JP2007240424A5 - - Google Patents
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- JP2007240424A5 JP2007240424A5 JP2006065742A JP2006065742A JP2007240424A5 JP 2007240424 A5 JP2007240424 A5 JP 2007240424A5 JP 2006065742 A JP2006065742 A JP 2006065742A JP 2006065742 A JP2006065742 A JP 2006065742A JP 2007240424 A5 JP2007240424 A5 JP 2007240424A5
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- 238000002866 fluorescence resonance energy transfer Methods 0.000 claims 98
- 239000000523 sample Substances 0.000 claims 87
- 238000001514 detection method Methods 0.000 claims 66
- 238000003260 fluorescence intensity Methods 0.000 claims 66
- 238000005259 measurement Methods 0.000 claims 19
- 230000001678 irradiating Effects 0.000 claims 13
- 230000005284 excitation Effects 0.000 claims 7
- 238000004364 calculation method Methods 0.000 claims 5
- 230000004044 response Effects 0.000 claims 4
- 230000000051 modifying Effects 0.000 claims 3
- 238000002165 resonance energy transfer Methods 0.000 claims 2
- 238000009795 derivation Methods 0.000 claims 1
- 230000002123 temporal effect Effects 0.000 claims 1
Claims (22)
レーザ光の強度を所定の周波数で時間変調して前記測定対象サンプルに照射し、このときの前記測定対象サンプルの発する蛍光を受光波長帯域の異なる複数の検出センサで受光することにより、前記測定対象サンプルの蛍光の蛍光強度情報および位相情報を含む検出値を収集するステップと、
予め記憶手段に記憶されている情報であって、前記測定対象サンプルの前記蛍光のうち前記第１分子が発する第１分子蛍光成分の、前記受光波長帯域における蛍光強度の比を表す第１の強度比率と、前記レーザ光の時間変調に対する前記第１分子蛍光成分の位相情報と、前記蛍光のうち前記第２分子が発する第２分子蛍光成分の、前記受光波長帯域の蛍光強度の比を表す第２の強度比率と、前記レーザ光の時間変調に対する前記第２分子蛍光成分の位相情報と、レーザ光によって励起された第１分子が発する蛍光が１次遅れ系の緩和応答であるとしたとき定義される寿命であって前記ＦＲＥＴが発生しない状態における前記第１分子蛍光成分の非ＦＲＥＴ蛍光寿命と、を少なくとも含むキャリブレーション情報を読み出して取得するステップと、
各検出センサから収集された前記検出値から、前記受光波長帯域毎に、前記測定対象サンプルの蛍光の蛍光強度情報および位相情報を求めて、求めた前記蛍光強度情報および前記位相情報と、前記第１の強度比率と、前記第１分子蛍光成分の前記位相情報と、前記第２の強度比率と、前記第２分子蛍光成分の前記位相情報と、を用いて、レーザ光によって励起された第１分子が発する蛍光が１次遅れ系の緩和応答であるとしたとき定義される前記第１分子蛍光成分のＦＲＥＴ蛍光寿命を求めるステップと、
前記第１分子蛍光成分のＦＲＥＴ蛍光寿命と、前記第１分子蛍光成分の前記非ＦＲＥＴ蛍光寿命との比を用いてＦＲＥＴ発生情報を求めるステップと、を有することを特徴とするＦＲＥＴ検出方法。 FRET (Fluorescence) in which the energy of the first molecule is transferred to the second molecule by irradiating the measurement target sample labeled with the first molecule and the second molecule with laser light and receiving the fluorescence emitted from the measurement target sample at this time A FRET detection method for detecting (Resonance Energy Transfer),
The intensity of the laser beam is time-modulated at a predetermined frequency to irradiate the sample to be measured, and the fluorescence emitted from the sample to be measured at this time is received by a plurality of detection sensors having different light receiving wavelength bands, thereby the object to be measured Collecting detected values including fluorescence intensity information and phase information of the fluorescence of the sample;
A information stored in advance in the storage unit, a first intensity representing the first molecule fluorescence component emitted by the first molecule of the fluorescence, the ratio of the fluorescence intensity at the light receiving wavelength range of the measurement target sample The ratio , the phase information of the first molecule fluorescence component with respect to the time modulation of the laser light, and the ratio of the fluorescence intensity in the light receiving wavelength band of the second molecule fluorescence component emitted by the second molecule of the fluorescence . Defined when the intensity ratio of 2, the phase information of the second molecular fluorescent component with respect to the time modulation of the laser beam, and the fluorescence emitted by the first molecule excited by the laser beam are relaxation responses of the first-order lag system obtaining by reading the calibration information including non-FRET fluorescence lifetime of the first molecule fluorescence component in a state in which the FRET does not occur a lifetime that is at least ,
From the detection values collected from each detection sensor, the fluorescence intensity information and the phase information of the fluorescence of the measurement target sample are obtained for each of the light reception wavelength bands, the obtained fluorescence intensity information and the phase information, and the first The first intensity excited by laser light using the intensity ratio of 1, the phase information of the first molecular fluorescent component, the second intensity ratio, and the phase information of the second molecular fluorescent component. Determining the FRET fluorescence lifetime of the first molecule fluorescence component defined when the fluorescence emitted by the molecule is a first order lag relaxation response;
FRET generation information is obtained by using the ratio of the FRET fluorescence lifetime of the first molecule fluorescence component to the non-FRET fluorescence lifetime of the first molecule fluorescence component.
