A team of scientists at Sandia National Laboratories in Livermore, Calif., has shown that, with the right photonic equipment, a molecule can tell two versions of the same story at once. The group, according to technical staff member A. Khai Luong, used advanced microscopy technology to simultaneously measure multiple fluorescence characteristics of individual fluorophores.Because these characteristics are affected by the fluorophore’s surroundings, being able to monitor changes in the fluorescence spectrum and lifetime of a molecule allows researchers to probe the immediate nanoscale area around it. That is the goal of much work in single-molecule spectroscopy, Luong said, and the ability to extract more information from the limited number of emitted photons could be useful.The scientists used fast, custom-built electronics based on the latest semiconductor technology, a state-of-the-art Hamamatsu Photonics KK photomultiplier tube, a novel readout scheme for which the lab has submitted a patent, and a modified Nikon confocal microscopy setup. They sent picosecond pulses of 532-nm light from a Time-Bandwidth Products Inc. laser operating at 50 MHz through a dichroic filter and a 1.4-NA, 60× objective to create a diffraction-limited spot on a sample. They collected the resulting fluorescence through imaging optics and a 75-μm pinhole, resulting in a confocal image that restricted the emission signal to a small volume, and sent the fluorescent signal through a dispersing prism to the detector.The photomultiplier detector had 32 discrete anodes, and the researchers used custom electronics to time-stamp each arriving photon to within 50 ns. Their scheme recorded anode position and, hence, the wavelength of fluorescence emission. At the same time, the equipment captured the photon’s emission time relative to the excitation pulse. The setup had a 140-nm spectral range, with resolution of 3 nm at 540 nm and of 5 nm at 650 nm. The emission time of a photon relative to the excitation pulse could be tracked to about 250 ps.Using this system, the investigators simultaneously measured the fluorescence emission wavelength and time for every photon from single molecules of rhodamine 6G, tetramethylrhodamine and Cy3 embedded in thin films of polymethyl methacrylate. They distinguished among the various fluorophores by looking at the captured characteristics. They also examined many single-molecule time traces under various conditions.“The goal in all these cases is to measure the distribution of molecular properties in a sample rather than just the average value of a property,” Luong explained. Gathering such statistics, she added, can be challenging even before measurements begin because sample preparation for single molecules can be difficult. In some cases, the task involves only changing a parameter, such as the acidity of the environment. In others, it may be necessary to immobilize the molecules prior to taking a reading, so procedures to do so may have to be worked out.The researchers plan to benchmark fluorophores in known protein, DNA and RNA environments, and to use this information to probe more complex and less well understood environments. The addition of a photon antibunching technique will heighten confidence that only one fluorophore is being excited. The method depends on the fact that there is a reduced probability of detecting two photons within an interval less than the excited-state lifetime of the fluorophore if only a single molecule is observed.Journal of Physical Chemistry B, Aug. 25, 2005, pp. 15691-15698.