Table 1 Relationships between parameter combinations of the StrExp function fitted to experimental FLIP data and the time hierarchy of photobleaching and possible intracellular transport processes
From: Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation
Scenario | Parameters recovered from the fit of the StrExp function to experimental fluorescence loss in FLIP image sets | |||
|---|---|---|---|---|
Stretching (h) | Time constant (τ) | Rate coefficient (k(t)) | Explanation | |
Diffusion-limited FL | Diffusion of molecules to the bleached ROI is slower than the bleaching process. This results in a ’depletion zone’ around the bleached area. Binding and release, if any, are faster than diffusion. | |||
Inside ROI | 1 < h < 2 | Dependent on bleach rate constant and diffusion constant*. | Decreasing over time. | Fluorophores get depleted by the bleaching acting as diffusion-limited reaction. |
Outside ROI | ~0.5 < h < 1 | Dependent on diffusion constant and bleach rate constant*. | Increasing over time. | Compressed decay, because diffusion to the bleach ROI causes delayed response. |
Bleach-limited FL | Diffusion of molecules to the bleached ROI as well as eventual binding and release are faster than the bleaching process. The only process causing FL is the repeated bleaching inside the ROI. | |||
Inside ROI | ~ 1 | Dependent on bleach rate constant only. | Constant over time and equal to 1/τ. | Approx. mono-exponential decay determined by the bleach rate. |
Outside ROI | ~ 1 | Dependent on bleach rate constant only. | Constant over time and equal to 1/τ. | Approx. mono-exponential decay determined by the bleach rate. |
Binding # -limited FL | Diffusion is fast but molecules are hindered by binding to cellular organelles or by obstacles. Any spatial fluorescence gradient is rapidly equilibrated and binders/barriers become visible. | |||
Inside ROI | ~ 1 | Dependent on bleach rate constant only. | Constant over time and equal to 1/τ. | Approx. mono-exponential decay determined by the bleach rate. |
Outside ROI | ~0.8 < h < 1 | Dependent on release rate constant and bleach rate constant*. | Increasing over time. | Compressed decay, because slow release causes delayed response. |