前記ＦＲＥＴ発生情報として、１−（τd *／τd）で表されるＦＲＥＴ効率Ｅtを求める請求項１に記載のＦＲＥＴ検出方法。 Wherein in the step of determining a FRET occurrence information, the non-FRET fluorescence lifetime tau d, wherein when the the FRET fluorescence lifetime tau d *,
The FRET as occurrence information, FRET detection method according to claim 1 for obtaining a FRET efficiency E t represented by 1- (τ d * / τ d ).
前記ＦＲＥＴ蛍光寿命を求めるステップでは、各検出値に基づいて、前記測定対象サンプルそれぞれの蛍光強度情報および位相情報を求め、求めた複数の蛍光強度情報および位相情報から前記ＦＲＥＴ蛍光寿命を求める請求項１または２に記載のＦＲＥＴ検出方法。 In the step of collecting the detection value, irradiating the laser light into a plurality of the measuring sample, collects the detection values for the plurality of the measurement target sample, respectively in the sensor,
The step of obtaining the FRET fluorescence lifetime includes obtaining fluorescence intensity information and phase information of each measurement target sample based on each detection value, and obtaining the FRET fluorescence lifetime from the obtained plurality of fluorescence intensity information and phase information. 3. The FRET detection method according to 1 or 2.
前記ＦＲＥＴ蛍光寿命を求めるステップでは、
前記第１波長帯域の前記検出センサにて収集される検出値によって表される第１波長帯域における前記ベクトルと前記第２の強度比率とを少なくとも用いて、前記ＦＲＥＴが生じた状態における前記第１分子蛍光成分の蛍光強度情報および位相情報を求め、
前記第２波長帯域の前記検出センサにより収集される検出値によって表される第２波長帯域における前記ベクトルと前記第１の強度比率とを少なくとも用いて、前記ＦＲＥＴ成分の蛍光強度情報および位相情報を求める請求項４に記載のＦＲＥＴ検出方法。 The light receiving wavelength band is centered on a first wavelength band centered on a peak wavelength at which the fluorescence intensity of the first molecule fluorescent component is maximized and a peak wavelength at which the fluorescence intensity of the second molecule fluorescence component is maximized. and a second wavelength band,
In the step of obtaining the FRET fluorescence lifetime,
The first in a state where the FRET has occurred using at least the vector in the first wavelength band represented by the detection value collected by the detection sensor in the first wavelength band and the second intensity ratio . Obtain fluorescence intensity information and phase information of molecular fluorescent components,
Using at least the vector in the second wavelength band represented by the detection value collected by the detection sensor in the second wavelength band and the first intensity ratio, the fluorescence intensity information and phase information of the FRET component The FRET detection method according to claim 4 to be obtained.
前記キャリブレーション情報を取得するステップでは、前記記憶手段に記憶されている前記直接励起蛍光成分の蛍光強度情報および位相情報を呼び出して、直接励起蛍光成分の情報をベクトルで表し、
前記ＦＲＥＴ蛍光寿命を求めるステップでは、前記直接励起蛍光成分の前記ベクトルと、前記第２波長帯域における前記ベクトルとを少なくとも用いて、前記ＦＲＥＴが生じた状態における前記第２分子蛍光成分の蛍光強度情報および位相情報を求める請求項５に記載のＦＲＥＴ検出方法。 The storage means stores in advance fluorescence intensity information and phase information of a directly excited fluorescence component generated by the second molecule being directly excited by the laser light in the second molecule fluorescence component,
In the step of acquiring the calibration information, the fluorescence intensity information and the phase information of the directly excited fluorescence component stored in the storage unit are called, and the information of the directly excited fluorescence component is represented by a vector,
Wherein in the step of determining a FRET fluorescence lifetime, and the vector of the direct excitation fluorescence component, with at least the said vector in said second wavelength band, the fluorescence intensity information of the second molecule fluorescence component in a state in which the FRET occurs The FRET detection method according to claim 5, wherein the phase information is obtained.
