FCS Helps to Track Individual Molecules
During Polystyrene Formation
Lynn M. Savage
Polymers are very important, whether they are artificial (plastics) or natural (proteins). Various techniques have been used to explore the processes involved in the formation of polymers from simpler molecules. Nuclear magnetic resonance, electron spin resonance and fluorescence spectroscopies can provide information about polymerization kinetics, for example, but only by averaging information from bulk ensembles of monomers as they convert to polymers.
Images acquired with an inverted microscope and a CCD camera indicate the movement of dye molecules amid styrene molecules as they polymerize. Experiments were conducted without a cross-linker (left), with the cross-linker 1,4-divinylbenzene (center) and with a similar dye that included two styrenyl groups (right). The images were acquired when polymerization was 64, 65 or 62 percent completed, respectively. Yellow = molecules slow enough for tracking by wide-field microscopy; red = very slow or completely immobile molecules. Reprinted with permission of Angewandte Chemie International.
Now researchers at Katholieke Universiteit Leuven in Heverlee, Belgium, and at Max Planck Institut für Polymerforschung in Mainz, Germany, have combined fluorescence correlation spectroscopy (FCS) with wide-field microscopy to watch polymerization transpire at the single- molecule level.
They studied styrene, polymerizing the material either with or without a cross-linker, and using probes comprising derivatives of perylenediimide, a very stable fluorescent dye with high quantum yield. The scientists followed the polymerization of the styrene molecules by detecting changes in the diffusion constant of the dye molecules as they moved first among the monomers in solution, then within the matrices formed by the emerging polymer.
For FCS measurements, they used a 543-nm HeNe laser from Melles Griot (now CVI Melles Griot) of Carlsbad, Calif., coupled to an Olympus inverted optical microscope equipped with a 1.3-NA, 100× oil-immersion objective, also from Olympus, and filters made by Chroma Technology Corp. of Rockingham, Vt. They collected fluorescence emission from the dye with a single-photon avalanche diode from PicoQuant GmbH of Berlin.
As polymerization proceeded, the motion of the dye molecules slowed because they had less freedom of movement. When the diffusion of the dye slowed to about 10-12 m2/s, the investigators imaged the individual molecules using a JDSU solid-state diode laser operating at 532 nm for excitation; a 512 ×512-pixel CCD camera made by Princeton Instruments Inc. of Trenton, N.J., for detection; and the same inverted microscope as before. Imaging with wide-field microscopy became possible when about half of the styrene molecules had polymerized.
The researchers found that FCS enabled them to determine the diffusion constants of freely moving dye molecules amid the monomers and expanding polymer matrices, but that wide-field microscopy permitted visualization of the motion of dyes that were entangled — and eventually stopped — by the polymers. Using 1,4-divinylbenzene as a cross-linker or using a derivative of perylenediimide that included a pair of styrenyl groups, which enabled the dye itself to be incorporated into the polymer chain and to act as a cross-linker, allowed the scientists to compare different polymerization conditions on a single-molecule level.
Angewandte Chemie International, Jan. 11, 2008, pp. 783-787.
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