Method for detecting time-resolved fluorescence based on principle of phase balanced frequency multiplication modulation

A method for detecting time-resolved fluorescence based on a principle of phase balanced frequency multiplication modulation, wherein the method comprises the following steps:

• step 1-1:
• generate a regular wave signal in a frequency X as a baseband signal by using a baseband signal generator, wherein the regular wave signal in the frequency X is used to modulate a stimulating light source, the light source emits stimulating light, and the stimulating light acts on a to-be-measured target to trigger fluorescence;
• step 1-2:
• control a photoelectric sensor to perform, by using N times the frequency X, sampling on the triggered fluorescence, so as to obtain a fluorescence intensity signal Ai at each moment in a sampling period, wherein i>0 and 30≦N≦80;
• step 1-3:
• perform signal filtering and AD conversion on the fluorescence intensity signal Ai at each moment in the sampling period to obtain a fluorescence intensity digital signal Di at each moment corresponding to the fluorescence intensity signal Ai at each moment, wherein i>0;
• step 1-4:
• plot the fluorescence intensity digital signal Di at each moment according to sampling time to obtain a spectral curve, wherein the spectral curve comprises a fluorescence emission period S and a fluorescence decay period R;
• step 1-5:
• perform frequency multiplication modulation on the baseband signal in the frequency X to produce a frequency-multiplied signal in a frequency X′, wherein X′=2X;
• divide the fluorescence emission period S into a fluorescence emission period S1 and a fluorescence emission period S2 that have an equal duration and divides the fluorescence decay period R into a fluorescence decay period R1 and a fluorescence decay period R2 that have an equal duration;
• step 1-6:
• perform integral operation on the fluorescence emission periods S1 and S2 and the fluorescence decay periods R1 and R2 on the spectral curve to obtain an integral area of the fluorescence emission period S1 and mark the integral area as Φ1, obtain an integral area of the fluorescence emission period S2 and mark the integral area as Φ2, an integral area of the fluorescence decay period R1 and mark the integral area as Φ3, and an integral area of the fluorescence decay period R2 and mark the integral area as Φ4, wherein Φ2>Φ1>0 and Φ3>Φ4>0;
• step 1-7:
• subtract the integral area Φ1 of the fluorescence emission period S1 from the integral area Φ2 of the fluorescence emission period S2 to obtain S′=Φ2−Φ1, wherein S′ is marked as a fluorescence intensity value of a fluorescence emission period;
• subtract the integral area Φ4 of the fluorescence decay period R2 from the integral area Φ3 of the fluorescence decay period R1 to obtain R=Φ3-Φ4, wherein R′ is marked as a fluorescence intensity value of a fluorescence decay period;
• add the fluorescence intensity value S′ of the fluorescence emission period to the fluorescence intensity value R′ of the fluorescence decay period to obtain ΔΦ=Φ2-Φ1+Φ3-Φ4 and mark the ΔΦ as a total fluorescence intensity value of fluorescence pigment emission, wherein S′>0 and R′>0;
• and
• step 1-8:
• obtain a concentration value of the to-be-measured target by using a calibration method by using the total fluorescence intensity value ΔΦ of the fluorescence pigment emission to represent a fluorescence signal measurement value of the to-be-measured target.