Method for measuring fluorescence resonance energy transfer
First Claim
1. A method of measuring energy transfer between donor fluorophores and acceptor fluorophores under energy-transfer condition included in a sample, the sample also including donor fluorophores and acceptor fluorophores under non-energy-transfer condition, the method comprising the steps of:
- irradiating, with excitation light, a sample including donor fluorophores and acceptor fluorophores both under non-energy-transfer condition and under energy-transfer condition, so that the donor fluorophores and the acceptor fluorophores emit fluorescence, the donor fluorophores and the acceptor fluorophores emitting fluorescence of different wavelength bands which are partly overlapped with each other, the donor fluorophores having fluorescence lifetime longer than a fluorescence lifetime of the acceptor fluorophores,wherein intensity of fluorescence from the acceptor fluorophores attenuates over time after the irradiation of the excitation light, in accordance with its fluorescent lifetime, the attenuation occurring so that the intensity of fluorescence from the acceptor fluorophores is below a predetermined threshold at a first timing, intensity of fluorescence from the donor fluorophores attenuates over time after the irradiation of the excitation light, in accordance with its fluorescent lifetime, the attenuation occurring so that the intensity of fluorescence from the donor fluorophores is below a second predetermined threshold at a second timing, the fluorescence lifetime of the donor fluorophores varies when energy transfer occurs from the donor fluorophores to the acceptor fluorophores so that the intensity of fluorescence from the donor fluorophores attenuates over time when the energy transfer occurs after the irradiation of the excitation light, in accordance with its varied fluorescent lifetime, the attenuation occurring so that the intensity of fluorescence from the donor fluorophores is below a third predetermined threshold at a third timing different from the second timing;
dividing light, emitted from the sample, into a first light of a first wavelength and a second light of a second wavelength, the first wavelength region being only within the fluorescence wavelength band of the donor fluorophores, the second wavelength region being set not only within the fluorescence wavelength band of the acceptor fluorophores but also within a predetermined amount of a part of the fluorescence wavelength band of the donor fluorophores;
measuring an intensity of the first light and an intensity of the second light over a first time period, which is determined between the first timing and the third timing, to thereby obtain a total intensity ID1 of the first light over the first time period and a total intensity IA1 of the second light over the first time period;
measuring an intensity of the first light and an intensity of the second light over a second time period, which is determined between the third timing and the second timing, to thereby obtain a total intensity ID2 of the first light over the second time period and a total intensity IA2 of the second light over the second time period; and
determining energy transfer, occurring between the donor fluorophores and the acceptor fluorophores based on a ratio between the amounts ID2 and ID1 and a ratio between the amounts IA2 and IA1.
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Abstract
A plurality of light emission molecules, having different light emission lifetimes, are irradiated with excitation light so that the light emission molecules emit light. The emitted light is divided into at least two different wavelength regions. Temporal changes in each of the at least two wavelength regions of the light emission is measured over at least two different time periods. Information on energy transfer generated between the plurality of light emission molecules is determined based on the measured results in each of the wavelength regions over each of the time periods.
70 Citations
26 Claims
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1. A method of measuring energy transfer between donor fluorophores and acceptor fluorophores under energy-transfer condition included in a sample, the sample also including donor fluorophores and acceptor fluorophores under non-energy-transfer condition, the method comprising the steps of:
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irradiating, with excitation light, a sample including donor fluorophores and acceptor fluorophores both under non-energy-transfer condition and under energy-transfer condition, so that the donor fluorophores and the acceptor fluorophores emit fluorescence, the donor fluorophores and the acceptor fluorophores emitting fluorescence of different wavelength bands which are partly overlapped with each other, the donor fluorophores having fluorescence lifetime longer than a fluorescence lifetime of the acceptor fluorophores, wherein intensity of fluorescence from the acceptor fluorophores attenuates over time after the irradiation of the excitation light, in accordance with its fluorescent lifetime, the attenuation occurring so that the intensity of fluorescence from the acceptor fluorophores is below a predetermined threshold at a first timing, intensity of fluorescence from the donor fluorophores attenuates over time after the irradiation of the excitation light, in accordance with its fluorescent lifetime, the attenuation occurring so that the intensity of fluorescence from the donor fluorophores is below a second predetermined threshold at a second timing, the fluorescence lifetime of the donor fluorophores varies when energy transfer occurs from the donor fluorophores to the acceptor fluorophores so that the intensity of fluorescence from the donor fluorophores attenuates over time when the energy transfer occurs after the irradiation of the excitation light, in accordance with its varied fluorescent lifetime, the attenuation occurring so that the intensity of fluorescence from the donor fluorophores is below a third predetermined threshold at a third timing different from the second timing; dividing light, emitted from the sample, into a first light of a first wavelength and a second light of a second wavelength, the first wavelength