Scanning concentration correlation spectroscopy using the confocal laser microscope

Abstract

Concentration correlation spectroscopy allows the assessment of molecular motions in complex systems. The technique generally monitors concentration fluctuations by means of some method such as the intensity of fluorescent molecules (fluorescence correlation spectroscopy). We describe here the use of scanning confocal laser microscopy to measure correlation functions in both space and time. This methodology offers two major advantages over conventional methods. First, collecting data from different regions of the sample significantly increases the signal-to-noise ratio. Second, molecular motions of colloidal gold can be analyzed by correlation methods with high temporal and spatial resolution. Using a MRC 600 laser scanning system, we collect data from an ensemble of 768 independent subvolumes and determine the space-time correlation function. We demonstrate the technique using two different types of samples, fluorescently labeled DNA molecules in solution and colloidal gold-tagged lipids in a planar bilayer. This approach, which we term "scanning concentration correlation spectroscopy," provides a straightforward means of performing high resolution correlation analysis of molecular motions with available instrumentation. © 1994, The Biophysical Society. All rights reserved.