The predominant population of dimeric native NBCe1-A was shown by SpIDA in kidney tissue. the width of the point-spread-function (PSF). The intensity brightness of a fluorescent oligomer (quantal brightness) is used to differentiate different oligomerization says. For example, a dimer with two fluorescent subunits emits on average twice as many photons as a monomer does, when quenching is usually negligible. For a single species, the amplitude of the image autocorrelation function is usually inversely proportional to the number of particles and the molecular brightness can be decided from the average intensity of the image divided by the number of particles. In the case of two populations present in the system, the overall integrated intensity will simply be the sum of the contributions from two oligomeric says: (1) where particles inside BA can be calculated by weighting each density configuration with its proper probability assuming a Poisson distribution. is usually defined as the probality of measuring an intensity of when having exactly fluorescent particles with an average brightness of in the PSF. The fitted function becomes: (3) where . is usually normalized over all intensity values so the integral over yields unity. The histogram fitted function is usually calculated by computing the fluorescence intensity of all possible configurations of particles in a given volume defined by the PSF. Each configuration is usually weighted according to their probability considering the Poisson spatial distribution of particles. This allows for the fit of an image intensity histogram to be performed. The fluorescent particle density, and mean quantity of fluorescent particles per BA and quantal brightness for the with the corresponding variance equal to which was measured in the control calibration experiment (see Physique 1). The standard deviations of the recovered mean values for both types of analysis were CA-4948 obtained from the analysis of CA-4948 multiple simulated or LSM/TIRF images of the same sample type. Results PMT shot noise characterization The ability of a CLSM to optically discriminate out-of-focus light makes it an ideal tool for p54bSAPK basal membrane imaging of cells. However, with a PMT equipped CLSM, we do not directly measure the fluorescence photon counts, but detect the analog photoelectric current which is converted to an intensity. The moments experimentally measured from single CLSM images are not identical to those of the photon counts because of the shot noise contributions. The number density and brightness are strongly affected by this contribution. Therefore, it is necessary to take into account the shot noise and the detector noise in a separate control measurement. We placed a mirror in the focus of the microscope and measured the reflection signal which provided uniform illumination of the detector. The acquisition parameters were set to be constant for all samples and controls so that valid comparisons could be made between measurements from different data sets. From the acquired point scan recording for this control, we calculated the variance in the measured intensity time traces. The plot CA-4948 of the mean variance as a function of the mean intensity is shown in Figure 1. Only the initial part of the data were taken into account for linear fitting. The values of the slopes for this control are used CA-4948 in spatial fluorescence intensity fluctuation analysis applied to all CLSM image sets for the experiments presented in this work. Control measurement of the molecular brightness of monomeric EGFP In order to carry out an independent measurement of the quantal brightness of monomeric EGFP, we transiently transfected CHO-K1 cells with monomeric EGFP (mEGFP) targeted to the membrane by attachment of a GPI moiety . This version of GFP has been modified such that the probability of its oligomerization is minimized. The cells were chemically fixed and imaged with CLSM. The collected data sets were analyzed with fluorescence image moment analysis and SpIDA assuming one population of fluorescent entities was present. The average brightness from cells expressing monomeric EGFP was used as a control and the results were normalized to 1 1 EGFP.