前記キャリブレーション情報を取得するステップでは、前記第２分子蛍光成分のうち、前記レーザ光によって第２分子が直接励起されることで発生する直接励起蛍光成分の蛍光強度情報および位相情報を呼び出して、前記直接励起蛍光成分のベクトルで表し、
前記ＦＲＥＴ蛍光寿命を求めるステップでは、前記第１分子蛍光成分のＦＲＥＴ寿命を前記キャリブレーション情報を用いて求めるのに加えて、前記第２波長帯域における前記ベクトルと、前記第１の強度比率とを少なくとも用いて前記ＦＲＥＴ成分の位相情報を求め、
求めた前記ＦＲＥＴ成分の位相情報と前記直接励起蛍光成分の前記ベクトルとを用いて、前記ＦＲＥＴが生じた状態における前記第２分子蛍光成分のＦＲＥＴ蛍光寿命と、前記ＦＲＥＴが生じない状態における前記第２分子蛍光成分の非ＦＲＥＴ蛍光寿命とを求め、
前記第２分子蛍光成分のＦＲＥＴ蛍光寿命と、前記第２分子蛍光成分の前記非ＦＲＥＴ蛍光寿命とを用いて、前記第１分子蛍光成分のＦＲＥＴ蛍光寿命を求める請求項５に記載のＦＲＥＴ検出方法。 The storage means stores in advance fluorescence intensity information and phase information of a directly excited fluorescence component generated by the second molecule being directly excited by the laser light in the second molecule fluorescence component,
In the step of acquiring the calibration information, the fluorescence intensity information and the phase information of the directly excited fluorescence component generated by the second molecule being directly excited by the laser light among the second molecule fluorescence components are called up , It represents a vector of the direct excitation fluorescence component,
In the step of obtaining the FRET fluorescence lifetime, in addition to obtaining the FRET lifetime of the first molecular fluorescence component using the calibration information, the vector in the second wavelength band and the first intensity ratio are obtained. Using at least the phase information of the FRET component,
Using the phase information of the FRET components obtained with the said vector of the direct excitation fluorescence component, and FRET fluorescence lifetime of the second molecule fluorescence component in a state in which the FRET occurs, the in state in which the FRET does not occur first Obtaining the non-FRET fluorescence lifetime of the bimolecular fluorescent component,
6. The FRET detection method according to claim 5, wherein the FRET fluorescence lifetime of the first molecule fluorescence component is obtained using the FRET fluorescence lifetime of the second molecule fluorescence component and the non-FRET fluorescence lifetime of the second molecule fluorescence component. .
前記記憶手段には、前記自家蛍光サンプル体に前記レーザ光を照射することにより発する前記自家蛍光サンプル体の蛍光の、前記受光波長帯域毎の蛍光強度情報および位相情報が予め記憶されており、
前記キャリブレーション情報を取得するステップでは、前記記憶手段に記憶されている、前記自家蛍光サンプル体の蛍光の前記受光波長帯域毎の蛍光強度情報および位相情報を呼び出して前記自家蛍光サンプル体の蛍光をベクトルで表し、
前記ＦＲＥＴ蛍光寿命を求めるステップでは、前記受光波長帯域毎の前記測定対象サンプルの前記ベクトルのそれぞれから、前記自家蛍光サンプル体の蛍光の前記ベクトルを減算し、この減算で得られたベクトルを用いて、前記ＦＲＥＴ蛍光寿命を求める請求項４〜７のいずれかに記載のＦＲＥＴ検出方法。 The sample to be measured is a sample labeled with the first molecule and the second molecule on an autofluorescent sample body that emits autofluorescence when excited by the laser beam,
In the storage means, fluorescence intensity information and phase information for each light receiving wavelength band of fluorescence of the autofluorescence sample body emitted by irradiating the autofluorescence sample body with the laser light is stored in advance,
In the step of obtaining the calibration information, the fluorescence intensity information and the phase information for each light receiving wavelength band of the fluorescence of the autofluorescence sample body, which is stored in the storage means, are called to obtain the fluorescence of the autofluorescence sample body . Represented as a vector ,
In the step of determining the FRET fluorescence lifetime, from said each of said vector of said measuring sample of each photosensitive wavelength range, said subtracting the vector of fluorescence autofluorescence sample material, using a vector obtained by the subtraction The FRET detection method according to claim 4, wherein the FRET fluorescence lifetime is obtained.
前記自家蛍光キャリブレーションでは、
前記自家蛍光サンプル体を測定対象物として所定の周波数で時間変調したレーザ光を照射することにより、各検出センサから、前記受光波長帯域毎の蛍光強度情報および位相情報を含む検出値を収集して、前記自家蛍光サンプル体の蛍光の、前記受光波長帯域毎の蛍光強度情報および位相情報を求め、求めた情報を前記記憶手段に記憶する請求項８に記載のＦＲＥＴ検出方法。 In the step of acquiring the calibration information, the information obtained by autofluorescence calibration is called from the storage means and acquired,
In the autofluorescence calibration,
By irradiating laser light that is time-modulated at a predetermined frequency with the autofluorescence sample body as a measurement object, detection values including fluorescence intensity information and phase information for each light reception wavelength band are collected from each detection sensor. 9. The FRET detection method according to claim 8, wherein fluorescence intensity information and phase information of the fluorescence of the autofluorescence sample body for each light receiving wavelength band are obtained, and the obtained information is stored in the storage means.
