Control of the Single Molecule Fluorescence Cycle - A Feasibility Study

单分子荧光的控制（或生物分子的道路安全）在脉冲或连续波激光的重复（连续）激发下，单个生物分子探针的寿命有点类似于一个行人随机地穿过（相当）安静的一段道路。给定一个推力（一束光子穿过样品），行人开始穿越道路的概率是有限的，成功的结果是到达另一边——这相当于光子的发射和分子返回到其电子基态的安全。然而，在行进过程中，行人可能会不幸遇到路面上的坑洞或缺陷，这相当于处于一种兴奋的电子状态，其不幸的后果是他们的脚被困住，而前往另一侧的进程也就停止了。行人无法到达安全的人行道，而且他们要花更长的时间才能把脚抽出来，而不是用其他方式穿过人行道。因此，行人往返的旅程会被打断，在分子的情况下，它会变得“黑暗”，在相当长的时间内（大约毫秒）不会发出荧光光子。不幸的是，这并不是故事的结尾，当被困在坑里的行人更有可能被公共汽车或其他类似的危险车辆撞到（在分子被困在“黑暗”状态的情况下，致命的碰撞是与氧分子或其他一些化学反应物质发生的变化）。行人随意走上道路的旅程结束了，在分子世界里，分子在光化学上已经死亡。我们建议进行的研究是帮助“行人”通过等待一段合理的时间来安全过马路（平均过马路时间），这与行人绊倒在坑里的平均时间相比通常是短的。然后我们添加第二个“push”；这将推动行人（如果他们还没有到达它）迅速到安全的人行道上。判断引导行人过马路的最佳机会的条件取决于行人的基本特性、陷阱发生的可能性和从陷阱中解脱出来的难度。我们为行人提供的推动是一种光学推动，也需要仔细地为行人量身定制。这需要我们对行人的随机行走有详细的了解，我们将从时间分辨的单分子荧光和荧光相关研究中获得这一点。我们还需要确定行人对被推的敏感程度（平均而言）。所有这些数据将被输入到随机行走模拟中，其中包括我们的光学“推动”，从中我们可以看到什么样的推动是合适的，并建立一个双脉冲激光实验，其中“街道”是倒置显微镜的共聚焦体积，我们通过敏感的单光子探测器产生的电信号来观察行人的重复旅程，该探测器由英国癌症研究中心捐赠给该项目。我们有好几种“行人”需要处理；我们在法国的合作者在开发可以毫不费力地推到街上的分子方面处于世界领先地位，我们发现有些分子也很容易被推到街上，但我们还没有看到它们单独的行为。我们在英国癌症研究中心的合作者使用绿色和黄色荧光蛋白作为天然标记来监测细胞信号蛋白的构象变化，我们也将研究光学控制荧光周期（更安全的过马路）的可能性。

Control of Single Molecule Fluorescence (or Road Safety for Biomolecules)The life of a single bio-molecular probe that is undergoing repetitive (continuous) excitation by a pulsed or continuous wave laser is somewhat akin to a pedestrian randomly setting out to cross a (fairly) quiet stretch of road. Given a push (a burst of photons passing through the sample) there is a finite probability that the pedestrian starts their journey across the road, the successful outcome of which is reaching the other side -this is equivalent to the emission of a photon and the molecule returning to the safety of its electronic ground state. However whilst in transit, equivalent to being in an excited electronic state, the pedestrian may unfortunately encounter a pot hole or imperfection in the road surface, with the unfortunate consequence that their foot becomes trapped and that progress to the other side is halted. The pedestrian cannot reach the safety of the pavement and it takes considerably longer for them to extricate their foot than the average time it would otherwise take them to cross. There are thus periods where the pedestrian's journeys to and fro are interrupted, in the case of a molecule it becomes 'dark' and no fluorescence photons are emitted for considerable (c.a. milliseconds) periods of time. Unfortunately this is not the end of the story, whilst trapped in the pot hole the pedestrian is in greater danger of being struck by a bus or some other similarly dangerous vehicle (in the case of a molecule trapped in a 'dark' state the fatal collision is with an oxygen molecule or some other reactive species that changes it chemically). The pedestrian's random journeys into the road are over and in the molecular world the molecule is photochemically dead.The research we propose to undertake is to help the 'pedestrian' make it across the road safely by waiting for a reasonable time for the journey to be made (the average crossing time) this is generally short in comparison to the average time it takes for the pedestrian to stumble into a pothole. We then add a second 'push'; this propels the pedestrian (if they have not already reached it) swiftly to the safety of the pavement. Judging the conditions by which we can optimise our chances of guiding the pedestrian across the road depends on the fundamental properties of the pedestrian, the probability of trapping taking place and the difficulty of extrication from the trap. The push that we provide to the pedestrian is an optical one and needs to be carefully tailored to the pedestrian as well. This requires us to have detailed knowledge of the pedestrian's random walk and we will obtain this from time resolved single molecule fluorescence and fluorescence correlation studies. We will also need to determine how susceptible (on average) the pedestrian is to being pushed. All this data will be input into a random walk simulation which includes our optical 'push', from this we can see what kind of push is appropriate and to set up a two pulse laser experiment in which the 'street' is the confocal volume of an inverted microscope and our observation of the pedestrian's repeated journeys is via the electrical signals produced by sensitive single photon detectors which have been donated to the project by Cancer Research UK.We have several kinds of 'pedestrians' to work with; our collaborators in France lead the world in developing molecules who can be propelled into the street with little effort, we have found that some are also very amenable to being pushed but we have yet to see how they behave individually. Our collaborators at Cancer Research UK use green and yellow fluorescent proteins as natural labels to monitor conformational changes in cell-signaling proteins and we will investigate the possibility of optically controlling the fluorescence cycle (safer road crossing) in these as well.