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Development of Super Sensitive Position Detector and Its Application on the Studies of Plasma Membrane Proteins

超灵敏位置探测器的研制及其在质膜蛋白研究中的应用

基本信息

批准号：
08558075
负责人：
SAKO Yasushi
金额：
$ 5.7万
依托单位：
Osaka University (1997)The University of Tokyo (1996)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

A new-type super-sensitive position detector was developed using a quadrant avalanche photodiode (4APD) as the photo sensor. This position detector was interfaced to a newly-developed reflective polarisation optical microscope with a near-JR (810 nm) external cavity diode Laser as the light source, In this system, temperature and voltage of 4APD can be optimised to achieve maximum S/N ratio.Using colloidal gold particles with a diameter of 10-100 nm as standard samples, 0.5-2 V output signal with>100 S/N was obtained. At this range of signal intensity, bandwidth of position measurement was 0-17 MHz (-3dB).Spatial resolution of the system was examined by moving the standard sample by a PZT stage. At 375x magnification of the microscope, spatial resolution better than 3 nm was achieved. At this time, the spatial resolution is determined by a fluctuation of the power supply, and using more stable power supply, it should be improved to more than 1 nm. Movement within a 200 nm-phi area can be traced.Using this system, lateral movement of plasma membrane proteins were measured. Although membrane proteins which is bound to the cytoskeleton was undetectable in 33 ms video-rate time window, rapid and Browninan diffusion movement restricted within an area of 300-500 nm was observed by more than 1 ms time resolution of the newly developed position detector. Such restricted movement of cytoskeleton-bound proteins suggests flexibility of the cytoskeletal filaments.

采用象限雪崩光电二极管（4APD）作为光传感器，研制了一种新型超灵敏位置探测器。该位置检测器以近jr （810 nm）外腔二极管激光器为光源，与新研制的反射偏光光学显微镜对接，通过优化4APD的温度和电压，实现最大信噪比。以直径为10-100 nm的胶体金颗粒为标准样品，获得0.5-2 V的输出信号，输出功率为>100 S/N。在此信号强度范围内，位置测量带宽为0-17 MHz （-3dB）。通过PZT平台移动标准样品来检测系统的空间分辨率。在375倍放大显微镜下，空间分辨率优于3 nm。此时的空间分辨率是由电源的波动决定的，使用更稳定的电源，应该提高到1nm以上。可以追踪200 nm-phi区域内的运动。利用该系统，测量了质膜蛋白的横向运动。虽然在33 ms视频速率时间窗内无法检测到与细胞骨架结合的膜蛋白，但新开发的位置检测器在超过1 ms的时间分辨率下，可以观察到限制在300-500 nm区域内的快速布朗尼曼扩散运动。细胞骨架结合蛋白的这种受限运动表明细胞骨架细丝具有柔韧性。