Fluorescence Lifetime Microscope Offers Picosecond Resolution

Daniel S. Burgess

Scientists at Riken research institute in Wako, Japan, have developed a fluorescence lifetime imaging microscope that incorporates a Kerr cell to offer picosecond temporal and micrometer spatial resolution. The apparatus, suggested Tahei Tahara, chief scientist at the institute and director of its molecular spectroscopy laboratory, may be used in conjunction with a fluorescence upconversion technique to achieve femtosecond resolution, enabling users to investigate samples by what he and his colleagues have termed fluorescence dynamics imaging.

Ultrafast time-resolved fluorescence images produced using a Kerr-gated microscope reveal the emissions of the free and self-trapped excitons in α-perylene microcrystals. Images courtesy of Tahei Tahara.

In the setup, a train of 110-fs pulses of 800-nm radiation from a Spectra-Physics Ti:sapphire laser is frequency-doubled in an LBO crystal. The 400-nm light passes through a 40×, 0.75-NA Nikon objective and is defocused to excite an approximately 80-μm-diameter region on the sample. The resulting fluorescence is collected by the objective and routed to a Kerr gate, comprising a pair of crossed polarizers, a pair of camera lenses and a 1-mm-long cell filled with the Kerr medium, carbon disulfide.

The gate is analogous to a mechanical shutter in a camera, Tahara explained. “The difference is speed,” he said. “The response of the Kerr cell is ultrafast, and the ‘shutter speed’ is a picosecond.”

Images of yeast cells labeled with rhodamine B show how the dye’s fluorescence changes with time.

The Kerr cell is triggered by the 800-nm pulses from the femtosecond laser, and the fluorescent signal that passes the cell is filtered and, finally, detected using a Princeton Instruments CCD camera. Based on measurements of the instantaneous rise of fluorescence in coumarin, the researchers determined that the temporal resolution of the instrument is 1.4 ps. Based on the spatial intensity profile of a 4.85-μm-diameter fluorescent bead, they estimated the spatial resolution to be on the order of 1 μm.

The ability to collect fluorescence data at a picosecond temporal resolution, Tahara said, enables one to probe phenomena such as the energy transfer rate, solvation and molecular rotation. The instrument should be suitable for use with almost all types of samples currently studied by fluorescence microscopy, he said, but its application to biological imaging and the characterization of microstructured devices is of particular interest to the team.

The scientists hope to use the nonscanning Kerr-gated microscope with a technique they developed that offers diffraction-limited spatial resolution and femtosecond temporal resolution. That approach currently requires X-Y scanning, which results in measurement time lags and longer measurement runs.

Applied Physics Letters, Sept. 26, 2005, 131105.