Confocal-FRAP Analysis of ECM Molecular Interactions - Methods in Molecular Biology: Extracellular Matrix Protocols - page 81

Fig. 2. Relative lateral tracer diffusion coefficients of FITC-dextrans in aggrecan
(2000 kDa) (Gribbon and Hardingham, unpublished data). The relative lateral tracer-
diffusion coefficient is the ratio of the tracer-diffusion coefficient at a finite concen-
tration ( D t ) to that at zero concentration ( D 0 ). These data show aggrecan networks
influence solute transport by acting as size-selective molecular sieves.
plex mixtures of macromolecules on the characteristics of the matrix to be
determined by measurements of a single-labeled probe.
In its original development, the nonconfocal FRAP technique was applied
to measure solute mobility in tissues and within single cells (18) . Experimen-
tally, this provided a number of constraints on the choice of bleach mode,
geometry, and recovery analysis, which were also limited by the fluorophore
concentration and the time-scale of diffusion. Fortunately, with homogenous
solutions of biological macromolecules there are fewer experimental con-
straints and the technique is well suited to a confocal microscope. Using the
scanning facility of a confocal microscope, the bleach dimension can be
adjusted to give bleach-recovery times suited to the macromolecule being
investigated. ECM macromolecules frequently have a high molecular mass and
relatively low translational-diffusion coefficients. But with appropriate
manipulation of the bleach and recovery conditions, the translational diffusion
of both low-molecular-weight probes and of concentrated- macromolecules-
forming networks are open to investigation.
In this chapter, the practical use of an unmodified commercial confocal
microscope for determining the diffusion properties of macromolecular ECM
components and of matrix-probe molecules will be outlined. The preparation
and characterization of aggrecan and hyaluronan solutions will be used as
examples, although the technique can be generally applied to the investigation
of a wide range of polymer-polymer and polymer-solute interactions.