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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.

The diffusive motion of DNA-containing chromatin in live cells and isolated nuclei is investigated using a two-photon standing wave fluorescence photobleaching experiment with 100 nm spatial resolution. The chromatin is labeled using the minor groove binding dye Hoechst 33342. In live cells, the mean diffusion rate is 5 x 10(-4) micro m2/s, with considerable cell-to-cell variation. This diffusion is highly constrained and cannot be observed in a standard, single beam fluorescence recovery after photobleaching experiment. To determine the chemical origin of the diffusion, we study motion in isolated nuclei and vary the strength of the histone-DNA interactions by changing the ionic strength and using chemical and photocross-linking experiments. At higher NaCl concentrations, we see increased chromatin diffusion as the histone-DNA interaction is weakened due to ionic screening, whereas photocross-linking the core histones to the DNA results in a complete absence of diffusive motion. These trends are consistent with the 100 nm scale motion being correlated with the interactions of histone proteins with the DNA. If chromatin diffusion is connected to the nucleosomal dynamics on much smaller length scales, this may provide a way to assay biochemical activity in vivo based on larger scale macromolecular dynamics observed via fluorescence microscopy.

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The photochemical effects of near‐UV light on chromatin labeled with the vital DNA dye Hoechst 33342 (H33342) are studied. Several types of experiments demonstrate that illumination at both 365 and 410 nm results in significant cross‐linking of proteins with the DNA. Fluorescence microscopy of dye‐stained Xenopus XTC‐2 nuclei shows that UV illumination has effects similar to chemical fixation by formaldehyde. At 365 nm a dose of ∼70 J/cm2 results in 50% of the DNA being cross‐linked, as measured by chloroform–sodium dodecyl sulfate extraction. At 410 nm the efficiency of cross‐linking was smaller by a factor of 3. Gel electrophoresis of the cross‐linked proteins shows them to be predominantly core histones. The implications of these results for experiments on live cells stained with H33342, for example, fluorescence microscopy of nuclear dynamics or cell sorting, are discussed.