Measuring fast dynamics in solutions and cells with a laser scanning microscope

¹ University of Illinois at Urbana-Champaign
² University of Virgina, Charlottesville, VA
³ McGill University, Canada
⁴ University of Virginia, Charlottesville, VA

^* To whom correspondence should be addressed. E-mail: enrico{at}scs.uiuc.edu.

Submitted on March 13, 2005
Revised on April 27, 2005
Accepted on 10 May 2005

	Abstract

Single point fluorescence correlation spectroscopy (FCS) allows measurements of fast diffusion and dynamic processes in the µs-ms time range. For measurements on living cells, image correlation spectroscopy (ICS) and temporal ICS extend the FCS approach to diffusion times as long as seconds to minutes and simultaneously provide spatially resolved dynamic information. However, ICS is limited to very slow dynamics due to the frame acquisition rate. Here we develop novel extensions to ICS that probe spatial correlations in previously inaccessible temporal windows. We show that using standard laser confocal imaging techniques (raster scan mode) we can not only reach the temporal scales of single point FCS but also have the advantages of ICS in providing spatial information. This novel method, called raster image correlation spectroscopy (RICS) rapidly measures during the scan many focal points within the cell providing the same concentration and dynamic information of FCS as well as information on the spatial correlation between points along the scanning path. Longer time dynamics are recovered from the information in successive lines and frames. We exploit the hidden time structure of the scan method in which adjacent pixels are a few microseconds apart thereby accurately measuring dynamic processes such as molecular diffusion in the microseconds to seconds time scale. In conjunction with simulated data, we show that a wide range of diffusion coefficients and concentrations can be measured by RICS. We used RICS to determine for the first time spatially resolved diffusions of paxillin-EGFP stably expressed in CHOK1 cells. This new type of data analysis has a broad application in biology and it provides a powerful tool for measuring fast as well as slower dynamic processes in cellular systems using any standard laser confocal microscope.

Key Words: fluctuation spectroscopy, fluorescence, image correlation

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