Molecular relativity: tracking single molecule movement relative to cell structures

荧光显微镜是细胞生物学家的重要工具，因为它允许他们用荧光分子(荧光团)标记不同的蛋白质，并在活细胞中观察它们。这些信息可以帮助我们了解疾病，并找到治疗疾病的新药。为了了解细胞中分子的行为，可以对标有荧光团的单个分子进行成像，并测量它们的速度。然而，目前这只能在细胞膜上完成，或者在细胞的非常静止的部分上完成。如果分子所连接的细胞部分移动，就不可能找出分子运动的原因和分子连接的结构的运动。我们建议解决这个问题，这样就可以在移动的结构中进行跟踪研究。要做到这一点，我们将使用可光开关荧光团的性质。这些是荧光团，可以在发射两种不同颜色的光之间切换。通常只记录一种颜色，其中只有几个荧光团发光。这意味着单个分子可以被追踪。我们想开发一种同时成像两种发射态的系统。这将使我们可以想象样品在一个通道中的结构，以及单个分子在另一个通道中的结构。这可以通过同时用两个激光照射样品，并将发射的光分成两部分，根据颜色进行分割来完成。为了从这个系统中获得有用的信息，我们需要开发一种新的数据分析方法。单分子的位置和样品的结构都需要进行拟合和跟踪。然后，可以利用样品结构的运动来校正单个分子的轨迹。我们将尝试两种不同的方法来模拟该结构，首先是一种简单的方法，将其近似为一系列直线，然后是一种更精确的模型，将其拟合为曲线。样本结构的图像包含额外的信息，我们也将尝试提取这些信息。这是因为这些图像中的分子虽然密集堆积，但随着时间的推移，它们会在发光或不发光的状态之间切换。这些信息可以通过两种方式使用。首先，通过分析短序列图像并使用来自荧光团波动的信息来改进对荧光团位置的了解，以获得对样品结构的增强分辨率。第二，了解分子在细胞结构中的平均运动方式。这是通过比较不同帧之间图像补丁的亮度来实现的。这两种方法将允许我们更准确地可视化细胞结构，并将单个分子如何运动与结构中分子的平均运动方式进行比较。这种方法将使细胞生物学家能够跟踪快速运动结构中的单分子。单分子跟踪已经为那些可以使用它的结构产生了重要的结果，我们的工作将使这项技术适用于更广泛的细胞生物学问题。

Fluorescence microscopy is a crucial tool for cell biologists because it allows them to label different proteins with fluorescent molecules (fluorophores) and observe them in live cells. This yields information which can help us to understand diseases, and find new drugs to treat them. To understand how molecules in a cell behave, individual molecules labelled with fluorophores can be imaged and their speed measured. However, currently this can only be done on the membrane of cells, or on parts of the cell that stay very still. If the part of the cell which the molecule is attached to moves, it isn't possible to work out what is due to the movement of the molecule and what is due to the movement of the structure the molecule is attached to.We propose to solve this problem, so that tracking studies can be carried out in moving structures. To do this we will use the properties of photoswitchable fluorophores. These are fluorophores which can be switched between emitting light in two different colours. Normally only one colour is recorded, in which only a few fluorophores emit light. This means that individual molecules can be tracked. We want to develop a system to simultaneously image both of the emitting states. This will let us image the structure of the sample in one channel, and individual molecules in another. This can be done by illuminating the sample with two lasers simultaneously and splitting the emitted light across two sections of a camera chip, with the split being based on colour.To get useful information from this system, we will need to develop a new data analysis method. Both the positions of the single molecules and the structure of the sample will need to be fitted and tracked. The movement of the structure of the sample can then be used to correct the tracks of individual molecules. We will try two different methods to model the structure, first a simple method where it is approximated as a series of straight lines, and then a more accurate model where it is fitted as a curve.The images of the structure of the sample contain additional information which we will also try to extract. This is because the molecules in these images, while densely packed, are moving over time and switching between states in which they emit or don't emit light. This information can be used in two ways. First, to get enhanced resolution of the structure of the sample by analysing a short sequence of images and using information from fluorophore fluctuations to improve knowledge of the fluorophore positions. Second, to get information about how the molecules are moving around the cell structure on average. This is done by comparing the brightness of image patches between different frames. These two methods will allow us to visualise the cell structure more accurately, and to compare how individual molecules move to how the molecules in the structure are moving on average.This method would allow cell biologists to track single molecules in rapidly moving structures. Single molecule tracking has produced important results for those structures with which it can be used, and our work would make this technique applicable to a much wider range of cell biology problems.