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Photobleaching-based method for quantitatively measuring fluorescence resonance energy efficiency

A technique for quantitative measurement of fluorescence resonance energy, applied in measuring devices, fluorescence/phosphorescence, instruments, etc., can solve the problems of difficult separation of donors and acceptors, difficult quantitative analysis of fluorescence resonance energy transfer efficiency, complexity, etc.

Inactive Publication Date: 2009-11-18
SOUTH CHINA NORMAL UNIVERSITY
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Problems solved by technology

However, due to the influence of autofluorescence and spectral crosstalk, it is difficult to quantitatively analyze the fluorescence resonance energy transfer efficiency with fluorescence resonance energy imaging microscopy.
There are many methods for calculating FRET efficiency, Gordon [Gordon G W, Berry G, Liang XH, Levine B, Herman B. Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophysical Journal, 1998, 74: 2702-2713.] one A method for quantitatively calculating FRET efficiency that can correct emission crosstalk, using three different sets of filters, respectively measuring the fluorescence intensity of the separated donor and acceptor and the donor-acceptor pair in the three sets of filter sets, so The method is very complicated
In addition, this method is not suitable for immobilized donor-acceptor pairs because it is difficult to obtain separated donor and acceptor
Traditional receptor photobleaching methods need to bleach all receptors, which can cause great damage to living cells

Method used

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  • Photobleaching-based method for quantitatively measuring fluorescence resonance energy efficiency
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  • Photobleaching-based method for quantitatively measuring fluorescence resonance energy efficiency

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Embodiment Construction

[0049] The present invention will be further described below in conjunction with the accompanying drawings and examples, but the embodiments of the present invention are not limited thereto.

[0050] 1. Plasmid source: Plasmid SCAT3 was purchased from the Shanghai office of Merck, Germany.

[0051] 2. Laser confocal scanning fluorescence microscope (LSM510, Zeiss, Germany)

[0052] LSM510 is configured with three lasers, among which the argon ion laser produces laser light with wavelengths of 458, 477, 488, and 514nm, and two helium-neon lasers produce laser light of 543nm and 633nm respectively. It is equipped with objective lenses with magnifications of 10×, 20×, 63× and 100×, and a scanning module. A micro CO 2 Incubator, always keep the same environment throughout the measurement: 37 °C constant temperature, 5% CO 2 .

[0053] 3. Cell culture

[0054] Human lung adenocarcinoma cells (ASTC-a-1) were grown in DMEM medium containing 10% newborn bovine serum, 50units / ml p...

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Abstract

The invention relates to a method for measuring conversion efficiency of fluorescence resonance energy, in particular to a photobleaching-based method for quantitatively measuring fluorescence resonance energy efficiency. In the method, the fluorescence resonance energy efficiency is calculated by acquiring the average fluorescence intensity of a donor channel and donee channel before and after the photobleaching in living cells transfected with donor-donee pair with a laser confocal microscopy before and after the photobleaching of the donor. The method can effectively solve the problem that the living cells are damaged seriously by the conventional photobleaching method of donee due to the overlong time of photobleaching. By the method, FRET efficiency can be obtained quantitatively by only bleaching small part of donee so as to quantitatively monitor the interactions between the proteins of the living cells. Because of the short time only in the order of seconds, the method can reduce the damages to the living cells considerably.

Description

technical field [0001] The invention relates to a method for measuring fluorescence resonance energy transfer efficiency, in particular to a quantitative measurement method for fluorescence resonance energy efficiency based on photobleaching. Background technique [0002] Various fluorescence-based techniques have become important techniques for real-time detection of molecular regulatory mechanisms of cell signal transduction, apoptosis, and proliferation in living cells. Fluorescence spectroscopy is widely used in the analysis of chemical compounds and protein composition and structure, and is also widely used in the analysis of tumor characteristics, components of traditional Chinese medicine and the mechanism of action of traditional Chinese medicine. Fluorescence Resonance Energy Transfer (FRET) is a technique used to quantitatively measure the distance between two different luminescent groups. FRET is a process of transferring energy from the excited state of the dono...

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Application Information

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IPC IPC(8): G01N21/64G01N33/574
Inventor 陈同生王龙祥
Owner SOUTH CHINA NORMAL UNIVERSITY
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