenthemaskiscenteredonthatpixel:imfilter q=OriginalImage Filtermask

imfilt(q,filt)

TryapplyingaGaussianfiltertoyourimage(hint:Gaussiansarereadilymadeusingfspecial) J_gaus=imfilt(J,fspecial(gaussian,11,3));

Anotherexample morphologicaloperations
Readthestockimagerice.png:rice=mat2gray(imread(rice.png)); Viewtheimage,noticethebackgroundisnoteven:imtool(rice,[]); Wewillopenthisimage,usingthefunctionimopen,toremove thesmallfeaturesandgetalookatthebackground. imopen isamorphologicalerosionfollowedbyadilation imclose isthereverse Asanexample,loadtheimagesticksandballs.tif Wecanusetheimopen featuretoremovethesticks Tryopeningthisimageandremovingthestickswithadisc shapedstructuralelement(strel)

Anotherexample morphologicaloperations
Readthestockimagerice.png:rice=mat2gray(imread(rice.png)); Viewtheimage,noticethebackgroundisnoteven:imtool(rice,[]); Wewillopenthisimage,usingthefunctionimopen,toremove thesmallfeaturesandgetalookatthebackground. imopen isamorphologicalerosionfollowedbyadilation imclose isthereverse Asanexample,loadtheimagesticksandballs.tif Wecanusetheimopen featuretoremovethesticks Tryopeningthisimageandremovingthestickswithadisc shapedstructuralelement(strel) sb_open=imopen(sb,strel(disk,5)); Nowletsreturntoourunevenlyilluminatedrice: opentheimagesuchthatonlythebackgroundremains

Anotherexample morphologicaloperations
Readthestockimagerice.png:rice=mat2gray(imread(rice.png)); Viewtheimage,noticethebackgroundisnoteven:imtool(rice,[]); Wewillopenthisimage,usingthefunctionimopen,toremove thesmallfeaturesandgetalookatthebackground. imopen isamorphologicalerosionfollowedbyadilation imclose isthereverse Asanexample,loadtheimagesticksandballs.tif Wecanusetheimopen featuretoremovethesticks Tryopeningthisimageandremovingthestickswithadisc shapedstructuralelement(strel) sb_open=imopen(sb,strel(disk,5)); Nowletsreturntoourunevenlyilluminatedrice: opentheimagesuchthatonlythebackgroundremains rice_open=imopen(rice,strel(disk,20));

Evenriceyeahbaby!
Lookattheoriginalimagewiththeunevenbackgroundsubtracted

Evenriceyeahbaby!
Lookattheoriginalimagewiththeunevenbackgroundsubtracted rice_nobg=ricerice_open imshow(rice_nobg) Butwearentusingtheentirecontrastrangenow,adjusttheimage rice_nobg=imadjust(rice_nobg);

Evenriceyeahbaby!
Lookattheoriginalimagewiththeunevenbackgroundsubtracted rice_nobg=ricerice_open imshow(rice_nobg) Butwearentusingtheentirecontrastrangenow,adjusttheimage rice_nobg=imadjust(rice_nobg); Segmentationisacommonneedinbiologyimageprocessing;letssegmentthericeusinga simplethresholdasacriterionforwhatisriceandwhatisnot. imhist givesahistogramoftheintensityoftheimagepixels choseathresholdbetweenthebackground(around0)andtherice(around1) Usingim2bw makethepixelsbelowthethresholdblackandtheonesabovewhite

Evenriceyeahbaby!
Lookattheoriginalimagewiththeunevenbackgroundsubtracted rice_nobg=ricerice_open imshow(rice_nobg) Butwearentusingtheentirecontrastrangenow,adjusttheimage rice_nobg=imadjust(rice_nobg); Segmentationisacommonneedinbiologyimageprocessing;letssegmentthericeusinga simplethresholdasacriterionforwhatisriceandwhatisnot. imhist givesahistogramoftheintensityoftheimagepixels choseathresholdbetweenthebackground(around0)andtherice(around1) Usingim2bw makethepixelsbelowthethresholdblackandtheonesabovewhite rice_bw=im2bw(rice_nobg,0.6); Eachconnectedregionof1scanbegivenitsownlabelwithbwlabel.