前記ｎｏｎ−ＦＲＥＴキャリブレーションでは、
前記第１分子および前記第２分子がサンプルに付着されて、ＦＲＥＴが生じない処理がなされたｎｏｎ−ＦＲＥＴサンプルを測定対象物として所定の周波数で時間変調したレーザ光を照射することにより、各検出センサから、前記受光波長帯域毎の蛍光強度情報および位相情報を含む検出値を収集して、前記ｎｏｎ−ＦＲＥＴサンプルの蛍光の、前記受光波長帯域毎の蛍光強度情報および位相情報を求め、
求めた前記受光波長帯域毎の蛍光強度情報および位相情報と、前記記憶手段に予め記憶された前記第１の強度比率と、前記第１分子蛍光成分の位相情報と、前記記憶手段に予め記憶された前記第２の強度比率と、前記第２分子蛍光成分の位相情報と、を用いて、レーザ光によって第２分子が直接励起される直接励起蛍光成分の蛍光強度情報および位相情報を求め、求めた情報を前記記憶手段に記憶する請求項１〜９のいずれかに記載のＦＲＥＴ検出方法。 In the step of acquiring the calibration information, the information obtained by non-FRET calibration is retrieved from the storage means and acquired.
In the non-FRET calibration,
Each detection is performed by irradiating the first-molecule and the second molecule attached to the sample and irradiating the non-FRET sample, which has been processed so as not to generate FRET, with time-modulated laser light at a predetermined frequency. from the sensor, said by collecting detected values including the fluorescence intensity information and phase information of each light-receiving wavelength band, of the fluorescence of the non-FRET sample, determine the fluorescence intensity information and phase information of each of the light receiving wavelength bands,
The obtained fluorescence intensity information and phase information for each received wavelength band, the first intensity ratio stored in advance in the storage means, the phase information of the first molecular fluorescence component, and stored in the storage means in advance. said second intensity ratio was using, and phase information of the second molecule fluorescence component, determine the fluorescence intensity information and phase information of the direct excitation fluorescence component second molecule is excited directly by the laser beam, determined The FRET detection method according to claim 1, wherein the stored information is stored in the storage unit.
前記キャリブレーション情報を取得するステップでは、自家蛍光キャリブレーションによって得られた情報を前記記憶手段から呼び出して取得し、
前記自家蛍光キャリブレーションは、前記ｎｏｎ−ＦＲＥＴキャリブレーションに先がけて行われるキャリブレーションであって、前記自家蛍光サンプル体を測定対象物として所定の周波数で時間変調したレーザ光を照射することにより、各検出センサから、前記受光波長帯域毎の蛍光強度情報および位相情報を含む検出値を収集して、前記自家蛍光サンプル体の蛍光の、前記受光波長帯域毎の蛍光強度情報および位相情報を求め、求めた情報を前記記憶手段に記憶し、
前記ｎｏｎ−ＦＲＥＴキャリブレーションでは、前記ｎｏｎ−ＦＲＥＴサンプルの蛍光の、前記受光波長帯域毎の蛍光強度情報および位相情報をベクトルで表したｎｏｎ−ＦＲＥＴサンプルベクトルから、前記自家蛍光キャリブレーションで求められた前記自家蛍光の情報をベクトルで表した自家蛍光ベクトルを減算し、この減算で得られたベクトルと、前記記憶手段に予め記憶された前記第１の強度比率と、前記第１分子蛍光成分の位相情報と、前記記憶手段に予め記憶された前記第２の強度比率と、前記第２分子蛍光成分の位相情報と、を用いて前記直接励起蛍光成分ベクトルを導出し、導出した結果を前記記憶手段に記憶する請求項１０に記載のＦＲＥＴ検出方法。 The non-FRET sample is a sample labeled with the first molecule and the second molecule on an autofluorescent sample body that emits autofluorescence when excited by the laser beam,
In the step of acquiring the calibration information, the information obtained by autofluorescence calibration is called from the storage means and acquired,
The autofluorescence calibration is a calibration performed prior to the non-FRET calibration, and each of the autofluorescence calibrations is irradiated with laser light that is time-modulated at a predetermined frequency using the autofluorescence sample body as a measurement object. from the detection sensor, collects the detection values including fluorescence intensity information and phase information of each of the light receiving wavelength band, of the fluorescence of the autofluorescence sample material, determine the fluorescence intensity information and phase information of each of the light receiving wavelength band, determined Information stored in the storage means,
In the non-FRET calibration , the auto-fluorescence calibration was obtained from the non-FRET sample vector in which the fluorescence intensity information and phase information for each light receiving wavelength band of the fluorescence of the non-FRET sample were expressed as vectors . Subtracting the autofluorescence vector representing the autofluorescence information as a vector, the vector obtained by this subtraction, the first intensity ratio stored in advance in the storage means, and the phase of the first molecular fluorescence component information and the previously stored second intensity ratio in the memory means, the second and the phase information of the molecule fluorescence component, and deriving the direct excitation fluorescence component vector using said storage means the derived results The FRET detection method according to claim 10, wherein the FRET detection method is stored .
