Abstract
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 two-dimensional maps of diffusion coefficients. We show the applicability of msICS to fluorophores in solution and to free EGFP in living cells.
Highlights
Since its inception in the 1970s [1, 2] fluorescence correlation spectroscopy (FCS) has emerged as a very useful method for probing transport, diffusion and interactions of biomolecules in vitro and in vivo [3,4,5]
This forms the foundation of confocal laser scanning microscopy (CLSM), a mode of fluorescence microscopy widely used in modern biology to visualize cells and cellular components
To cover a broad dynamic range of diffusion properties we have developed an extended approach referred to as multiple scan speed image correlation spectroscopy: the idea is to measure several time series with varying pixel dwell times and otherwise constant settings
Summary
Since its inception in the 1970s [1, 2] fluorescence correlation spectroscopy (FCS) has emerged as a very useful method for probing transport, diffusion and interactions of biomolecules in vitro and in vivo [3,4,5]. Due to the suppression of scattered light and the diffraction-limited resolution in three dimensions, a confocal setup allows to probe the spatial distribution of fluorescent molecules by raster-scanning pixel by pixel and line by line the focus over the sample using rotary galvanometer-driven mirrors effectively located in the back focal plane of the objective lens [6,7,8]. This forms the foundation of confocal laser scanning microscopy (CLSM), a mode of fluorescence microscopy widely used in modern biology to visualize cells and cellular components
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