Pulsed Interleaved Excitation Fluctuation Imaging: Method and Application to HIV-1 Assembly

Abstract

Pulsed interleaved excitation (PIE) is the method of nanosecond alternating excitation with time-resolved detection and allows accurate, independent and quasi-simultaneous determination of fluorescence intensities and lifetimes of different fluorophores. We have combined PIE with fluctuation imaging methods (PIE-FI) such as raster image correlation spectroscopy (RICS) and number and brightness analysis (N&B) on a home-built multicolor laser/sample scanning microscope. PIE-FI is a novel method that allows robust and accurate measurements of concentrations (down to pM), diffusion and stoichiometry, crosstalk-free dual-color measurements to probe protein-protein interactions, dual-color fluorescence lifetime measurements to quantify Forster resonance energy transfer and even allows resolving different species in images on the basis of their fluorescence lifetime rather than their color. We have applied PIE-FI to study the HIV-1 Gag protein inside live cells. During de novo synthesis of HIV particles in infected cells, this protein assembles at the plasma membrane and creates the shell that ultimately defines the size and shape of budded virions. Little is known about interactions of HIV Gag with cellular and/or viral components inside the cytosol of infected cells. We investigated the wild-type HIV Gag as well as some critical site-specific mutants that influence membrane and/or RNA and/or auto-interactions of Gag to gain more insight into these early stages of HIV assembly.