| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Department of Physics and Department of Chemistry, McGill University, Montreal, Quebec, Canada
Correspondence: Address reprint requests to Paul W. Wiseman, E-mail: paul.wiseman{at}mcgill.ca.
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 µm–2 within a 121 x 98 pixel subregion of a single cell.
This article has been cited by other articles:
 |
|
 |
|
  S. Semrau and T. Schmidt Particle Image Correlation Spectroscopy (PICS): Retrieving Nanometer-Scale Correlations from High-Density Single-Molecule Position Data Biophys. J., January 15, 2007; 92(2): 613 - 621. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Brown, B. Hebert, D. L. Kolin, J. Zareno, L. Whitmore, A. R. Horwitz, and P. W. Wiseman Probing the integrin-actin linkage using high-resolution protein velocity mapping J. Cell Sci., December 15, 2006; 119(24): 5204 - 5214. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. W. D. Comeau, S. Costantino, and P. W. Wiseman A Guide to Accurate Fluorescence Microscopy Colocalization Measurements Biophys. J., December 15, 2006; 91(12): 4611 - 4622. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach Spatially Resolved Fluorescence Correlation Spectroscopy Using a Spinning Disk Confocal Microscope Biophys. J., December 1, 2006; 91(11): 4241 - 4252. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Sergeev, S. Costantino, and P. W. Wiseman Measurement of Monomer-Oligomer Distributions via Fluorescence Moment Image Analysis Biophys. J., November 15, 2006; 91(10): 3884 - 3896. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. L. Kolin, D. Ronis, and P. W. Wiseman k-Space Image Correlation Spectroscopy: A Method for Accurate Transport Measurements Independent of Fluorophore Photophysics Biophys. J., October 15, 2006; 91(8): 3061 - 3075. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. L. Kolin, S. Costantino, and P. W. Wiseman Sampling Effects, Noise, and Photobleaching in Temporal Image Correlation Spectroscopy Biophys. J., January 15, 2006; 90(2): 628 - 639. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
