Multicolor Particle Tracking of Nanocrystals

Typically the resolution of any optical imaging system is limited by the Rayleigh criterium to approximately the wavelength of light, which is far too large to isolate the location and motion of single molecules. It is, however, feasible to locate the position of a single macromolecule through the use of fluorescence. As the diffraction pattern from a point source is well known it is possible to locate the center position of it within approximately 5 nm. However, if there is more than one point source, their relative positions can no longer be determined as accurately, and the resolution reverts to the standard Rayleigh criterion, set by the wavelength of light. Consequently for a large macromolecule, it is not possible to determine its configuration by placing fluorescent tags along its length. Similarly, it is not possible to use fluorescence to study the interaction of two different molecules using a single set of fluorescent tags. Some of these limitations can be overcome through the use of several different tags, which separately label different molecules or different parts of a macromolecule; then each wavelength can be resolved to within 5 nm, and even nearly overlapping probes can be simultaneously resolved. Thus, multicolor tagging offers the possibility for simultaneously measuring both the position and configuration of single molecules.

Fig. 2.: (A) The Rayleigh creterium can be "beaten" using the additional information of emission wavelength. (B) Setup for the two-color tracking. The image is split by a dichroic mirror and refolded onto a single CCD chip.

This principal has been applied using conventional fluorescence molecules (Schmidt et al., 2000; van Oijen et al., 1999). However, the application has been limited by three main disadvantages: first, conventional fluorophores with distinct emission spectra have to be excited with different wavelengths, second the very low intensity of single fluorescence molecules and third the bleaching of the fluorophores allows the observations only for short periods of time.
Here we use nanocrystals as tags, particles with a few nanometers of diameter. The work is done in close collaboration with Inhee Chung in Moungi Bawendi’s lab. They nanocrystals can be excited with the same wavelength, they are emitting at different wavelengths depending on their size, and they do not have an observable photobleaching effect (Dabbousi et al., 1997). This allows the observation over extended periods of time with a simple optical pathway as shown in Fig. 2.

A B
Fig. 3: (A) Sample image of co-localized single nanocrystals with different spectral characteristic, the image is split by a dichroic at 590nm. (B) Particle tracking results of co localized nanocrystals, immobilized onto a cover glass with a lag time of 30 ms over 10 minutes. The measured distance between the nanocrystals is 40nm

One of the technical difficulties, which had to be overcome, was the irregular blinking of the nanocrystals which makes the tracking very difficult. We are now able to track the position of colocalized nanocrystals with a time resolution of 30ms over time periods up to hours (Fig. 3).
In a next, though very difficult step we will functionalize the nanocrystals as tags for specific sites inside of single proteins or a single DNA molecule. Because of their small size they are well suited to track internal protein motion without affecting the measurement. Multicolor tagging of biological molecules will offer new possibilities to observe their interaction dynamics. Unlike the current static colocalization studies used in biology, this technique will facilitate the study of actual dynamics of protein interactions.

References:
Dabbousi, B. O., J. RodriguezViejo, F. V. Mikulec, J. R. Heine, H. Mattoussi, R. Ober, K. F. Jensen, and M. G. Bawendi. 1997. (CdSe)ZnS core-shell quantum dots: Synthesis and characterization of a size series of highly luminescent nanocrystallites. Journal of Physical Chemistry B. 101:9463-9475.
Schmidt, M., M. Nagorni, and S. W. Hell. 2000. Subresolution axial distance measurements in far-field fluorescence microscopy with precision of 1 nanometer. Rev. of Sc. Instr. 71:2742-2745.
van Oijen, A. M., J. Kohler, J. Schmidt, M. Muller, and G. J. Brakenhoff. 1999. Far-field fluorescence microscopy beyond the diffraction limit. J. Opt. Soc. Am. A. 16:909-915.

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