TN348: Openlab Module - Colocalzaton Topc The Colocalzaton module provdes the faclty to vsualze and quantfy colocalzaton between pars of mages. The Colocalzaton wndow contans a prevew of the two mages overlad, a scatter plot and a dsplay of statstcal analyss. Ths techncal note descrbes the use of ths dalog and the Automator tasks for colocalzaton. The Colocalzaton module s avalable as an opton wth Openlab 3.1 and hgher. Dscusson What s colocalzaton? Colocalzaton n the context of dgtal fluorescence magng can be descrbed as the detecton of sgnal at the same pxel locaton n each of two mages. The two mages are two dfferent fluorescent channels of the same sample area. Vewng colocalzaton In the context of ths Techncal Note, a par of mages s consdered to be two grayscale mages of the same sample locaton maged wth two separate fluorescence channels. It wll be much easer to vsualze colocalzaton f each mage has a dfferent colour look up table appled. The mages that you wsh to analyze must be n the same Openlab mage document. Begn by selectng the par of mages n the Layers Manager. Select Show Colocalzaton from the Image menu. www.mprovson.com Page 1 of 8
The Colocalzaton wndow, unque to the mage document, appears: Image overlay Scatter plot Statstcs The Colocalzaton wndow The wndow conssts of 3 parts: Overlay mage The two channels supermposed. For mages wth dfferent CLUTs appled colocalzaton wll appear as a new color n ths mage, for example red and green wll make yellow. It s also n ths mage that the bnary mask, created when an ROI s drawn on the scatter plot, wll be prevewed. Scatter plot The scatter plot s a graphcal representaton of the ntensty values found wthn the mages. One mage of the par s assgned the x axs, and the other mage s assgned the y axs. Each axs represents the ascendng gray levels of the mages. For example f a pxel n mage x has an ntensty value of 100 and the correspondng pxel n mage y has an ntensty value of 30, a pont wll be plotted at co-ordnates 100,30. It does not matter how may pxels n the mage par have ntensty values of 100,30, only one pont wll be plotted on the scatter plot. Two perfectly colocalzed mages,.e. dentcal, wll generate a scatter plot where the ponts fall n a lne at 45 degrees to ether axs. Statstcs Pearson s Correlaton and Overlap Coeffcents are generated for the whole mage and Colocalzaton Coeffcents for a regon of nterest drawn on the scatter plot. See Calculatng colocalzaton statstcs secton below for more nformaton. www.mprovson.com Page 2 of 8
Usng the Colocalzaton wndow The frst step n usng the colocalzaton wndows s to set thresholds for each axs so that the lower threshold s above background. It may not be necessary to change the upper threshold. If thresholds are not set pxels where both mages show background ntensty levels wll be nterpreted as colocalzed. Set thresholds by draggng the ponts on the color ramp for each mage. Any pxels of ntensty outsde threshold wll be removed from the overlay mage for that channel. Any pont on the scatter plot that s outsde threshold for ether channel wll be removed from the scatter plot leavng only ponts whch are postve for both x and y,.e.colocalzed. After settng thresholds select the whole of the scatter plot (Select all from the Edt menu or Apple A). A colored, bnary mask s produced showng the areas of colocalzaton over the overlad mages. When the cursor s placed over the scatter plot t becomes a freehand ROI tool. Use ths to select a porton of the colocalzed pxels and vew ther locaton n the overlad mages. Clck and drag the mouse to draw an ROI. Use shft-clck and drag to add to the ROI, use shft+opton-clck to subtract from the ROI. Clck outsde the ROI wthout movng the mouse to deselect t. Select the color of the bnary mask usng the color pcker at the top of the wndow: www.mprovson.com Page 3 of 8
www.mprovson.com Page 4 of 8
