Using two-photon standing waves and patterned photobleaching to measure diffusion from nanometers to microns in biological systems

A method of measuring molecular diffusion rates in microscopic sample volumes is described. This method utilizes the standing wave interference created by colliding two counterpropagating laser beams at the focus of two opposing microscope objectives, creating a periodic light distribution in a volume on the order of 1 fl. By using a Pockels cell to vary the laser intensity with a time resolution of milliseconds, we show how this experimental geometry can be used to perform ultrahigh resolution fluorescence recovery after patterned photobleaching ~FRAPP! experiments. A mathematical treatment of the experiment shows that the laser excitation profile has two characteristic length scales, the width of the focal spot and the period of the standing wave, which permits the simultaneous measurement of dynamics on two separate length scales. This feature may be used to determine whether the measured diffusion is anomalous. We present experimental results using a femtosecond Ti:sapphire laser to create a two-photon excitation profile with a fringe visibility on the order of 100. This standing wave is used to demonstrate FRAPP in both model dye/polymer systems and in more complex systems like living cells stained with a fluorescent dye. By combining the advantages of standing wave microscopy and two-photon fluorescence recovery after photobleaching, this technique permits the measurement of very short length motions in localized sample volumes, which should be useful in both biology and the study of diffusion in microscopically heterogeneous systems. © 2002 American Institute of Physics. @DOI: 10.1063/1.1464656#