前記第１分子キャリブレーションでは、
前記第１分子のみで標識された第１分子サンプルを測定対象物として所定の周波数で時間変調したレーザ光を照射することにより、各検出センサから、前記第１分子サンプルの蛍光の、前記受光波長帯域毎の蛍光強度情報および位相情報を含む検出値を収集して、前記第１分子サンプルの蛍光の、前記受光波長帯域毎の蛍光強度情報および位相情報を求め、
前記第１の強度比率として、前記第１分子サンプルの、前記受光波長帯域の蛍光強度の比を求め、
求めた前記第１の強度比率と求めた前記位相情報とを前記記憶手段に記憶する請求項１〜１１のいずれかに記載のFRET検出方法。 In the step of obtaining the calibration information, the information obtained by the first molecule calibration is retrieved from the storage means and obtained,
In the first molecule calibration,
By irradiating a first molecular sample labeled only with the first molecule with a laser beam time-modulated at a predetermined frequency using the first molecular sample as a measurement object, the light receiving wavelength of the fluorescence of the first molecular sample is detected from each detection sensor. Collecting detection values including fluorescence intensity information and phase information for each band, and obtaining fluorescence intensity information and phase information for each light receiving wavelength band of the fluorescence of the first molecule sample,
As the first intensity ratio , the ratio of the fluorescence intensity of the light reception wavelength band of the first molecular sample is obtained,
FRET detection method according to any one of claims 1 to 11 for storing said phase information obtained from the first intensity ratio obtained in the memory means.
前記キャリブレーション情報を取得するステップでは、自家蛍光キャリブレーションによって得られた情報を前記記憶手段から呼び出して取得し、
前記自家蛍光キャリブレーションは、前記第１分子キャリブレーションに先がけて行われるキャリブレーションであって、前記自家蛍光サンプル体を測定対象物として所定の周波数で時間変調したレーザ光を照射することにより、各検出センサから、前記受光波長帯域毎に蛍光強度情報および位相情報を含む検出値を収集して、前記自家蛍光サンプル体の蛍光の、前記受光波長帯域毎の蛍光強度情報および位相情報を求め、求めた情報を前記記憶手段に記憶し、
前記第１分子キャリブレーションでは、前記第１分子サンプルの蛍光の、前記受光波長帯域毎の蛍光強度情報および位相情報をベクトルで表した第１分子サンプルベクトルから、前記自家蛍光キャリブレーションで求められた前記自家蛍光サンプル体の蛍光の情報をベクトルで表した自家蛍光ベクトルを減算し、この減算で得られたベクトルを用いて、前記第１分子サンプルの蛍光の、前記受光波長帯域毎の蛍光強度情報および位相情報を求め、求めた情報を前記記憶手段に記憶する請求項１２に記載のＦＲＥＴ検出方法。 The first molecule sample is a sample labeled with the first molecule on an autofluorescence sample body that emits autofluorescence when excited by the laser beam,
In the step of acquiring the calibration information, the information obtained by autofluorescence calibration is called from the storage means and acquired,
The autofluorescence calibration is a calibration performed prior to the first molecule calibration, and each autoirradiation sample is irradiated with a laser beam time-modulated at a predetermined frequency with the autofluorescence sample body as a measurement object. By collecting detection values including fluorescence intensity information and phase information for each light reception wavelength band from a detection sensor , the fluorescence intensity information and phase information for each light reception wavelength band of the fluorescence of the autofluorescence sample body is obtained and obtained. Information stored in the storage means,
In the first molecule calibration, the fluorescence of the first molecule sample was obtained by the autofluorescence calibration from the first molecule sample vector representing the fluorescence intensity information and the phase information for each light receiving wavelength band as vectors . Subtracting the autofluorescence vector representing the fluorescence information of the autofluorescence sample body as a vector, and using the vector obtained by this subtraction, fluorescence intensity information of the fluorescence of the first molecule sample for each light receiving wavelength band 13. The FRET detection method according to claim 12, wherein phase information is obtained and the obtained information is stored in the storage means .
前記第２分子キャリブレーションでは、
前記第２分子のみで標識された第２分子サンプルを測定対象物として所定の周波数で時間変調したレーザ光を照射することにより、各検出センサから、前記第２分子サンプルの蛍光の、前記受光波長帯域毎の蛍光強度情報および位相情報を含む検出値を収集して、前記第２分子サンプルの蛍光の、前記受光波長帯域毎の蛍光強度情報および位相情報を求め、
前記第２の強度比率として、前記第２分子サンプルの、前記受光波長帯域の蛍光強度の比を求め、
求めた前記第２の強度比率および求めた前記位相情報を前記記憶手段に記憶する請求項１〜１３のいずれかに記載のＦＲＥＴ検出方法。 In the step of acquiring the calibration information, the information obtained by the second molecule calibration is called from the storage means and acquired,
In the second molecule calibration,
By irradiating a second molecular sample labeled only with the second molecule with a laser beam time-modulated at a predetermined frequency using the second molecular sample as a measurement object, the light receiving wavelength of the fluorescence of the second molecular sample is detected from each detection sensor. Collecting detection values including fluorescence intensity information and phase information for each band, and obtaining fluorescence intensity information and phase information for each light receiving wavelength band of the fluorescence of the second molecule sample,
As the second intensity ratio , the ratio of the fluorescence intensity of the light receiving wavelength band of the second molecular sample is obtained,
The FRET detection method according to claim 1, wherein the obtained second intensity ratio and the obtained phase information are stored in the storage unit.