Evenriceyeahbaby!
Lookattheoriginalimagewiththeunevenbackgroundsubtracted rice_nobg=ricerice_open imshow(rice_nobg) Butwearentusingtheentirecontrastrangenow,adjusttheimage rice_nobg=imadjust(rice_nobg); Segmentationisacommonneedinbiologyimageprocessing;letssegmentthericeusinga simplethresholdasacriterionforwhatisriceandwhatisnot. imhist givesahistogramoftheintensityoftheimagepixels choseathresholdbetweenthebackground(around0)andtherice(around1) Usingim2bw makethepixelsbelowthethresholdblackandtheonesabovewhite rice_bw=im2bw(rice_nobg,0.6); Eachconnectedregionof1scanbegivenitsownlabelwithbwlabel. rice_label=bwlabel(rice_bw); Viewtheresultingimage Findtheareaandperimeterofeachlabeledregion withregionprops

Evenriceyeahbaby!
Lookattheoriginalimagewiththeunevenbackgroundsubtracted rice_nobg=ricerice_open imshow(rice_nobg) Butwearentusingtheentirecontrastrangenow,adjusttheimage rice_nobg=imadjust(rice_nobg); Segmentationisacommonneedinbiologyimageprocessing;letssegmentthericeusinga simplethresholdasacriterionforwhatisriceandwhatisnot. imhist givesahistogramoftheintensityoftheimagepixels choseathresholdbetweenthebackground(around0)andtherice(around1) Usingim2bw makethepixelsbelowthethresholdblackandtheonesabovewhite rice_bw=im2bw(rice_nobg,0.6); Eachconnectedregionof1scanbegivenitsownlabelwithbwlabel. rice_label=bwlabel(rice_bw); Viewtheresultingimage Findtheareaandperimeterofeachlabeledregion withregionprops rice_dims=regionprops(rice_label,Area,Perimeter);

Segmentedrice
Nowwecananswerquestionslike: Whatisthemeanareaoftherice?Thevarianceinthemean?Theperimeter?

Segmentedrice
Nowwecananswerquestionslike: Whatisthemeanareaoftherice?Thevarianceinthemean?Theperimeter? areas=[rice_dims.Area] mean(areas) var(areas) hist(areas) andonandon Forfun,trysegmentingthesuppliedimagefish.tif

PointSpreadFunction
Foranidealoptical system,apointsource isnotapointatthe imageplane. Instead,itappearsasa PointSpreadFunction (PSF) akatheimpulse responsefunctionfor theopticalsystem.

Put experimental onehere

PSF:Why?

PSF:Why?
Theobjectivecannotcaptureallofthelight radiatedbythepointsource. Equivalently:highfrequencyFourier componentsarelost. MissingcomponentsintheFourierdomain correspondtouncertaintyinpositionin physicalspace. Howmuchlightiscaptured?Quantifyby NumericalAperture (NA)

NumericalAperture

NA=nsin() n:indexofrefractionofmedium betweenspecimenandobjective. WhydoesNAdependonn?

Puttingittogether:Resolution
RayleighCriterion: conventionfordefining resolution
r=1.22/(NAobj+NAcond) r250nmfortypical imagingconditions

FluorescenceMicroscopy:Howare imagesformed?
TheimagedetectedbyyourCCDisthe convolution ofthePSFwiththepointsources (e.g.,fluorescentproteinmolecules,quantum dots,fluorescentdyemolecules)withinthe fieldofview. Wellexplorethisideainthenextfewslides.

Space:TheFinalFrontier
Loadprovidedimageof pointsources(points.tif)

Space:TheFinalFrontier
Loadprovidedimageof pointsources(points.tif)
P=imread(points.tif); imshow(P)

ExperimentalPSF
Loadprovidedimageof pointspreadfunction (psf.tif)
psf =imread(psf.tif); imshow(psf)

[Aside:improfile]
Useimprofile toestimate theminimumresolvable distanceaccordingto Rayleighcriterion

[Aside:improfile]
Useimprofile toestimate theminimumresolvable distanceaccordingto Rayleighcriterion(recall thisisthedistancefrom centralpeaktofirst minimum).

About7pixelsorso.

Convolvingthepointsources
Nowconvolveyourimageofpoint sourceswiththepointspread function,usingimfilter. (Hint:itsusuallyagoodideato convertallyourimagestodoubles beforefilteringthem,soyoumay wanttouseim2double)

Convolvingthepointsources
Nowconvolveyourimageofpoint sourceswiththepointspread function,usingimfilter. (Hint:itsusuallyagoodideato convertallyourimagestodoubles beforefilteringthem,soyoumay wanttouseim2double)

PD=im2double(P); psfd =im2double(psf); J=imfilter(PD,psfd); imshow(J,[])

BeatingtheLimit:Deconvolution
Weveseenthatevenforanidealoptical system,thereexisthardlimitson performance. Isthereanywaywecouldcleverlygetaround theselimits?

