Fluorescence Recovery after Photobleaching with the Leica TCS SP2

Among all photobleaching experiments which have been described, fluorescence recovery after photobleaching (FRAP) is the most popular. It employs irradiation of a fluorophore in a living sample with a short laser pulse to degrade it and thereby abolish fluorescence followed by time-resolved image recording of the sample. If the fluorophore (or a so-called mobile fraction of it, see below) is able to freely diffuse through the sample a recovery of fluorescence can be observed. Photobleaching experiments can be conducted with modern laser scanning microscopes, where the laser is used at high intensity for bleaching and low intensity for image recording. The LCS software provided by Leica Microsystems contains a FRAP application wizard that guides the user ot the Leica TCS SP2 through the steps of a FRAP experiment.

Content of this application letter:

• Qualitative vs. Quantitative FRAP
• Preparation

  • Establish experimental conditions
  • Initial experiments

• FRAP experiments

  • Photobleaching a fluorescein-labelled polymer in solution
  • FRAP of a nuclear protein in live cells

• Data processing

  • Background subtraction
  • Correct for fluorescence loss due to photobleaching
  • Normalization

• Estimation of parameters

  • Calculation of the mobile fraction
  • Calculation of t_1/2
  • Estimation of the diffusion coefficient D
  • Modeling

• Evaluation of demo experiments

  • FD 464
  • H1-GFP

• Final remarks