region being only within the fluorescence wavelength band of the donor fluorophores, the second wavelength region being set not only within the fluorescence wavelength band of the acceptor fluorophores but also within a predetermined amount of a part of the fluorescence wavelength band of the donor fluorophores; measuring an intensity of the first light and an intensity of the second light over a first time period, which is determined between the first timing and the third timing, to thereby obtain a total intensity ID1 of the first light over the first time period and a total intensity IA1 of the second light over the first time period; measuring an intensity of the first light and an intensity of the second light over a second time period, which is determined between the third timing and the second timing, to thereby obtain a total intensity ID2 of the first light over the second time period and a total intensity IA2 of the second light over the second time period; and determining energy transfer, occurring between the donor fluorophores and the acceptor fluorophores based on a ratio between the amounts ID2 and ID1 and a ratio between the amounts IA2 and IA1. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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18. A method of measuring energy transfer occurring in a sample which includes donor fluorophores and acceptor fluorophores both under non-energy-transfer condition and under energy-transfer condition, the method comprising the steps of:
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measuring a lifetime of fluorescence from acceptor fluorophores under non-energy-transfer condition to determine a first timing, at which intensity of fluorescence from the non-energy-transfer acceptor fluorophores attenuates below a predetermined threshold after irradiation of excitation light; measuring a lifetime of fluorescence from donor fluorophores under non-energy-transfer condition to determine a second timing, at which intensity of fluorescence from the non-energy-transfer donor fluorophores attenuates below a second predetermined threshold after irradiation of excitation light, the fluorescence lifetime of the donor fluorophores being longer than the fluorescence lifetime of the acceptor fluorophores; measuring a lifetime of fluorescence from the acceptor fluorophores and the donor fluorophores under energy-transfer condition to determine a third timing, at which intensity of fluorescence from the donor and the acceptor fluorophores attenuates below a third predetermined threshold after irradiation of excitation light; determining a first time period between the first timing and the third timing and a second time period between the third timing and the second timing; irradiating, with excitation light, a sample including the donor fluorophores and the acceptor fluorophores under both non-energy-transfer condition and energy-transfer condition, so that the donor fluorophores and the acceptor fluorophores emit fluorescence, dividing light, emitted from the sample, into a first light of a first wavelength and a second light of a second wavelength, the first wavelength region being only within the fluorescence wavelength band of the donor fluorophores, the second wavelength region being not only within the fluorescence wavelength band of the acceptor fluorophores but also within a predetermined amount of a part of the fluorescence wavelength band of the donor fluorophores; measuring an intensity of the first light and an intensity of the second light over the first time period to thereby obtain a total intensity ID1 of the first light over the first time period and a total intensity IA1 of the second light over the first time period; measuring an intensity of the first light and an intensity of the second light over the second time period to thereby obtain a total intensity ID2 of the first light over the second time period and a total intensity IA2 of the second light over the second time period; and determining energy transfer, occurring between the donor fluorophores and the acceptor fluorophores based on a ratio between the amounts ID2 and ID1 and a ratio between the amounts IA2 and IA1. - View Dependent Claims (19, 20)
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21. A method of measuring energy transfer occurring in a sample which includes donor fluorophores and acceptor fluorophores both under non-energy-transfer condition and under energy-transfer condition, the method comprising the steps of:
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measuring a lifetime of fluorescence from acceptor fluorophores under non-energy-transfer condition to determine a first timing, at which intensity of fluorescence from the non-energy-transfer acceptor fluorophores attenuates below a predetermined threshold after irradiation of excitation light; measuring a lifetime of fluorescence from non-energy-transfer donor fluorophores to determine a second timing, at which intensity of fluorescence from the non-energy-transfer donor fluorophores attenuates below a second predetermined threshold after irradiation of excitation light, the fluorescence lifetime of the donor fluorophores being longer than the fluorescence lifetime of the acceptor fluorophores; measuring a lifetime of fluorescence from the acceptor fluorophores and the donor fluorophores under energy-transfer condition to determine a third timing, at which intensity of fluorescence from the donor and acceptor fluorophores attenuates below a third predetermined threshold after irradiation of excitation light; determining a first time period between the first timing and the third timing and a second time period between the third timing and the second timing; irradiating, with excitation light, a test sample including the donor fluorophores and the acceptor fluorophores only under non-energy-transfer condition so that the donor fluorophores and the acceptor fluorophores emit fluorescence, measuring an intensity of the fluorescence emitted from the test sample in a first wavelength region and a second wavelength region during the first time period, to thereby obtain an intensity ID1 of the first wavelength region and intensity IA1 of the second wavelength region, the first wavelength region being set only within the fluorescence