BeatingtheLimit:Deconvolution
Weveseenthatevenforanidealoptical system,thereexisthardlimitson performance. Isthereanywaywecouldcleverlygetaround theselimits? Yes:Deconvolution! Basicidea:weknowwhataPSFlookslike,so extrapolatebackwards

TheMath

Howmanyspots?
Openhowmany.tifinthe tutorialfolder.

Howmanyspots?
Openhowmany.tifinthe tutorialfolder. J=imread(howmany.tif);

Howmanyspots?
Openhowmany.tifinthe tutorialfolder. J=imread(howmany.tif); Now,deconvolve theimage usingdeconvwnr,which performsWiener deconvolutionn. Syntax:deconvwnr(I,psf)

Howmanyspots?
Openhowmany.tifinthe tutorialfolder. J=imread(howmany.tif); Now,deconvolve theimage usingdeconvwnr,which performsWiener deconvolutionn. Syntax:deconvwnr(I,psf) JD=deconvwnr(J,psf);

LeastSquaresFitting
Fittinginvolvesfindingthecurvewhichminimizesthesumofthesquaresoftheoffsets("the residuals")ofthegivendatapointsfromthecurve:

f(x)
offset IncludedinthedistributedfilesisD1Resids.mfor fittinga1DGaussiantodata.Letstryandfitoneto a1Dintensityprofileoftheimagepsf.tif.

}
x

Firstmaketheintensityprofilewhichyouwillfit:Ichoseahorizontalslicethroughthemiddle

LeastSquaresFitting
Fittinginvolvesfindingthecurvewhichminimizesthesumofthesquaresoftheoffsets("the residuals")ofthegivendatapointsfromthecurve:

f(x)
offset IncludedinthedistributedfilesisD1Resids.mfor fittinga1DGaussiantodata.Letstryandfitoneto a1Dintensityprofileoftheimagepsf.tif.

}
x

Firstmaketheintensityprofilewhichyouwillfit:Ichoseahorizontalslicethroughthemiddle psf=mat2gray(imread(psf.tif')); psf_profile=psf(18,:); Now,tryminimizingtheresidualswithD1Resids.m

LeastSquaresFitting
Fittinginvolvesfindingthecurvewhichminimizesthesumofthesquaresoftheoffsets("the residuals")ofthegivendatapointsfromthecurve:

f(x)
offset IncludedinthedistributedfilesisD1Resids.mfor fittinga1DGaussiantodata.Letstryandfitoneto a1Dintensityprofileoftheimagepsf.tif.

}
x

Firstmaketheintensityprofilewhichyouwillfit:Ichoseahorizontalslicethroughthemiddle psf=mat2gray(imread(psf.tif')); psf_profile=psf(18,:); Now,tryminimizingtheresidualswithD1Resids.m fitp =fminsearch(@(x)D1Resids(x,inputdata),[17,3,.04,.01]) Overlayyourfittedfunctionwithpsf.tif

LeastSquaresFitting
Fittinginvolvesfindingthecurvewhichminimizesthesumofthesquaresoftheoffsets("the residuals")ofthegivendatapointsfromthecurve:

f(x)
offset IncludedinthedistributedfilesisD1Resids.mfor fittinga1DGaussiantodata.Letstryandfitoneto a1Dintensityprofileoftheimagepsf.tif.

}
x

Firstmaketheintensityprofilewhichyouwillfit:Ichoseahorizontalslicethroughthemiddle psf=mat2gray(imread(psf.tif')); psf_profile=psf(18,:); Now,tryminimizingtheresidualswithD1Resids.m fitp =fminsearch(@(x)D1Resids(x,inputdata),[17,3,.04,.01]) Overlayyourfittedfunctionwithpsf.tif x=[1:35] y=fitp(3)*exp((xfitp(1)).^2/fitp(2))+fitp(4) plot(psf_profile,'k');hold;plot(x,y,'r')

Fittingin2D
Youshouldnowhavethepropertoolstofita2DGaussiantopsf.tif Giveitatry! Canyoulocalizethequantumdotinpsf.tiftosubpixelresolutiohn?