Applications of Pulsed Interleaved Excitation in Live Cell Experiments

Abstract

To make Fluorescence Correlation Spectroscopy (FCS) measurements more viable in living cells, various new methods have been developed. Among these are Raster Image Correlation Spectroscopy (RICS), and Scanning Fluorescence Correlation Spectroscopy (SFCS).In RICS, a confocal raster scanning image of a sample is evaluated to extract concentration, diffusion, or colocalization information of fluorescently labeled molecules using both the temporal and spatial information.Another advantageous possibility, especially in small organisms, is to use SFCS. There, the confocal laser spot is rotated through a small area, effectively increasing the focal volume and reducing fluorophore bleaching.Especially in live cell measurements, where fluorescent proteins are typically used, signal levels are often weak, and spectral crosstalk can be a significant problem. Therefore we combined both RICS and SFCS with Pulsed Interleaved Laser Excitation (PIE), a technique we developed to avoid the introduction of artifacts by spectral crosstalk. The sensitivity of both RICS and SFCS to detect dually labeled molecules could be significantly improved, rendering them much more useful for biologically relevant applications both in live cells and in vitro.The principles of PIE-RICS and PIE-SFCS will be presented along with applications on calcium channels and protein interactions in yeast cells.