Colocalization of Fluorescent Probes

Transcription

1 Colocalzaton of Fluorescent Probes APPLICATION NOTE #1 1. Introducton Fluorescence labelng technques are qute useful to mcroscopsts. Not only can fluorescent probes label sub-cellular structures wth a hgh degree of specfcty, they can also do so wth a very hgh degree of senstvty. Ths combnaton of specfcty and senstvty permts observatons of cellular events that are not always vsble usng tradtonal methodologes, such as brghtfeld mcroscopy. Labelng specmens can occur usng one or more fluorochromes. Sngle-wavelength fluorochromes are often used as labels to reveal structures, such as mcrotubules, mtochondra or nucle. Ths technques works well when all a researcher s nterested n s a specfc structure wthn the feld of vew. However, t often the case when two or more labels are used to elucdate multple structures. The reason for usng multple probes, n general, s to vew two or more dfferent types of sub-cellular structures smultaneously, but mantanng them vsually dstnct from one another. To ad wth mage analyss, each fluorescent wavelength s usually acqured ndependently and then combned wth the others to create a composte mage. After mage acquston and processng, there may be some overlap when two dscrete wavelengths of lght occupy the same physcal locaton wthn the mage. Ths nformaton, known as colocalzaton, can be of specal nterest to the researcher. It then becomes necessary to provde a vsual representaton as to the locaton and degree of overlap between two wavelengths.. Acqurng Images Fluorescence exctaton occurs through ether confocal scannng or wde feld epfluorescence mcroscopy usng standard llumnaton methods. Image capture s accomplshed through standard CCD acquston technques. Acquston of ndvdual wavelengths s usually performed, followed by the applcaton of look-up tables (color values to the mage. Afterwards, a composte mage s created from the colorzed consttuents. It s mportant to note that mage acquston can, and often does, occur through several optcal sectons. The physcal methods of exctaton and acquston are beyond the scope of ths applcaton note and are not dscussed n any detal. After dgtzaton, mages are mported to Image Pro Plus v. 4.5 for colocalzaton analyss. 3. A Defnton for Colocalzaton Colocalzaton descrbes the presence of two or more types of molecules at the same physcal locaton. Wthn the context of a cell or sub-cellular t may be the molecules are attached to the same receptor, whle n the context of dgtal magng t means the colors emtted by the fluorescent molecules occupy the same pxel n the mage. Measurng the degree of colocalzaton s performed through the use of the Color Composte and Colocalzaton functons n Image Pro Plus (verson 4.5. Colocalzaton does not refer to the lkelhood that fluorochromes wth smlar emsson spectra wll appear as overlapped n the composte mage. It s mportant to note that cross-talk or bleed-through may occur f the emsson spectra of the two fluorochromes are smlar. Accurate colocalzaton determnaton can only occur f emsson spectra are suffcently separated between fluorochromes and the correct flter sets are used durng the acquston step. To acheve ths am, red and green wavelengths are usually selected, and usually those dyes representng these wavelengths are carefully matched to the power spectrum of the llumnaton source to obtan maxmum exctaton wavelengths whle stll mantanng a degree of separaton between the emsson wavelengths. 4. How Does All Ths Work? The degree of colocalzaton s measured by comparng the equvalent pxel postons n each of the acqured mages. To acheve ths, Image Pro Plus uses a combnaton of the Process Color Composte and Measure Colocalzaton commands. 1

2 So how do we measure colocalzaton? What s a scatterplot? What do all those generated numbers mean anyway? And what are all those sold colors that appear on the mage, and what do they mean? 4.1 Generatng the Colocalzaton Scatterplot The frst step n the process nvolves dsplayng the mage upon whch the colocalzaton analyss wll be performed. The mage may be created as a composte from two sngle wavelength acqustons (the preferred method or a mult-labeled specmen acqured as a sngle mage (Fg. 1. Then, t s determned whch par of colors wll be evaluated for colocalzaton. Typcally, red-green pars are selected, although t s possble to select other combnatons. Afterward, a scatterplot (Fg. of the ndvdual pxels from the pared mages s generated. Dmmer pxels n the mage are located toward the orgn of the scatterplot, whle brghter pxels are located farther out. Pure red and pure green pxels are colored as such, and tend to cluster more toward the axes of the plot. If colocalzed pxels are present, they appear as orange to yellow, dependng on the degree of colocalzaton, toward the mddle of the plot. Fg 1.Mouse Fbroblast Cells. Mcrotubules staned wth FITC, mtochondra staned wth Texas Red, nucle staned wth DAPI. Fluorochromes were acqured separately and merged usng the Process Color Composte command n Image Pro Plus v Fg. The scatterplot dsplays the ntensty range of red and green pxels n the mage, as well as the varous degrees of colocalzaton, as shown n orange and yellow. 4. Analyzng the Scatterplot and Generatng a Mask Followng the generaton of the scatterplot, t s now possble to quanttatvely evaluate the presence and level of colocalzaton n the pared mages. In ths process, the scatterplot s used n conjuncton wth the mage to defne the locaton of the colocalzed molecules. An area of nterest (AOI s drawn on the scatterplot to ndcate threshold levels of sgnal to be ncluded n the analyss (Fg 3. The vertcal and horzontal edges of the AOI are algned such that they exclude background sgnal (clustered on the plot along the x- and y- axes. Only sgnal ncluded wthn the boundares of the AOI wll be ncluded n the estmaton of colocalzaton. By clckng wthn the area of nterest, t s possble to see on the mage the color ntenstes that wll be ncluded n the analyss. Three threshold masks (red, green, yellow appear n the mage when the left mouse button s depressed. Red and green masks ndcate those pxels wth no color mxng, whle a yellow mask ndcates regons contanng colocalzed nformaton. The colocalzaton mask (Fg. 4 may then be supermposed on the mage as a colocalzaton map (Fg. 5.

