Imaging and tracking of single hyaluronan molecules diffusing in solution
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- 作者：Tim Kaminski ; Jan-Peter Siebrasse ; Volkmar Gieselmann ; Ulrich Kubitscheck and Joachim Kappler
- 关键词：Hyaluronan ; Single molecule microscopy ; Biophysics
- 刊名：Glycoconjugate Journal
- 出版年：2008
- 出版时间：August, 2008
- 年：2008
- 卷：25
- 期：6
- 页码：555-560
- 全文大小：237.6 KB
文摘
Hyaluronan is an important soluble component of the extracellular matrix of many tissues with well known space-filling, lubricating and signaling functions. As such, hyaluronan can regulate cell adhesion, migration, differentiation and proliferation. Ultrastructural studies showed the existence of fibers and networks of hyaluronan molecules at surfaces, while bulk studies of hyaluronan in solution indicated that the polymer forms random coils. Here, we show that single hyaluronan molecules can be visualized and tracked in three-dimensional samples at room temperature in aqueous buffer. Using a wide-field fluorescence microscope equipped with laser excitation and an sensitive and fast EMCCD camera for fluorescence detection, single FITC-labeled hyaluronan molecules from rooster comb were detected in aqueous solutions. Freely moving hyaluronan-FITC could be tracked over up to 20 images acquired at a frame rate of 98 Hz. Analysis of the trajectories revealed Brownian motion of hyaluronan in tris-buffered saline with an average diffusion coefficient D = 3.0 ¡À 0.2 μm2/s. These observations confirm the concept that hyaluronan molecules form random coils in solution. The possibility of following the tracks of single hyaluronan molecules in solution facilitates the analysis of processes that lead to the formation of more organized forms of hyaluronan and its interactions with cells with very high spatial and temporal accuracy.