Once satsfed wth the selecton made t s possble to create a bnary layer n the mage document by clckng on the make Bnary Layer button. Make Bnary Layer Update Colocalzaton Ths bnary layer can then be used wth Openlab measurements to provde more detaled morphologcal and ntensty nformaton on the regons selected. The Advanced Measurements module s requred for ths type of analyss. Calculatng the statstcs The statstcs presented n the Colocalzaton wndow are calculated as follows: x = ntensty of pxel n mage unless x s outsde threshold n whch case x = 0 y = ntensty of pxel n mage y unless y s outsde threshold n whch case y =0 Pearson's Correlaton = (x x aver ) (y y aver ) (x x aver ) 2 (y y aver ) 2 Pearson s Correlaton s a statstcal test for a lnear relatonshp between two varables. Pearson s Correlaton can vary between 1 and 1. A correlaton of 0 means there s no lnear relatonshp between the varables. 1 and 1 ndcate perfect negatve and perfect postve lnear relatonshps respectvely. Interpretng the degree of colocalzaton from negatve values s dffcult. Overlap Coeffcent (R) = x y (x ) 2 (y ) 2 www.mprovson.com Page 5 of 8
Overlap Coeffcent (kx) = Overlap Coeffcent (ky) = Colocalzaton Coeffcent (Mx) = Colocalzaton Coeffcent (My) = x y (x ) 2 x y (y ) 2 x,coloc x y,coloc Where x, coloc = x f y s wthn the ntensty range defned by the ROI, x, coloc = 0 f y s outsde the ntensty range and y,coloc = y f x s wthn the ntensty range defned by the ROI, y,coloc = 0, f x s outsde the ntensty range. Colocalzaton Coeffcents may be values between 0 and 1. 0 ndcates that none of the sgnal wthn thresholds n that channel exsts as colocalzed wth the other channel. A value of 1 ndcates that all of the sgnal wthn thresholds n that channel exsts as colocalzed wth the other channel. For further explanaton of these statstcs see the reference gven at the end of ths techncal note. Exportng from the Colocalzaton wndow At any pont, whle workng wth the Colocalzaton wndow, the overlay mage, scatter plot or statstcs may be coped to the clpboard. Clck on the relevant area of the wndow to hghlght t and chose Copy from the Edt menu or Apple C. These can then be pasted nto any applcaton that accepts pasted text and mages. y To vew the colocalzaton of a dfferent par of mages select the new par of mages n the Layers Manager and choose Update colocalzaton from the Image menu or clck the Update colocalzaton button at the top of the colocalzaton wndow. Colocalzaton Automator tasks The followng Automator tasks are avalable wthn the Automator under the Colocalzaton secton headng. www.mprovson.com Page 6 of 8
Update colocalzaton Ths task has no set up dalog. Followng mage selecton t wll prompt the generaton of overlay mage, scatter plot and statstcs. Update Colocalzaton must be performed n an automaton for each par of mages before Colocalzaton nformaton s extracted. Set Colocalzaton nfo Ths task sets the x and y thresholds on the scatter plot. Get Colocalzaton Info Store any of the colocalzaton statstcs produced as varables. For Selected pxels and Correlaton coeffcents a ROI must be drawn on the Colocalzaton wndow. Ths cannot be done by the Automator. www.mprovson.com Page 7 of 8
Make Colocalzaton overlay Makes the current colored overlay n the overlay mage nto a bnary layer n the mage document. Ths requres an ROI drawn on the scatter plot. Ths cannot be done by the automator. Use the Select Layers tasks to choose the layers to be colocalzed wthn the automaton. Important consderatons It s mportant to have adequate separaton of the two fluorescence sgnals, wth no overlap between emsson of one and exctaton of the other. Ths affects choce of flters as well as fluorochromes themselves. The Colocalzaton wndow can only analyze the pxel values presented n the selected mages. It s the users responsblty to ensure the mages are vald for the study they are carryng out. When analyzng 2D mages users should be aware that fluorescence from other planes wthn ther sample may be affectng the apparent degree of colocalzaton. Users may wsh to deconvolve mages before processng. The Openlab module 3D Restoraton s approprate for ths applcaton snce t uses teratve deconvoluton preservng pxel ntensty values. The module assumes that there has been no pxel shft between the capture of the two channels and that they are perfectly algned. The Openlab module Regstraton allows users to correct for measured pxel shft between channels. Adequate controls are also essental to avod the possblty of false colocalzaton, for example generated by non-specfc labellng. References M.M. Manders, P. J. Verbeek & J. A. Aten Measurement of co-localzaton of objects n dual colour confocal mages Journal of Mcroscopy. Vol. 169, Pt 3, March 1993, pp. 375-382 www.mprovson.com Page 8 of 8