Method and system for multiplexed time-resolved fluorescence detection

A method for multiplexed time-resolved fluorescence (TRF) detection, the method comprising the steps of:

• a) providing a sample on a sample support, the sample comprising a first fluorescent label bound to a first analyte and a second fluorescent label bound to a second analyte, wherein the first fluorescent label has a first fluorescence emission lifetime, a first excitation wavelength, and a first emission wavelength, and the second fluorescent label has a second fluorescence emission lifetime, a second excitation wavelength different from the first excitation wavelength, and a second emission wavelength, and the first fluorescence emission lifetime is at least 3 times longer than background fluorescence emission lifetimes;
• b) exciting the first fluorescent label with a first excitation light having the first excitation wavelength, whereby the first fluorescent label emits a first detection signal having the first emission wavelength;
• c) exciting, thereafter, the second fluorescent label with a second excitation light having the second excitation wavelength, whereby the second fluorescent label emits a second detection signal having the second emission wavelength;
• d) directing the first detection signal through an emission filter to filter the first detection signal, and then to a detector;
• e) directing the second detection signal through the emission filter to filter the second detection signal, and then to the detector;
• f) measuring intensity of the first detection signal at the detector, wherein the intensity of the first detection signal is correlated with the amount of the first analyte in the sample;
• and
• g) measuring intensity of the second detection signal at the detector, wherein the intensity of the second detection signal is correlated with the amount of the second analyte in the sample, and wherein:
• the second fluorescence emission lifetime is at least 5 times longer than the first fluorescence emission lifetime;
• the first fluorescent label is selected from a first group consisting of transition metal chelates of ruthenium (Ru(II)), osmium (Os(II)), and rhenium (Re(I));
• the second fluorescent label is selected from a second group consisting of lanthanide chelates of samarium (Sm(III)), dysprosium (Dy(III)), europium (Eu(III)), and terbium (Tb(III)), wherein the first fluorescent label and the second fluorescent label are each selected from the first group and second group respectively, rather than both being selected from the same group.