前記キャリブレーション情報を取得するステップでは、自家蛍光キャリブレーションによって得られた情報を前記記憶手段から呼び出して取得し、
前記自家蛍光キャリブレーションは、前記第２分子キャリブレーションに先がけて行われるキャリブレーションであって、前記自家蛍光サンプル体を測定対象物として所定の周波数で時間変調したレーザ光を照射することにより、各検出センサから、前記受光波長帯域毎に蛍光強度情報および位相情報を含む検出値を収集して、前記自家蛍光サンプル体の蛍光の、前記受光波長帯域毎の蛍光強度情報および位相情報を求め、求めた情報を前記記憶手段に記憶し、
前記第２分子キャリブレーションでは、前記第２分子サンプルの蛍光の、前記受光波長帯域毎の蛍光強度情報および位相情報をベクトルで表した第２分子サンプルベクトルから、前記自家蛍光サンプル体キャリブレーションで求められた前記自家蛍光サンプルの蛍光の情報をベクトルで表した自家蛍光ベクトルを減算し、この減算で得られたベクトルを用いて、前記第２分子サンプルの蛍光の、前記受光波長帯域毎の蛍光強度情報および位相情報を求め、求めた情報を前記記憶手段に記憶する請求項１４に記載のＦＲＥＴ検出方法。 The second molecule sample is a sample labeled with the second molecule on an autofluorescent sample body that emits autofluorescence when excited by the laser beam,
In the step of acquiring the calibration information, the information obtained by autofluorescence calibration is called from the storage means and acquired,
The autofluorescence calibration is a calibration performed prior to the second molecule calibration, and each autoirradiation sample is irradiated with laser light that is time-modulated at a predetermined frequency with the autofluorescence sample body as a measurement object. By collecting detection values including fluorescence intensity information and phase information for each light reception wavelength band from a detection sensor , the fluorescence intensity information and phase information for each light reception wavelength band of the fluorescence of the autofluorescence sample body is obtained and obtained. Information stored in the storage means,
In the second molecule calibration, the fluorescence of the second molecule sample, the second molecule sample vector representing the fluorescence intensity information and phase information of each of the light receiving wavelength band vector, determined by the autofluorescence sample material calibration The autofluorescence vector representing the fluorescence information of the autofluorescence sample obtained as a vector is subtracted, and using the vector obtained by this subtraction, the fluorescence intensity of the fluorescence of the second molecule sample for each light receiving wavelength band 15. The FRET detection method according to claim 14, wherein information and phase information are obtained , and the obtained information is stored in the storage means .
レーザ光の強度を所定の周波数で時間変調して前記測定対象サンプルについて照射し、このときの前記測定対象サンプルの蛍光を受光波長帯域の異なる複数の検出センサで受光することにより、前記測定対象サンプルの蛍光の蛍光強度情報および位相情報を含む検出値を取得する検出情報取得部と、
前記測定対象サンプルの前記蛍光のうち前記第１分子が発する第１分子蛍光成分の、前記受光波長帯域の蛍光強度の比を表す第１の強度比率と、前記第１分子蛍光成分の位相情報と、前記第２分子が発する第２分子蛍光成分の、前記受光波長帯域の蛍光強度の比を表す第２の強度比率と、前記第２分子蛍光成分の位相情報と、レーザ光によって励起された第１分子が発する蛍光が１次遅れ系の緩和応答であるとしたときに定義される寿命であって、前記ＦＲＥＴが発生しない状態における前記第１分子蛍光成分の非ＦＲＥＴ蛍光寿命と、を少なくとも含むキャリブレーション情報を予め記憶しておく記憶手段と、
前記検出情報取得部が取得した前記検出値から、前記受光波長帯域それぞれについて、前記測定対象サンプルの蛍光の蛍光強度情報および位相情報を求めて、求めた前記蛍光強度情報および前記位相情報と、前記記憶手段から読み出した前記第１の強度比率と、前記第１分子蛍光成分の前記位相情報と、前記第２の強度比率と、前記第２分子蛍光成分の前記位相情報と、を用いて、レーザ光によって励起された第１分子が発する蛍光が１次遅れ系の緩和応答であるとしたときに定義される前記第１分子蛍光成分のＦＲＥＴ蛍光寿命を求めるＦＲＥＴ蛍光寿命算出部と、
前記第１分子蛍光成分のＦＲＥＴ蛍光寿命と、前記第１分子蛍光成分の前記非ＦＲＥＴ蛍光寿命との比を用いて表されるＦＲＥＴ発生情報を求めるＦＲＥＴ発生情報算出部と、を有することを特徴とするＦＲＥＴ検出装置。 The sample to be measured labeled with the first molecule and the second molecule is irradiated with laser light, and at this time, the fluorescence emitted from the sample to be measured is received, thereby transferring the energy of the first molecule to the second molecule. A FRET detection device for detecting (Resonance Energy Transfer),
The measurement target sample is irradiated with the measurement target sample after time-modulating the intensity of the laser beam at a predetermined frequency, and the fluorescence of the measurement target sample at this time is received by a plurality of detection sensors having different reception wavelength bands. A detection information acquisition unit for acquiring a detection value including fluorescence intensity information and phase information of the fluorescence of
Of the fluorescence of the sample to be measured, a first intensity ratio representing a ratio of fluorescence intensity of the light reception wavelength band of a first molecule fluorescence component emitted by the first molecule, and phase information of the first molecule fluorescence component The second intensity ratio representing the ratio of the fluorescence intensity of the light receiving wavelength band of the second molecule fluorescence component emitted by the second molecule, the phase information of the second molecule fluorescence component, and the first excited by laser light A lifetime defined when the fluorescence emitted from one molecule is a relaxation response of a first-order lag system, and includes at least the non-FRET fluorescence lifetime of the first molecule fluorescence component in a state where the FRET does not occur Storage means for storing calibration information in advance;
From the detection value acquired by the detection information acquisition unit, for each of the light receiving wavelength bands, to obtain fluorescence intensity information and phase information of the fluorescence of the measurement target sample, the obtained fluorescence intensity information and the phase information, Using the first intensity ratio read from the storage means, the phase information of the first molecule fluorescence component, the second intensity ratio, and the phase information of the second molecule fluorescence component, a laser is obtained. A FRET fluorescence lifetime calculation unit for obtaining a FRET fluorescence lifetime of the first molecule fluorescence component defined when the fluorescence emitted by the first molecule excited by light is a relaxation response of a first-order lag system;
A FRET generation information calculation unit that obtains FRET generation information expressed by using a ratio between the FRET fluorescence lifetime of the first molecule fluorescence component and the non-FRET fluorescence lifetime of the first molecule fluorescence component. FRET detection device.
