Determination of particle number and brightness using a laser scanning confocal microscope operating in the analog mode

Abstract

We describe a method to obtain the brightness and number of molecules at each pixel of an image stack obtained with a laser scanning microscope. The method is based on intensity fluctuations due to the diffusion of molecules in a pixel. For a detector operating in the analog mode, the variance must be proportional to the intensity. Once this constant has been calibrated, we use the ratio between the variance and the intensity to derive the particle brightness. Then, from the ratio of the intensity to the brightness we obtain the average number of particles in the pixel. We show that the method works with molecules in solution and that the results are comparable to those obtained with fluctuation correlation spectroscopy. We compare the results obtained with the detector operating in the analog and photon counting mode. Although the dynamic range of the detector operating in the photon counting mode is superior, the performance of the analog detector is acceptable under common experimental conditions. Since most commercial laser scanning microscopes operate in the analog mode, the calculation of brightness and number of particles can be applied to data obtained with these instruments, provided that the variance is proportional to the intensity. We demonstrate that the recovered brightness of mEGFP, independent of concentration, is similar whether measured in solution or in two different cell types. Furthermore, we distinguish between mobile and immobile components, and introduce a method to correct for slow variations in intensity. Microsc. Res. Tech. 71:69-81, 2008. V

Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus

“…Plasma membrane lipid binding by VP40 has been hypothesized to induce the structural arrangements needed for VP40 hexamerization and filament formation (16,21,24). We assessed the ability of VP40 to oligomerize on the plasma membrane inner leaflet with a combination of confocal microscopy and number and brightness (N&B) analysis (22,(41)(42)(43). Here we also employed a double mutant of VP40 (W95A/ E160A), which has previously been shown to significantly reduce VP40 plasma membrane localization (22,55), by encouraging formation of an alternate, perinuclear ring structure instead (21).…”

“…N&B analysis is based upon moment analysis and allows measurement of the average number of molecules as well as of the brightness in each pixel of a fluorescence microscopy image (22)(23)(24)(40)(41)(42)(43). Here, the average brightness of a particle is determined from the ratio (of variance to intensity) at each pixel.…”

Dynamics and Distribution of Klothoβ (KLB) and Fibroblast Growth Factor Receptor-1 (FGFR1) in Living Cells Reveal the Fibroblast Growth Factor-21 (FGF21)-induced Receptor Complex

“…of 3 or more experimental days with a minimum of 10 curves collected per day. N&B Analysis-N&B analysis was done as described previously (20,24). Briefly, N&B images were collected on an LSM710 confocal microscope (Carl Zeiss Microimaging) using a 63 ϫ 1.4 NA Plan-Apochromat oil immersion objective lens.…”

“…S1 and S2). The apparent brightness (B/S) of each pixel was then calculated using the following (24).…”

Multicolor fluctuation spectroscopy in cells

“…The fit of the autocorrelation curve using the equations for diffusion given in the Material and Method section provide the value of the diffusion coefficient and the G(0), from which we can calculate the number of particles in the volume of excitation. The particle brightens can be directly obtained using the Number and Brightness analysis (N&B) at each column 8,9 . Every column should be equivalent for a homogeneous sample, so that we can calculate the autocorrelation function for every column and then fit all the columns either globally or individually.…”