Accuracy and dynamic range of spatial image correlation and cross-correlation spectroscopy.

We present a comprehensive study of the accuracy and dynamic range of spatial image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS). We use simulations to model laser scanning microscopy imaging of static subdiffraction limit fluorescent proteins or protein clusters in a cell membrane. The simulation programs allow us to control the spatial imaging sampling variables and the particle population densities and interactions and introduce and vary background and counting noise typical of what is encountered in digital optical microscopy. We systematically calculate how the accuracy of both image correlation methods depends on practical experimental collection parameters and characteristics of the sample. The results of this study provide a guide to appropriately plan spatial image correlation measurements on proteins in biological membranes in real cells. The data presented map regimes where the spatial ICS and ICCS provide accurate results as well as clearly showing the conditions where they systematically deviate from acceptable accuracy. Finally, we compare the simulated data with standard confocal microscopy using live CHO cells expressing the epidermal growth factor receptor fused with green fluorescent protein (GFP/EGFR) to obtain typical values for the experimental variables that were investigated in our study. We used our simulation results to estimate a relative precision of 20% for the ICS measured receptor density of 64 microm(-2) within a 121 x 98 pixel subregion of a single cell.

[1]  N. Thompson,et al.  Imaging fluorescence correlation spectroscopy: nonuniform IgE distributions on planar membranes. , 1996, Biophysical journal.

[2]  J. R. Abney,et al.  On the measurement of particle number and mobility in nonideal solutions by fluorescence correlation spectroscopy. , 1990, Biophysical journal.

[3]  W. Webb,et al.  Fluorescence correlation spectroscopy. II. An experimental realization , 1974, Biopolymers.

[4]  H. Vogel,et al.  Factors influencing fluorescence correlation spectroscopy measurements on membranes: simulations and experiments , 2003 .

[5]  J. Lakowicz.  Topics in fluorescence spectroscopy , 2002 .

[6]  N O Petersen,et al.  An internalization-competent influenza hemagglutinin mutant causes the redistribution of AP-2 to existing coated pits and is colocalized with AP-2 in clathrin free clusters. , 1999, Biochemistry.

[7]  N O Petersen,et al.  Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application. , 1993, Biophysical journal.

[8]  W. Webb,et al.  Thermodynamic Fluctuations in a Reacting System-Measurement by Fluorescence Correlation Spectroscopy , 1972 .

[9]  Watt W. Webb,et al.  Fluorescence correlation spectroscopy , 2000 .

[10]  M E Martone,et al.  Counting dendritic spines in brain tissue slices by image correlation spectroscopy analysis , 2002, Journal of microscopy.

[11]  R. Rigler,et al.  The standard deviation in fluorescence correlation spectroscopy. , 2001, Biophysical journal.

[12]  N O Petersen,et al.  Image correlation spectroscopy. II. Optimization for ultrasensitive detection of preexisting platelet-derived growth factor-beta receptor oligomers on intact cells. , 1999, Biophysical journal.

[13]  Mark Ellisman,et al.  Spatial mapping of integrin interactions and dynamics during cell migration by Image Correlation Microscopy , 2004, Journal of Cell Science.

[14]  Jeffrey A. Squier,et al.  Two-photon image correlation spectroscopy and image cross-correlation spectroscopy , 2001, SPIE BiOS.

[15]  H. Qian,et al.  On the statistics of fluorescence correlation spectroscopy. , 1990, Biophysical chemistry.

[16]  R. Rigler,et al.  Resolution of fluorescence correlation measurements. , 1999, Biophysical journal.

[17]  Watt W. Webb,et al.  Fluorescence correlation spectroscopy. III. Uniform translation and laminar flow , 1978 .

[18]  E. Elson,et al.  Fluorescence correlation spectroscopy. I. Conceptual basis and theory , 1974 .

[19]  N O Petersen,et al.  An image correlation analysis of the distribution of clathrin associated adaptor protein (AP-2) at the plasma membrane. , 1998, Journal of cell science.

[20]  Dennis E. Koppel,et al.  Statistical accuracy in fluorescence correlation spectroscopy , 1974 .

[21]  R. Rigler,et al.  Fluorescence correlation spectroscopy , 2001 .

[22]  T M Jovin,et al.  Comparison of fixation protocols for adherent cultured cells applied to a GFP fusion protein of the epidermal growth factor receptor. , 1999, Cytometry.

[23]  Peet Kask,et al.  Statistical accuracy in fluorescence fluctuation experiments , 1997, European Biophysics Journal.

[24]  E. Elson,et al.  Fluorescence Correlation Spectroscopy Measures Molecular Transport in Cells , 2001, Traffic.