Pralle A, Prummer M, Florin E L, Stelzer E H, Hörber J K
European Molecular Biology Laboratory, Heidelberg, Germany.
Microsc Res Tech. 1999 Mar 1;44(5):378-86. doi: 10.1002/(SICI)1097-0029(19990301)44:5<378::AID-JEMT10>3.0.CO;2-Z.
A quadrant photodiode placed in the back-focal plane of the microscope of a laser trap provides a high-resolution position sensor. We show that in addition to the lateral displacement of a trapped sphere, its axial position can be measured by the ratio of the intensity of scattered laser light to the total amount of the light reaching the detector. The addition of the axial information offers true three-dimensional position detection in solution, creating, together with a position control, a photonic force microscope with nanometer spatial and microsecond temporal resolution. The measured position signals are explained as interference of the unscattered trapping laser beam with the laser light scattered by the trapped bead. Our model explains experimental data for trapped particles in the Rayleigh regime (radius a <0.2lambda) for displacements up to the focal dimensions. The cross-talk between the signals in the three directions is explained and it is shown that this cross-talk can be neglected for lateral displacements smaller than 75 nm and axial displacements below 150 nm. The advantages of three-dimensional single-particle tracking over conventional video-tracking are shown through the example of the diffusion of the GPI-anchored membrane protein Thy1.1 on a neurite.
置于激光阱显微镜后焦平面的象限光电二极管可提供高分辨率位置传感器。我们表明，除了被捕获球体的横向位移外，其轴向位置可通过散射激光强度与到达探测器的总光量之比来测量。轴向信息的加入可在溶液中实现真正的三维位置检测，并与位置控制一起创建具有纳米空间分辨率和微秒时间分辨率的光子力显微镜。所测量的位置信号被解释为未散射的捕获激光束与被捕获珠子散射的激光之间的干涉。我们的模型解释了处于瑞利区域（半径a <0.2λ）的捕获粒子在直至焦域尺寸位移情况下的实验数据。解释了三个方向信号之间的串扰，并表明对于小于75 nm的横向位移和低于150 nm的轴向位移，这种串扰可忽略不计。通过糖基磷脂酰肌醇（GPI）锚定膜蛋白Thy1.1在神经突上扩散的例子，展示了三维单粒子跟踪相对于传统视频跟踪的优势。