前記ＦＲＥＴ蛍光寿命算出部では、
前記第１波長帯域の検出センサによる前記検出値から求められる第１波長帯域におけるベクトルと、前記第２の強度比率とを少なくとも用いて、前記ＦＲＥＴが生じた状態における前記第１分子蛍光成分の蛍光強度情報および位相情報を求め、
前記第２波長帯域の前記検出センサによる前記検出値によって表される第２波長帯域におけるベクトルと、前記第１の強度比率とを少なくとも用いて、前記ＦＲＥＴ成分の蛍光強度情報および位相情報を求める請求項１６または１７に記載のＦＲＥＴ検出装置。 Each light receiving wavelength band of the sensor has a first wavelength band centered on a peak wavelength at which the fluorescence intensity of the first molecule fluorescence component is maximized, and a peak wavelength at which the fluorescence intensity of the second molecule fluorescence component is maximized. The second wavelength band in the center,
In the FRET fluorescence lifetime calculation unit,
A vector in the first wavelength band obtained from the value detected by the detection sensor of the first wavelength band, said second intensity ratio and using at least a fluorescence of said first molecule fluorescence component in a state in which the FRET occurs Find intensity information and phase information,
Wherein a vector in a second wavelength band represented by said value detected by said detection sensor of the second wavelength band, with at least a first intensity ratio, determining the fluorescence intensity information and phase information of the FRET component according Item 18. The FRET detection device according to Item 16 or 17.
前記自家蛍光キャリブレーション部は、前記レーザ光によって励起されて自家蛍光を発する自家蛍光を測定対象物として所定の周波数で時間変調したレーザ光を照射することにより、前記検出センサそれぞれから、前記受光波長帯域の蛍光強度情報および位相情報を含む検出値を収集し、前記自家蛍光サンプル体の蛍光の、前記受光波長帯域毎の蛍光強度情報および位相情報を求めて、前記記憶手段に記憶し、
前記ＦＲＥＴ蛍光寿命算出部では、前記受光波長帯域毎の前記測定対象サンプルの蛍光の情報をベクトルで表し、このベクトルから、前記自家蛍光サンプル体の前記蛍光強度情報および前記位相情報を表したベクトルを減算し、減算して得られたベクトルを用いて、前記ＦＲＥＴ蛍光寿命を求める請求項１６〜１８のいずれかに記載のＦＲＥＴ検出装置。 The FRET detection device further includes an auto fluorescence calibration unit,
The autofluorescence calibration unit, by irradiating a laser beam temporal modulation at a predetermined frequency autofluorescence emits autofluorescence being excited by the laser beam as a measuring object, from each of the detecting sensor, the light receiving wavelength Collecting detection values including fluorescence intensity information and phase information of the band, obtaining fluorescence intensity information and phase information of the fluorescence of the autofluorescence sample body for each of the reception wavelength bands , and storing them in the storage means ,
In the FRET fluorescence lifetime calculation unit, the fluorescence information of the measurement target sample for each light reception wavelength band is represented by a vector, and from this vector, the fluorescence intensity information and the phase information of the autofluorescence sample body are represented. The FRET detection apparatus according to any one of claims 16 to 18, wherein the FRET fluorescence lifetime is obtained using a vector obtained by subtraction and subtraction.
