Fluorescence Correlation Spectroscopy at Micromolar Concentrations without Optical Nanoconfinement

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• 作者：Ted A. Laurence ; Sonny Ly ; Feliza Bourguet ; Nicholas O. Fischer ; Matthew A. Coleman
• 刊名：Journal of Physical Chemistry B
• 出版年：2014
• 出版时间：August 14, 2014
• 年：2014
• 卷：118
• 期：32
• 页码：9662-9667
• 全文大小：349K
• ISSN：1520-5207

文摘

Fluorescence correlation spectroscopy (FCS) is an important technique for studying biochemical interactions dynamically that may be used in vitro and in cell-based studies. It is generally claimed that FCS may only be used at nM concentrations. We show that this general consensus is incorrect and that the limitation to nM concentrations is not fundamental but due to detector limits as well as laser fluctuations. With a high count rate detector system and applying laser fluctuation corrections, we demonstrate FCS measurements up to 38 渭M with the same signal-to-noise as at lower concentrations. Optical nanoconfinement approaches previously used to increase the concentration range of FCS are not necessary, and further increases above 38 渭M may be expected using detectors and detector arrays with higher saturation rates and better laser fluctuation corrections. This approach greatly widens the possibilities of dynamic measurements of biochemical interactions using FCS at physiological concentrations.