Verification of 3D Resolution in Nanometer Range in Evanescent Field
Date Issued
2005
Date
2005
Author(s)
Li, Jiang-Yi
DOI
zh-TW
Abstract
Single-molecule detection (SMD) provides a way to observe and measure the behavior of single molecule directly. In the past ensemble measurements involving a large number of molecules, the differences between individual molecule are ignored. Therefore, SMD provides powerful tools to look into biomolecules. However, fluorescent microscopy is limited in its resolution by the Rayleigh criterion at ~250nm. On the other end of the size spectrum, fluorescence resonance energy transfer (FRET) provides a way to probe the events under 10nm. Distance measurements in the range of 10-250nm is the choke point of most techniques. Motivation of this thesis is originated from verifying the three-dimensional resolution in nanometer range of total internal reflection fluorescent microscopy (TIRFM) by using atomic force microscopy (AFM).
Fluorescent beads are affixed to cantilever tip through chemical modification and use property of the piezoscanner which can resolve displacement in nanometer range. The tip is moved by piezoscanner and the images are collected by CCD simultaneously. With comparison of data of AFM and TIRFM, the resolution of TIRFM can be confirmed.
To quantify horizontal displacement, this paper use Gaussian curve to fit the distribution of intensity profile of single fluorescent molecule. The center position of fluorescent molecule can correspond to the peak of fitting curve. The change of vertical distance can be determined from the change of intensity and penetration depth.
To show that TIRFM is able to observe and quantify signal transduction in cell or between cells, we observe the phenomenon that GFP adsorb on the coverslip. Distinct from other fluorescent techniques that only observe the dynamic motion in the transverse plane, TIRFM is able to observe the three-dimensional motion of single molecule in real time.
Quantum dot has a variety of features such as lifetime long as ten hours and only one wavelength for excitation. To make use of the characteristic of quantum dot and excellent resolution of TIRFM, it will be revolutionary of single-molecule detection in cell and between cells.
Subjects
漸逝場
解析度驗證
全反射螢光顯微術
evanescent wave
3D resolution
TIRFM
Type
thesis
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