前記ｎｏｎ−ＦＲＥＴキャリブレーション部は、
前記第１分子および前記第２分子がサンプルに付着されて、ＦＲＥＴが生じない処理がなされたｎｏｎ−ＦＲＥＴサンプルを測定対象物として所定の周波数で時間変調したレーザ光を照射することにより、各検出センサから、前記受光波長帯域毎の蛍光強度情報および位相情報を含む検出値を収集した際、
前記ｎｏｎ−ＦＲＥＴサンプルの蛍光の、前記受光波長帯域毎の蛍光強度情報および位相情報を求め、求めた蛍光強度情報および位相情報と、前記第１の強度比率と、前記第１分子蛍光成分の前記位相情報と、前記第２の強度比率と、前記第２分子蛍光成分の前記位相情報と、を用いて、前記レーザ光によって第２分子が直接励起されることで発生する直接励起蛍光成分の蛍光強度情報および位相情報を求め、これらの情報をベクトルで表した直接励起蛍光成分ベクトルを導出し、この導出結果を前記記憶手段に記憶し、
前記ＦＲＥＴ蛍光寿命算出部は、前記直接励起蛍光成分ベクトルを用いて前記ＦＲＥＴ蛍光寿命を求める請求項１６〜１９のいずれかに記載のＦＲＥＴ検出装置。 The FRET detection device further includes a non-FRET calibration unit,
The non-FRET calibration unit is
Each detection is performed by irradiating the first-molecule and the second molecule attached to the sample and irradiating the non-FRET sample, which has been processed so as not to generate FRET, with time-modulated laser light at a predetermined frequency. When collecting detection values including fluorescence intensity information and phase information for each light receiving wavelength band from the sensor,
Fluorescence intensity information and phase information of the fluorescence of the non-FRET sample for each light receiving wavelength band is obtained, the obtained fluorescence intensity information and phase information, the first intensity ratio, and the first molecular fluorescence component by using the phase information, and the second intensity ratio, and a said phase information of said second molecule fluorescence component, fluorescence directly excited fluorescence component second molecule occurs by being excited directly by the laser beam Obtaining intensity information and phase information , deriving a direct excitation fluorescence component vector representing these information as vectors, storing the derivation result in the storage means ,
The FRET detection apparatus according to any one of claims 16 to 19, wherein the FRET fluorescence lifetime calculation unit calculates the FRET fluorescence lifetime using the direct excitation fluorescence component vector.
前記第１分子キャリブレーション部は、
前記第１分子のみで標識された第１分子サンプルを測定対象物として所定の周波数で時間変調したレーザ光を照射することにより、各検出センサから、前記第１分子サンプルの蛍光の、前記受光波長帯域毎の蛍光強度情報および位相情報を含む検出値を収集した際、
前記第１分子サンプルの蛍光の、前記受光波長帯域毎の蛍光強度情報および位相情報を求め、前記第１の強度比率として、前記第１分子サンプルの、前記受光波長帯域の蛍光強度の比を求め、求めた前記第１の強度比率及び求めた前記第１分子サンプルの蛍光の前記位相情報を前記記憶手段に記憶する請求項１６〜２０のいずれかに記載のＦＲＥＴ検出装置。 The FRET detection apparatus further includes a first molecule calibration unit,
The first molecule calibration unit includes:
By irradiating a first molecular sample labeled only with the first molecule with a laser beam time-modulated at a predetermined frequency using the first molecular sample as a measurement object, the light receiving wavelength of the fluorescence of the first molecular sample is detected from each detection sensor. When collecting detection values including fluorescence intensity information and phase information for each band,
Fluorescence intensity information and phase information of the fluorescence of the first molecule sample for each light reception wavelength band is obtained, and a ratio of the fluorescence intensity of the light reception wavelength band of the first molecule sample is obtained as the first intensity ratio. 21. The FRET detection apparatus according to claim 16, wherein the obtained first intensity ratio and the obtained phase information of the fluorescence of the first molecule sample are stored in the storage means .
前記第２分子キャリブレーション部は、
前記第２分子のみで標識された第２分子サンプルを測定対象物として所定の周波数で時間変調したレーザ光を照射することにより、各検出センサから、前記第２分子サンプルの蛍光の、前記受光波長帯域毎の蛍光強度情報および位相情報を含む検出値を収集した際、
前記第２分子サンプルの蛍光の、前記受光波長帯域毎の蛍光強度情報および位相情報を求め、前記第２の強度比率として、前記第２分子サンプルの、前記受光波長帯域の蛍光強度の比を求め、求めた前記第２の強度比率及び求めた前記第２分子サンプルの蛍光の前記位相情報を前記記憶手段に記憶する請求項１６〜２１のいずれかに記載のＦＲＥＴ検出装置。 The FRET detection apparatus further includes a second molecular calibration unit,
The second molecule calibration unit includes:
By irradiating a second molecular sample labeled only with the second molecule with a laser beam time-modulated at a predetermined frequency using the second molecular sample as a measurement object, the light receiving wavelength of the fluorescence of the second molecular sample is detected from each detection sensor. When collecting detection values including fluorescence intensity information and phase information for each band,
Fluorescence intensity information and phase information of the fluorescence of the second molecule sample for each light reception wavelength band is obtained, and a ratio of the fluorescence intensity of the second molecule sample in the light reception wavelength band is obtained as the second intensity ratio. The FRET detection device according to any one of claims 16 to 21, wherein the obtained second intensity ratio and the obtained phase information of the fluorescence of the second molecular sample are stored in the storage means .
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