Image Correlation Spectroscopy

Image correlation spectroscopy (ICS) is a fluorescence fluctuation analysis that analyzes the spatial fluctuations within an image instead of the temporal fluctuations [1,2]. Assuming an ergodic system, the ensemble (or spatial) average and time average are equivalent. Hence, the spatial autocorrelation function also provides information regarding the average number of particles in the observed volume. Data from any microscope that is capable of taking images, such as TIRF or confocal microscopes, can be used for ICS. The correlation of higher order moments like the skewness (3nd) and the kurtosis (4th) of the image intensity distribution allows separation of two molecular species (e.g. monomers versus dimers) within cells [3]. ICS can be applied to either live or fixed cell samples and, by selecting certain regions of interest (ROIs), the characteristics of molecular complexes between different cellular components e.g. between focal adhesions and the nucleus can be compared. The method can be further extended by using both the spatial and temporal information available in a series of images (temporal Image Correlation Spectroscopy (tICS, [4]) or Raster Image Correlation Spectroscopy (RICS, [5]) to study the dynamics of the formation of molecular complexes. Depending on the image acquisition scheme, slow (tICS of image data) or fast movements (RICS from scanning data) can be addressed. Cross-correlation spectroscopy (ICCS, [4]) of two different labeled species contains information about molecular interactions.

[1] Petersen, N. O., et al. "Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application." Biophysical journal 65 (1993): 1135-1146.

[2] Wiseman, P. W., and N. O. Petersen. "Image correlation spectroscopy. II. Optimization for ultrasensitive detection of preexisting platelet-derived growth factor-b receptor oligomers on intact cells." Biophysical Journal 76 (1999): 963-977.

[3] Sergeev M., et al. "Measurement of monomer-oligomer distributions via fluorescence moment image analysis." Biophysical Journal 91 (2006): 3884-96.

[4] Srivastava, Mamta and N.O. Petersen. "Image cross-correlation spectroscopy: A new experimental biophysical approach to measurement of slow diffusion of fluorescent molecules." Methods in Cell Science 18 (1996): 47-54.

[5] Digman, M. A., et al. "Fluctuation Correlation Spectroscopy with a Laser-Scanning Microscope: Exploiting the Hidden Time Structure." Biophysical Journal 88 (2005): L33-L36.