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Method for measuring three-way fluorescent resonance energy displace microscope G parameter

A technology of fluorescence resonance energy and measurement method, which is applied in the cross field of optoelectronics, biotechnology and life science, and can solve the problems of difficult imaging, weak fluorescence and large error

Inactive Publication Date: 2007-09-26
SHANGHAI INST OF OPTICS & FINE MECHANICS CHINESE ACAD OF SCI
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Problems solved by technology

[0013] 1. Cells need to be fixed. The luminescent properties of CFP and YFP fluorescent proteins in a fixed cell environment and a living cell environment may be different, so the G value measured in a fixed cell environment may not represent the G value in a living cell environment.
[0014] 2. The method of partially bleaching the receptor generally requires a long time for the fluorescence bleaching of the receptor, so that the fluorescence of the receptor will become very weak, and it is not easy to image in a three-channel FRET microscope, and the signal-to-noise ratio of the imaging is also very low , and then calculate the G parameter, the calculation error will be very large

Method used

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  • Method for measuring three-way fluorescent resonance energy displace microscope G parameter
  • Method for measuring three-way fluorescent resonance energy displace microscope G parameter

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Embodiment Construction

[0065] Plasmid:

[0066] The CFP-YFP fusion expression plasmid was donated by Professor Luo Jianhong from the Department of Neurobiology, Zhejiang University School of Medicine.

[0067] Cell culture and transfection:

[0068] CHO cells were cultured in RPMI1640 medium containing 10% calf serum in 5% CO 2 in a 37°C incubator. The experiment adopted the method of transient transfection, and the CHO cells were transfected when they grew to 80-90% confluence, and 0.5 μg of CFP-YFP plasmid and 1 μl of LipofectAMINE2000 (GIBCO) were added to each well of a 12-well plate, specifically For the experimental method, refer to the instruction manual of LipofectAMINE 2000.

[0069] Three-channel FRET microscope:

[0070] Imaging was achieved using an Olympus IX71 inverted microscope equipped with a 40x objective lens and a Cascade512F (Roper) CCD. This microscope is equipped with three imaging channels, which are CFP channel (excitation, 440 / 21nm; emission, 480 / 30nm), YFP channel (ex...

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Abstract

This invention relates to one three-channel fluorescence calibration energy transfer microscope G parameter test method, which comprises the following steps: a, testing three-channel fluorescence vibration energy transfer microscope filter parameters; b using three channel vibration energy to transfer microscope to test CFP-YFP protein even intensity in the YFP and FRET channel; c, computing the G parameter. This invention measures the G parameters with small standard error with good accuracy.

Description

technical field [0001] The invention belongs to the cross field of optoelectronics, biotechnology and life science. It is a method for measuring the G parameters of a three-channel fluorescence resonance energy transfer microscope. Background technique [0002] Fluorescence resonance energy transfer (FRET for short) is a non-radiative energy transfer process, during which the fluorophore (donor) in the excited state transfers The process by which energy is transferred to another molecule (the acceptor), resulting in the quenching of the donor's fluorescence and the enhancement of the acceptor's luminescence. See [G.W.Gordon, G.Berry, X.H.Liang, B.Levine, B.Herman, Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys. J.74(1998) 2702-2713]. In the process of FRET research, there are two main measures of FRET: apparent FRET efficiency and FRET index. For the calculation of apparent FRET efficiency, the method of receptor bl...

Claims

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Application Information

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IPC IPC(8): G01N21/64G06F19/00
Inventor 付国王桂英徐至展
Owner SHANGHAI INST OF OPTICS & FINE MECHANICS CHINESE ACAD OF SCI
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