Fluorescence Cross-Correlation Spectroscopy (FCCS) in Living Cells

Fluorescence cross-correlation spectroscopy (FCCS) is a single-molecule sensitive technique to quantitatively study interactions among fluorescently tagged biomolecules. Besides the initial implementation as dual-color FCCS (DC-FCCS), FCCS has several powerful derivatives, including single-wavelength FCCS (SW-FCCS), two-photon FCCS (TP-FCCS), and pulsed interleaved excitation FCCS (PIE-FCCS). However, to apply FCCS successfully, one needs to be familiar with procedures ranging from fluorescent labeling, instrumentation setup and alignment, sample preparation, and data analysis. Here, we describe the procedures to apply FCCS in various biological samples ranging from live cells to in vivo measurements, with the focus on DC-FCCS and SW-FCCS.