A cell may be seen (in a very simple model) as a compartmented space filled with liquid (cytoplasm) and containing numerous macro molecules (like proteins, RNA/DNA), single ions (sodium, chlorine...) and even bigger cellular organelles (like the cytoskeleton, mitochondria, the nucleus...). The function of the cell depends on chemical reactions of the molecules within it. As these reactions are often catalysed by proteins and are localized near dedicated cellular structures, the understanding of transport in the cell is essential. Many cellular reactions are diffusion controlled, i.e. the dominant transport process is diffusion. In addition there are also active transport mechanisms in the cell, like e.g. at membrane pores, or by kinesin which may drag particles along the cytoskeleton (see image below).
Here the diffusing particle is described by its hydrodynamic radius Rh, so the larger the the particle is, the slower it gets. The environment is characterized by its absolute temperature T (kB is Boltzma's constant) and its dynamic viscosity η.
As one can see the spacial structure of the cell and the characteristics of the cellular environment significantly influence the motion of particles in the cell. Thus also the diffusion-dominated chemical reactions in the cells strongly depend on the local cellular environment. The other way round, the motion of these particles may be used to measure these environment properties. This explains why it is important to measure these motion properties in a spacially resolved manner in order to better understand the function of the whole cell.
We use Fluorescence Correlation Spectroscopy (FCS), to generate a diffusion coefficient map with rather high accuracy and spacial resolution. A standard implementation of FCS is based on a confocal microscope and can thus only do a measurement on a single spot at a time. So in order to generate a complete map we have to do many single-spot measurements. We demonstrated the usability of this technique. The next image shows an example map of EGFP diffusing in a HeLa cell, created by single-spot FCS measurements (one measurement was done at each position marked with a white +):
N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, J. Langowski (2009): Mapping eGFP oligomer mobility in living cell nuclei. PLoS ONE 4(4): e5041. DOI: 10.1371/journal.pone.0005041This type of measurements leads to a long overall duration of the experiment and expose the cell to a lot of stress (laser illumination with rather high intensities) during this time. So we will now use a new technique, called Single Plane Illumination Microscopy Fluorescence Correlation Spectroscopy (SPIM-FCS) which allows us to do FCS measurements at different positions in the cell in parallel and also reduces the part of the cell that is illuminated to the actual measurement volume. In addition this technique allows us to also evaluate spacial data, which gives us acces to additional motion processes, like e.g. directed flow.