The intracellular mobility of biomolecules is determined by
transport and diffusion as well as molecular interactions and is crucial for
many processes in living cells. Methods of fluorescence microscopy like
confocal laser scanning microscopy (CLSM) can be used to characterize the
intracellular distribution of fluorescently labeled biomolecules.
Fluorescence correlation spectroscopy (FCS) is used to describe diffusion,
transport and photo-physical processes quantitatively. As an alternative to
FCS, spatially resolved measurements of mobilities can be implemented
using a CLSM by utilizing the spatio-temporal information inscribed into
the image by the scan process, referred to as raster image correlation
spectroscopy (RICS). Here we present and discuss an extended approach,
multiple scan speed image correlation spectroscopy (msICS), which
benefits from the advantages of RICS, i.e. the use of widely available
instrumentation and the extraction of spatially resolved mobility
information, without the need of a priori knowledge of diffusion properties.
In addition, msICS covers a broad dynamic range, generates correlation
data comparable to FCS measurements, and allows to derive twodimensional
maps of diffusion coefficients. We show the applicability of
msICS to fluorophores in solution and to free EGFP in living cells |
