Quantifying Green Fluorescent Protein Diffusion in Escherichia coli by Using Continuous Photobleaching with Evanescent Illumination

Abstract

Fluorescence recovery after photobleaching and fluorescence correlation spectroscopy are the primary means for studying translational diffusion in biological systems. Both techniques, however, present numerous obstacles for measuring translational mobility in structures only slightly larger than optical resolution. We report a new method using through-prism total internal reflection fluorescence microscopy with continuous photobleaching (TIR-CP) to overcome these obstacles. Small structures, such as prokaryotic cells or isolated eukaryotic organelles, containing fluorescent molecules are adhered to a surface. This surface is continuously illuminated by an evanescent wave created by total internal reflection. The characteristic length describing the decay of the evanescent intensity with distance from the surface is smaller than the structures. The fluorescence decay rate resulting from continuous evanescent illumination is monitored as a function of the excitation intensity. The data at higher excitation intensities provide apparent translational diffusion coefficients for the fluorescent molecules within the structures because the decay results from two competing processes (the intrinsic photobleaching propensity and diffusion in the small structures). We present the theoretical basis for the technique and demonstrate its applicability by measuring the diffusion coefficient, 6.3 ± 1.1 µm2/sec, of green fluorescent protein (GFP) in Escherichia coli cells.

Effects of Recombinant Protein Expression on Green Fluorescent Protein Diffusion in Escherichia coli

“…Despite these variations in protein size, shape, and concentration, D GFP remained within error of the commonly referenced GFP diffusion coefficient in E. coli , 7.7 ± 2.5 μ m 2 s −1 (14). D GFP was also consistent with the value recently measured by total internal reflection continuous photobleaching (38). Our observations show that GFP diffusion is independent of the type or amount of protein coexpressed, but several points need to be addressed.…”

Interaction of α-synuclein with vesicles that mimic mitochondrial membranes

“…Mito Tracker Red CMXRos (Invitrogen) was used to stain the active mitochondria after protein import and trypsinization. Fused silica and glass microscope slides were prepared as described [37]. Mitochondria containing α-synuclein were allowed to attach to the poly-L-lysine coated slide for 30 min and then rinsed with minimal media.…”

Macromolecule diffusion and confinement in prokaryotic cells

“…CP-TIR. Slade and co-workers have combined Continous Photobleaching (CP) with Total Internal Reflection (TIR) microscopy to determine diffusion coefficients of GFP under normal conditions [55] and in cells overexpressing proteins [56]. TIR illumination limits the laser light path in the axial direction and allows the photobleaching volume to be restricted to only a small subvolume of the cytoplasm, leaving enough non-photobleached GFP outside of the excitation region to observe fluorescence recovery with good signal-to-noise ratios (Figure 2e).…”