CORE B- HEART BIOLOGY CORE

在我们的实验条件下，我们是

In our experimental conditions, we are determining "colocalization" in the resolution limit of light microscopy. In this PPG, the "colocalization" term will imply that two molecules may share the same microenvironment and/or can be elements of the same set of macromolecular complexes which are resolved by the optical system. Forexample, two proteins forming part of two different molecular complexes may show positive "colocalization" tests due to the lack of the optical resolution to detect each molecular complex. Still, the positive test will indicate that the two proteins are located proximally within the optical resolution. Thus, in "colocalization" determinations it is critical to achieve the maximal x,y,z resolution. In this PPG, we will use developed methodologies in the laboratory to achieve the highest resolution attainable with optical microscopy. We have maximized the optical linearity and the quantity of light recorded by the detectors in conjunction with image restoration analysis to recover the light lost by the optical imperfections of the system. This methodology has allowed us to reach a resolution in the x,y plane of about 100-200 nm (see Figs. 1-3). Definition of "colocalization" and random "colocalization". In a cell labeled with two fluorophores, one red (R) and one green (G), the number of voxels above threshold that are labeled with the red fluorophore is nRand the number labeled with the green fluorophore is nG. The number of colocalized voxels, i.e. those voxels containing intensity signals above threshold from both fluorophores is ncoioc. The percentage of "colocalization" of G with R is given by 100*^^- (eq. 1); and the percentage of "colocalization" of R with G is given by 100*^^ (eq. 2). In n* nc these measurements we will estimate the probability that the measured "colocalization" could occur by chance by calculating % random "colocalization" from ((area_1x area_2)/total areaA2) x100, where area_1 and area_2 are the areas above threshold (eq. 3). The "colocalization" method is generally based on the user ability to set the intensity threshold, and has the major draw back of the lack of a mathematical model to adjust the threshold of both images that will define the degree of paired pixel overlap. Most importantly the pixel overlap method has the limitation of being a binary test that determines whether the two paired pixels in two images have or not intensities above the intensity threshold and does not consider whether the two stained proteins have a landscape of intensity staining with a high degree of correlation, as it would be expected if they are elements of a common complex. The application of a correlation measurement was recently described by Li et al. (2004)7. In the following sections, I will discuss and apply developed algorithms to quantify the degree of protein-protein association by two methods, the INTENSITY CORRELATION ANALYSIS and the INTENSITY THRESHOLD "COLOCALIZATION"ANALYSIS. Intensity correlation analysis. In the present analysis, we acquire high resolution images to compare the correlation of pixel intensities in equivalent x,y coordinates of paired images from cells double stained for two different proteins. The prediction is that if two proteins are elements of the same macromolecular complex, the intensity staining landscape of the two images should have a x,y pixel to pixel positive correlation. On the contrary, if the two proteins are localized in distinct compartments the result will be a negative correlation. Finally, if the proteins in the two images are labeled in a diffuse non structured pattern (random), the correlation will tend to 0. This method is based on the principle that for any set of values the sum of the differences from the mean equal zero, i.e., ?N(A-a)=0, where a is the mean of the distribution with N values of Al. In the experiment N is the number of pixels, and A is the intensity for each pixel. If we have two set of values in two arrays 1 and 2 with N pixels per array having a random distribution of intensities A-,and 6, for arrays 1 and 2 , the sum of the product of their differences will also tend to zero, thus IN(A-a)(S,-Jb)~0. On the other hand, if the two intensities are positively correlated, the product will tend to be a positive value (^(Ara)(Brb}>G) and if they are negatively correlated the product will tend to a negative value (LN(Ara)(Brb)<0).