Laboratoire de Spectrométrie Physique UMR 5588, Université de Grenoble I/CNRS, BP 87, 38402 Saint Martin d'Hères, France.
J Phys Chem B. 2010 Mar 4;114(8):2988-96. doi: 10.1021/jp910082h.
Determining the number of fluorescent entities that are coupled to a given molecule (DNA, protein, etc.) is a key point of numerous biological studies, especially those based on a single molecule approach. Reliable methods are important, in this context, not only to characterize the labeling process but also to quantify interactions, for instance within molecular complexes. We combined fluorescence correlation spectroscopy (FCS) and photobleaching experiments to measure the effective number of molecules and the molecular brightness as a function of the total fluorescence count rate on solutions of cDNA (containing a few percent of C bases labeled with Alexa Fluor 647). Here, photobleaching is used as a control parameter to vary the experimental outputs (brightness and number of molecules). Assuming a Poissonian distribution of the number of fluorescent labels per cDNA, the FCS-photobleaching data could be easily fit to yield the mean number of fluorescent labels per cDNA strand (approximately = 2). This number could not be determined solely on the basis of the cDNA brightness, because of both the statistical distribution of the number of fluorescent labels and their unknown brightness when incorporated in cDNA. The statistical distribution of the number of fluorophores labeling cDNA was confirmed by analyzing the photon count distribution (with the cumulant method), which showed clearly that the brightness of cDNA strands varies from one molecule to the other. We also performed complementary continuous photobleaching experiments and found that the photobleaching decay rate of Alexa Fluor 647 in the excited state decreases by about 30% when incorporated into cDNA, while its nonradiative decay rate is increased such that the brightness of individual Alexa labels is decreased by 25% compared to free Alexa dyes.
确定与给定分子（DNA、蛋白质等）偶联的荧光实体的数量是许多生物学研究的关键点，特别是那些基于单分子方法的研究。在这种情况下，可靠的方法不仅对于表征标记过程很重要，而且对于定量相互作用（例如在分子复合物内）也很重要。我们结合荧光相关光谱（FCS）和光漂白实验来测量有效分子数量和分子亮度作为 cDNA 溶液的总荧光计数率的函数（包含百分之几的用 Alexa Fluor 647 标记的 C 碱基）。在这里，光漂白被用作控制参数来改变实验输出（亮度和分子数量）。假设每个 cDNA 上荧光标记的数量呈泊松分布，那么 FCS-光漂白数据可以很容易地拟合，以得出每个 cDNA 链上的平均荧光标记数量（约为 2）。由于荧光标记数量的统计分布及其在掺入 cDNA 时的未知亮度，仅基于 cDNA 亮度无法确定这个数量。通过分析光子计数分布（用累积法），证实了 cDNA 上荧光团标记数量的统计分布，这清楚地表明 cDNA 链的亮度从一个分子到另一个分子变化。我们还进行了互补的连续光漂白实验，发现当 Alexa Fluor 647 在激发态中掺入 cDNA 时，其光漂白衰减率降低约 30%，而其非辐射衰减率增加，使得单个 Alexa 标记的亮度比游离 Alexa 染料降低 25%。
