Researchers at the Max Planck Institute for Biophysical Chemistry have introduced a microscope that images molecular dynamics at a temporal resolution of 380 fs or faster. The all-optical, steady-state technique is significantly faster than those that depend on fast electronic circuitry to increase temporal resolution. "We don't have to modulate or demodulate any signals in order to retrieve lifetime information," said Stefan Hell, who with Marcus Dyba and Thomas Klar reported the system in the July 24 issue of Applied Physics Letters. "We just need to record the fluorescence in the detector in the usual way and, in the simplest case, compare the intensity of two subsequently recorded images." Fluorescence confocal images show yeast cells labeled with the styryl dye Nile Red. Left, an intensity image. Right, a stimulated emission depletion image for a delay of 26 ps after excitation. The method is based on a pump-and-probe technique. The team created a reference image by exciting a dye molecule with an upconverted beam from a Ti:sapphire laser from Coherent Photonics Group, Laser Div., of Santa Clara, Calif. A Coherent optoparametric oscillator converted the beam to light at 550 to 555 nm. The researchers then re-imaged the sample, following the excitation pulse with a pulse of 735- to 750-nm light from the Ti:sapphire laser. This second pulse elicits stimulated emission from dye molecules in the low vibrational state -- the fluorescent level -- of excitation. "The idea behind our method is to probe the population of the low level of the excited state simply by checking the efficiency with which the stimulating beam switches off the fluorescence [point by point in the image]," Hell said. The technique also can probe the vibrational, solvent and orientational relaxations that a molecule goes through as it transitions down to the fluorescent state. He add-ed that the team can follow the fluorescence lifetime as well and that it should be able to increase the temporal resolution even further with faster laser pulses. The researchers used piezo scanning to create a diffraction-limited image of the various relaxational lifetimes; however, the method also can be implemented in a regular beam scanning confocal microscope. This may be particularly useful for biological applications where dye molecules convey information about cellular changes. Because the technique employs relatively low laser powers, Hell said, it should be compatible with living cells. The group plans to continue its work by investigating the excitation and stimulated-emission wavelength pairs for biological research dyes. The researchers hope to use this information to develop ways of retrieving biochemical information.