The space within living cells is often quite crowded and complex. To study and quantify the physical nature of these spaces we employ techniques of fluorescence microscopy. Our model system, Escherichia coli, is a Gram-negative bacterium. Unlike eukaryotes, prokaryotes do not have membrane bound organelles or a membrane enclosed nucleus. This, and their small size, have led some to claim that bacteria are merely sacks of biomolecules with little to no internal structure. This hypothesis has been largely discredited by careful studies by light and electron microscopists^[a]. Many of these studies have been unable to quantify dynamic properties of these complex media because their techniques often require fixing the cells and/or high doses of harmful radiation. We use the diffusion of tracer particles to study dynamics in these crowded and complex media. We use genetically encoded fluorescent proteins like GFP because they are easy to work with biologically.

The two general techniques that we employ are fluorescence recovery after photobleaching (FRAP) and time-resolved fluorescence anisotropy. In FRAP, a region of the bacterium is irreversibly bleached with a focused laser pulse. The whole cell is then exposed to a broad diffuse laser and the recovery of the fluorescence profile is monitored. Recovery occurs because the tracers that have not been photobleached diffuse throughout an enclosed space. The technique of FRAP is used to study translational diffusion of GFPs on the 1µm scale with a time resolution of at least 25ms. See figure 1 for a description of the analysis.

The other technique used, time resolved fluorescence anisotropy, measures the rotational diffusion of the tracer particle. By exciting fluorophores with linearly polarized light and measuring the polarization of the response as a function of time, the rotational dynamics of the fluorophore can be studied. The short time scale of rotational diffusion (~10-100ns) coupled with the use of diffraction limited excitation profiles allow the study of much shorter length scales, on the order of 200nm, thus, enabling us to probe the local environment of prokaryotic structural elements.

Armed with these techniques, we can study different phases of cell growth or subject cells to stresses. E. coli cells routinely encounter stress in the form of temperature change, nutrient deprivation, drugs, or osmotic shock. We can also tag biologically active molecules (RNAP, β-galactosidase, HU) or target the GFPs to a specific region in the cell (nucleoid, periplasm, cytoplasmic periphery, membranes). See figure 2 for an example of this targeting. Using all these methods together allows us to study quantitatively the physical nature of these biologically relevant crowded environments.