wavelength band of the donor fluorophores, the second wavelength region being set not only within the fluorescence wavelength band of the acceptor fluorophores but also within a predetermined amount of a part of the fluorescence wavelength band of the donor fluorophores; measuring an intensity of the fluorescence emitted from the test sample in the first wavelength region and the second wavelength region during the second time period, to thereby obtain an intensity ID2 of the first wavelength region and an intensity IA2 of the second wavelength region; determining energy transfer test information Z based on a ratio between the amounts ID2 and ID1 and a ratio between the amounts IA2 and IA1 ; irradiating, with excitation light, a sample including the donor fluorophores and the acceptor fluorophores under both non-energy-transfer condition and energy-transfer condition, so that the donor fluorophores and the acceptor fluorophores emit fluorescence; measuring an intensity of the fluorescence emitted from the sample in the first wavelength region and the second wavelength region during the first time period, to thereby obtain an intensity ID1 '"'"' of the first wavelength region and an intensity IA1 '"'"' of the second wavelength region; measuring an intensity of the fluorescence emitted from the sample in the first wavelength region and the second wavelength region during the second time period, to thereby obtain an intensity ID2 '"'"' of the first wavelength region and an intensity IA2 '"'"' of the second wavelength region; determining energy transfer information Z'"'"' based on a ratio between the amounts ID2 '"'"' and ID1 '"'"' and a ratio between the amounts IA2 '"'"' and IA1 '"'"'; and determining, based on the energy transfer test information Z and the energy transfer information Z'"'"', energy transfer occurring between the donor fluorophores and the acceptor fluorophores under energy-transfer condition in the sample. - View Dependent Claims (22, 23)
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24. A method of determining whether a certain substance exists in a test sample, the method comprising the steps of:
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preparing a donor fluorophore and an acceptor fluorophore, a donor fluorophore binding specifically to a certain substance, an acceptor fluorophore binding specifically to the certain substance, and energy transfer between the donor fluorophore and the acceptor fluorophore occurring when both of the donor fluorophore and the acceptor fluorophore bind to the certain substance; measuring a lifetime of fluorescence from acceptor fluorophores to determine a first timing, at which intensity of fluorescence from the acceptor fluorophores attenuates below a predetermined threshold after irradiation of excitation light; measuring a lifetime of fluorescence from donor fluorophores to determine a second timing, at which intensity of fluorescence from the donor fluorophores attenuates below a second predetermined threshold after irradiation of excitation light, the fluorescence lifetime of the donor fluorophores being longer than the fluorescence lifetime of the acceptor fluorophores; measuring a lifetime of fluorescence from the acceptor fluorophores and the donor fluorophores under energy-transfer condition to determine a third timing, at which intensity of fluorescence from the donor and acceptor fluorophores attenuates below a third predetermined threshold after irradiation of excitation light; determining a first time period between the first timing and the third timing and a second time period between the third timing and the second timing; preparing a control sample including only the donor fluorophores and the acceptor fluorophores; irradiating, with excitation light, the control sample so that the donor fluorophores and the acceptor fluorophores emit fluorescence, measuring an intensity of the fluorescence emitted from the control sample in a first wavelength region and a second wavelength region during the first time period, to thereby obtain an intensity ID1 of the first wavelength region and intensity IA1 of the second wavelength region, the first wavelength region being set only within the fluorescence wavelength band of the donor fluorophores, the second wavelength region being set not only within the fluorescence wavelength band of the acceptor fluorophores but also within a predetermined amount of a part of the fluorescence wavelength band of the donor fluorophores; measuring an intensity of the fluorescence emitted from the control sample in the first wavelength region and the second wavelength region during the second time period, to thereby obtain an intensity ID2 of the first wavelength region and an intensity IA2 of the second wavelength region; determining energy transfer test information Z based on a ratio between the amounts ID2 and ID1 and a ratio between the amounts IA2 and IA1 ; preparing a sample through adding the donor fluorophores and the acceptor fluorophores to the test sample, a part of the donor fluorophores and the acceptor fluorophores being under energy transfer condition if the certain substance exists in the test sample; irradiating, with excitation light, the sample so that the donor fluorophores and the acceptor fluorophores emit fluorescence; measuring an intensity of the fluorescence emitted from the sample in the first wavelength region and the second wavelength region during the first time period, to thereby obtain an intensity ID1 '"'"' of the first wavelength region and an intensity IA1 '"'"' of the second wavelength region; measuring an intensity of the fluorescence emitted from the sample in the first wavelength region and the second wavelength region during the second time period, to thereby obtain an intensity ID2 '"'"' of the first wavelength region and an intensity IA2 '"'"' of the second wavelength region; determining energy transfer information Z'"'"' based on a ratio between the amounts ID2 '"'"' and ID1 '"'"' and a ratio between the amounts IA2 '"'"' and IA1 '"'"'; and determining, based on the energy transfer test information Z and the energy transfer information Z'"'"', information on the substance in the test sample. - View Dependent Claims (25, 26)
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Specification