Mapping the Number of Molecules and Brightness in the Laser Scanning Microscope

Abstract

We describe a technique based on moment-analysis for the measurement of the average number of molecules and brightness in each pixel in fluorescence microscopy images. The average brightness of the particle is obtained from the ratio of the variance to the average intensity at each pixel. To obtain the average number of fluctuating particles, we divide the average intensity at one pixel by the brightness. This analysis can be used in a wide range of concentrations. In cells, the intensity at any given pixel may be due to bright immobile structures, dim fast diffusing particles, and to autofluorescence or scattering. The total variance is given by the variance of each of the above components in addition to the variance due to detector noise. Assuming that all sources of variance are independent, the total variance is the sum of the variances of the individual components. The variance due to the particles fluctuating in the observation volume is proportional to the square of the particle brightness while the variance of the immobile fraction, the autofluorescence, scattering, and that of the detector is proportional to the intensity of these components. Only the fluctuations that depend on the square of the brightness (the mobile particles) will have a ratio of the variance to the intensity >1. Furthermore, changing the fluorescence intensity by increasing the illumination power, distinguishes between these possible contributions. We show maps of molecular brightness and number of cell migration proteins obtained using a two-photon scanning microscope operating with a photon-counting detector. These brightness maps reveal binding dynamics at the focal adhesions with pixel resolution and provide a picture of the binding and unbinding process in which dim molecules attach to the adhesions or large molecular aggregates dissociate from adhesion.

Stimulated emission depletion-based raster image correlation spectroscopy reveals biomolecular dynamics in live cells

“…The enhanced optical resolution provided by STED-RICS is particularly beneficial to probe intracellular dynamics over small spatial scales. We note that raster-scan image data can also be subjected to other statistical methods such as number and brightness analysis 18 that likewise benefit from the increased spatial resolution provided by STED.…”

Quantitative single-molecule imaging by confocal laser scanning microscopy

“…The introduction of fluorescence intensity fluctuation analysis in imaging offers the possibility to achieve quantitative fluorescence imaging with single-molecule sensitivity, an accomplishment that would considerably advance our understanding of biological processes at the molecular level. To this point, introduction of fluorescence intensity fluctuation analysis in imaging was successfully pursued in the form of fluorescence fluctuation correlation in image correlation spectroscopy (ICS) (23), raster image correlation spectroscopy (RICS) (24,25), and photon-counting histogram analysis (PCH) (26,27).…”

“…The ensemble of photon counts acquired at time intervals of different lengths is analyzed to determine the average number of molecules and their mobility, without using temporal autocorrelation analysis. This is an alternative to RICS (24,25), which relies on temporal autocorrelation analysis.…”

Raster Image Correlation Spectroscopy and Number and Brightness Analysis

“…As a result of these differences with respect to DLS, FCS has developed as an independent method during the 1970s (Magde, Elson, & Webb, 1972, 1974. A major breakthrough in the FCS approach was due to the realization that FCS measurements can be performed in live cells (Berland, So, & Gratton, 1995;Schwille, 2003, 2007;Bacia et al, 2006;Haustein and Schwille, 2007;Kim et al, 2007;Schwille, 1999;Schwille et al, 1997a,b) and that scanning confocal microscopy methods could be exploited to obtain the map of fluctuations in entire cells and in tissues Digman, Dalal, Horwitz, & Gratton, 2008;Digman & Gratton, 2009a, 2009bDigman, Wiseman, Choi, Horwitz, & Gratton, 2009;Ries, Chiantia, & Schwille, 2009;Ries, Yu, Burkhardt, Brand, & Schwille, 2009). Although FCS still shares some technological and analysis approach of the DLS method, today, FCS analysis has diverged from DLS.…”

“…Instead, for the analysis of images, we use a method that utilizes only the first and second moment of the histogram of the amplitude fluctuations. This method is called the Brightness and Number analysis or N&B (Digman et al, 2008). In this original presentation of the method, the basic of the moment analysis was presented.…”