JP2007232559A5

JP2007232559A5 -

Info

Publication number: JP2007232559A5
Application number: JP2006054347A
Authority: JP
Filing date: 2006-03-01
Publication date: 2009-09-03
Anticipated expiration: 2026-03-01

Claims

A FRET detection method for detecting FRET (Fluorescence Resonance Energy Transfer) in which energy of a first molecule excited by laser light irradiation moves to a second molecule,
In order to excite the first molecule, the first molecule is irradiated with the first laser light whose intensity is modulated at the first frequency, and the second molecule is excited to be different from the first frequency. Irradiating the second molecule with a second laser beam modulated in intensity at a frequency of two;
Receiving the fluorescence emitted by the second molecule;
The first phase delay of the signal component of the first frequency in the fluorescence signal of the fluorescence emitted from the received second molecule with respect to the intensity modulation of the first laser beam, and the received second molecule emits. A second phase lag of the signal component of the second frequency in the fluorescence signal of the fluorescence with respect to the intensity modulation of the second laser light is extracted, and based on the first phase lag and the second phase lag. Te, FRET detection method characterized by comprising the steps of determining the presence or absence of energy transfer the energy of the first molecule is moved to a second molecule, the.

The FRET detection method according to claim 1, wherein the energy transfer is determined by a ratio of the first phase lag to the second phase lag.

The first laser beam is intensity-modulated using a first modulation signal having a first frequency for intensity modulation of the first laser beam, and the second laser beam is the second laser beam Intensity modulation is performed using a second modulation signal having a second frequency for intensity modulation of laser light, and the modulation signal of the first frequency is converted into the modulation signal of the second frequency by the difference frequency. It is a signal obtained by synthesizing the generated signal,
3. The FRET according to claim 1, wherein a fluorescence signal of fluorescence emitted from the received second molecule is sampled with a frequency that is an integral multiple of the difference frequency as a sampling frequency in synchronization with the generation signal of the difference frequency. Detection method.

The sampled fluorescence signal is subjected to frequency analysis with a frequency that is a fraction of an integer of the difference frequency as a frequency resolution, whereby the first frequency component of the fluorescence signal and the first of the fluorescence signals. 4. The FRET detection method according to claim 3, wherein a signal component having a frequency of 2 is extracted.

When receiving the fluorescence emitted by the second molecule, further receiving the fluorescence emitted by the first molecule;
A third phase lag with respect to the intensity modulation of the first laser light of the fluorescence signal emitted from the first molecule received is calculated, and the third phase lag is used to determine whether or not FRET has occurred. Item 5. The FRET detection method according to any one of Items 1 to 4.

A FRET detection device that detects FRET in which energy of a first molecule excited by laser light irradiation moves to a second molecule,
A laser light source that irradiates the first molecule with the first laser beam to excite the first molecule and irradiates the second molecule with the second laser beam to excite the second molecule. And
A light receiving portion for receiving fluorescence emitted by the second molecule;
The intensity of the first laser beam emitted from the laser light source unit is modulated at a first frequency, and the intensity of the second laser beam emitted from the laser light source unit is modulated at a second frequency different from the first frequency. A light source control unit that generates a modulation signal;
The first phase delay of the signal component of the first frequency in the fluorescence signal of the fluorescence emitted from the received second molecule with respect to the intensity modulation of the first laser beam, and the received second molecule emits. A second phase lag of the signal component of the second frequency in the fluorescence signal of the fluorescence with respect to the intensity modulation of the second laser light is extracted, and based on the first phase lag and the second phase lag. Te, FRET detection device, characterized in that it comprises a processing unit for energy of the first molecule to determine the presence or absence of energy transfer to move to a second molecule, the.

Wherein the processing unit, FRET detection device according to claim 6 to determine the energy transfer by the ratio of the first phase difference with respect to the second phase difference.

The light source control unit modulates the intensity of the first laser light using a first modulation signal having a first frequency for intensity modulation of the first laser light, and the second laser light. Is intensity-modulated using a second modulation signal having a second frequency for intensity modulation of the second laser beam, and the modulation signal of the first frequency is the modulation signal of the second frequency And a signal obtained by synthesizing the generated signal of the difference frequency,
The said processing part samples the fluorescence signal of the fluorescence which the said 2nd molecule | numerator received light synchronizes with the production | generation signal of the said difference frequency as a sampling frequency using the frequency of the integral multiple of the said difference frequency. The FRET detection device according to 1.

The processing unit performs frequency analysis on the sampled fluorescence signal using a frequency that is a fraction of the difference frequency as an integer, so that the signal component of the first frequency of the fluorescence signal and the fluorescence The FRET detection apparatus according to claim 8, wherein a signal component of the second frequency is extracted from a signal.

The light receiving unit receives the fluorescence emitted by the first molecule in addition to the fluorescence emitted by the second molecule,
The processing unit calculates a third phase delay of the fluorescence signal of the fluorescence emitted by the first molecule received by the light receiving unit with respect to the intensity modulation of the first laser beam, and calculates the third phase delay of the FRET. The FRET detection apparatus according to any one of claims 6 to 9, which is used to determine whether or not the occurrence has occurred .