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）通过脉冲或连续波激光进行重复（连续）激发的单个生物分子探针的寿命有点类似于行人随机穿越（相当）安静的道路。如果有一个推力（光子穿过样品的爆发），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-这相当于发射光子，分子返回到其电子基态的安全状态。然而，在运输过程中，相当于处于激发的电子状态，行人可能不幸地遇到路面上的坑洞或缺陷，其不幸的后果是他们的脚被卡住，并且停止向另一侧前进。行人无法到达安全的人行道，他们需要更长的时间来摆脱他们的脚比平均时间，否则将采取他们穿越。因此，存在行人的往返行程被中断的时期，在分子的情况下，它变得“黑暗”并且在相当长的时间内没有荧光光子被发射（c.a.毫秒）的时间段。不幸的是，这不是故事的结局，当被困在坑洞中时，行人被公共汽车或其他类似危险车辆撞到的危险更大（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性物质发生的）。行人随机进入道路的旅程已经结束，在分子世界中，分子是光化学死亡的。我们建议进行的研究是帮助“行人”通过等待合理的时间来完成旅程（平均过马路时间），这通常比行人绊倒到坑洞的平均时间要短。然后我们再加上第二个“推”，这会将行人（如果他们还没有到达）迅速推到安全的人行道上。判断我们可以优化引导行人过马路的机会的条件取决于行人的基本属性，发生陷阱的概率和从陷阱中解脱的难度。我们提供给行人的推动力是一种光学的，也需要仔细地为行人量身定制。这需要我们有详细的知识行人的随机行走，我们将获得这从时间分辨单分子荧光和荧光相关性的研究。我们还需要确定行人（平均）被推的可能性。所有这些数据将被输入到一个随机行走模拟，其中包括我们的光学'推'，由此我们可以看出什么样的推动是合适的，并建立了一个双脉冲激光实验，其中“街道”是倒置显微镜的共焦体积，我们对行人重复行程的观察是通过敏感的单光子探测器产生的电信号进行的，这些探测器已捐赠给该项目我们有几种“行人”可以与之合作;我们在法国的合作者在开发分子方面处于世界领先地位，这些分子可以毫不费力地被推到街上，我们发现有些分子也很容易被推，但我们还没有看到它们各自的行为。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签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循环（更安全的道路交叉）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.