Single Molecule Detection by Total Internal Reflection Fluorescence Microscopy
Date Issued
2004
Date
2004
Author(s)
Cheng, Shu-Ya
DOI
zh-TW
Abstract
Single molecule detection is a powerful and practical approach to explore bio-molecules, because it provides information about actual fluctuation and distribution of dynamic and kinetic parameters, enables relationship between inputs and outputs of events of proteins reaction to be qualified, and removes the need for synchronization. We use a laser of suitable wavelength to excite the selected fluorescent-labeled molecules, and then address the dynamic behaviors of target molecules by analysis and observation of the labeled molecules. This is namely single molecule fluorescence microscopy.
This work is mainly about the construction of a Prism-type Total Internal Reflection Fluorescence Microscopy system for the purpose of single molecule studies. The key of TIRFM is to reduce the background fluorescence effectively. When the light is totally reflected in the interface, the evanescent field is generated. The evanescent field is several times the intensity of the incident light, and diminishes exponentially with the distance form the interface. The penetration depth is quite narrow, around 100nm. Fluorescent-labeled molecules, which happen to be in this region, are excited selectively and emit fluorescence. The signal is collected by a high numerical aperture objective, and detected by CCD and APD. CCD is able to provide spatial information and extract statistics on numbers of individual molecules, while APD offers adequate temporal resolution. The signal pulses from one or double channels were counted and analyzed with time series.
The reporters that are chosen for ATP synthase are CyDye, including Cy3 and Cy5. We use a green laser (wavelength = 532nm) to pump the Cy3. The substrates are spin-coated with Cy3 in agarose solution of 99% water as a test specimen, with concentrations in the range of 10~100pM. Both the CCD visualization and the APD photon counting measurements demonstrate that the TIRFM does work and is capable of probing single molecules. The background fluorescence value in our experiments goes around 20 photo-electrons/sec and the SBR averages 13, 20 at most, and still could be enhanced. Before photo-bleaching, about 100,000∼1,000,000 photo-electrons were detected from each Cy3 molecule by APD.
Since the single molecule detection system has been built up successfully, we will carry on the investigation into the dynamic behaviors of bio-molecules and the study of the enzyme kinetic, such as fluorescence resonance energy transfer between Cy3 and Cy5, so as to elucidate the underlying mechanism of bio-motors from a chemist’s point of view.
Subjects
旋鍍
光子計數
漸逝場
ATP合成酶
全反射顯微螢光顯微術
單分子偵測
SMD
Cy3
spin coating
photon counting
TIRFM
evanescent field
single molecule detection
ATP synthase
Total Internal Reflection Fluorescence Microscopy
Type
thesis
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