Scanning total internal reflection fluorescence microscopy under one-photon and two-photon excitation: image formation.
Scanning total internal reflection fluorescence microscopy under one-photon and two-photon excitation: image formation.
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单光子和双光子激发下的扫描全内反射荧光显微镜：图像形成。
DOI：
10.1364/ao.43.001063
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发表时间：
2004
期刊：
影响因子：
1.9
通讯作者：
M. Gu
中科院分区：
文献类型：
--
作者：
J. Chon;M. Gu
我们提出了一种新型的全内反射荧光显微镜(TIRFM)，称为扫描TIRFM(STIRFM)，它使用了聚焦的环形光束照明和大数值孔径物镜(NA=1.65)。STIRFM产生的逝去场被横向聚焦，产生一个小的激发体积，可以诱导出双光子吸收等非线性效应。对CdSe量子点纳米晶和罗丹明6G掺杂微球的实验图像表明，在电流装置下获得了良好的横向和轴向分辨率。对STIRFM几何结构中产生的焦斑的理论模拟表明，由于大数值孔径物镜的退偏效应，聚焦的逝去光场分裂成两个峰。然而，单光子和双光子激发情况下的点扩散函数分析表明，分焦效应的检测依赖于检测针孔的大小。从理论上研究了针孔尺寸对成像的影响，并用纳米晶图像进行了实验验证。
We propose a new type of total internal reflection fluorescence microscopy (TIRFM) called scanning TIRFM (STIRFM) that uses a focused ring-beam illumination and a high-numerical-aperture objective (NA = 1.65). The evanescent field produced by the STIRFM is focused laterally, producing a small excitation volume that can induce a nonlinear effect such as two-photon absorption. Experimental images of CdSe quantum dot nanocrystals and Rhodamine 6G-doped microbeads show that good lateral and axial resolutions are achieved with the current setup. The theoretical simulation of the focal spot produced in STIRFM geometry shows that the focused evanescent field is split into two peaks because of the depolarization effect of a high numerical-aperture objective lens. However, the point-spread function analysis of both one-photon and two-photon excitation cases shows that the detection of the focus-splitting effect is dependent on the detection pinhole size. The effect of pinhole size on image formation is theoretically investigated and confirmed experimentally with the nanocrystal images.