3 Overlap Coeffcent k, Colocalzaton for each color channel (m1 and m Values calculated for sgnal ntenstes wthn the AOI nclude: Colocalzaton of the frst channel (M1 Colocalzaton of the second channel (M In the followng examples we call the frst channel red and the second channel green. Fg 3. Area of nterest (AOI wthn the scatterplot. The AOI has been set to nclude prmarly the colocalzed areas, seen as orange and yellow. Fg. 4 Colocalzaton mask showng overlappng regons of red and green Pearson s Correlaton Pearson s Correlaton s a well defned and commonly accepted means for descrbng the extent of overlap between mage pars. It s a value computed to be between -1 and 1, wth -1 beng no overlap whatsoever between mages and 1 beng perfect mage regstraton. Pearson s Correlaton takes nto account only the smlarty of shapes between mages and does not take nto account mage ntensty. Snce a negatve value can be reported usng ths method, t may be necessary to look at the data usng other coeffcents. Pearson s Correlaton s calculated accordng to the followng formula: R r ( ( ( S ( S S S Fg. 5. Composte mage showng FITC-labeled fbroblasts (green, Texas Red-labeled mtochondra (red and colocalzaton map (yellow 4.3 Generatng and Explanng Colocalzaton Coeffcents A more quanttatve assessment of the colocalzed areas may be performed usng the nformaton derved n the prevous step. Several values are calculated, each s explaned n more detal below. These values nclude calculatons for the entre scatterplot, as well as values for pxel values contaned wthn the AOI. Values calculated for the entre scatterplot nclude the followng: Pearson s Correlaton (R r, Overlap Coeffcent (R, Overlap Coeffcent k 1, s sgnal ntensty of pxels n the frst channel and S s sgnal ntensty of pxels n the second channel, and S - age ntensty of frst channel and second channels respectvely Overlap Coeffcent As Pearson s Correlaton does some agng of pxel nformaton and can return negatve values another method, the Overlap Coeffcent, s smultaneously used to descrbe overlap. Ths method does not perform any pxel agng functons, so correlatons are returned as values between 0 and 1. Ths method s not senstve to ntensty varatons n the mage analyss. Ths s especally mportant when consderng ssues typcal to fluorescence magng such as camera quantum 3

4 effcency, hybrdzaton effcency and sample photobleachng. R ( S ( S Table 1. Example of colocalzaton data derved from Fgs Overlap Coeffcents k 1 and k These coeffcents descrbe the dfferences n ntenstes of red and green. Smply put, the value k 1 s senstve to dfferences n ntensty for green whle k s senstve to dfferences n ntensty for red. S k1 ( S k ( S Colocalzaton Coeffcents m 1 and m Now that we have generated some nformaton about the degree of overlap and accounted for ntensty varaton between the color channels, we can estmate the contrbuton of one color channel n the colocalzed areas of the mage to the overall colocalzed fluorescence n the mage. m 1 s used to descrbe the contrbuton of green to the colocalzed area whle m s used to descrbe the contrbuton of red., coloc m1 m S, coloc S, coloc S, coloc S 1 f S f S >0 > Colocalzaton Coeffcents M 1 and M Colocalzaton coeffcents can be calculated for ntensty ranges defned by AOI on the scatter-plot. Now that we have generated some nformaton about the degree of overlap and accounted for ntensty varaton between the color channels, we can estmate the contrbuton of one color channel n the colocalzed areas of the mage to the overall colocalzed fluorescence n the mage. M 1 s used to descrbe the contrbuton of green to the colocalzed area whle M s used to descrbe the contrbuton of red. S 1, coloc M1 and M S, coloc S S 1, coloc f S s wthn thresholds defned by AOI (left and rght sdes of AOI n case of rectangular AOI, S 1, coloc 0 f S s outsde the threshold levels. S, coloc S f s wthn thresholds (top and bottom margns of AOI n case of rectangular AOI, S, coloc 0 f s outsde the AOI. These coeffcents, M1 and M, are proportonal to the amount of fluorescence of colocalzng objects n each component of the mage, relatve to the total fluorescence n that component. The components are descrbed as the red and green mages, respectvely. Table 1. Example of colocalzaton data derved from Fgs

5 5. References Manders, E.M.M., Verbeek, F.J., Aten, J.A. Measurement of co-localzaton of objects n dualcolor confocal mages. Journal of Mcroscopy 169: (1993. Smallcombe, A., McMllan, D. Bo-Rad Techncal Note 11. Co-localzaton; how t s determned, and how t s analysed wth the Bo-Rad LaserPx mage analyss software? Meda Cybernetcs, Inc. South Amerca: Phone: Georga Avenue Fax: Slver Sprng, MD 0910 USA Phone: Europe: Phone: Fax: Fax: Emal: nfo@medacy.com Asa Pacfc: Phone: Web: Fax: All products and servces mentoned are trademarks of ther respectve owners. 00 Meda Cybernetcs, Inc. 5