Compositions, systems and methods for biological analysis involving energy transfer dye conjugates and analytes comprising the same

A system, comprising:

• a first radiant source characterized by a first average excitation wavelength;
• a second radiant source characterized by a second average excitation wavelength that is different than the first average excitation wavelength;
• a sample disposed to receive radiation from the radiant sources, the sample comprising:
• a first dye;
• a second dye;
• and
• a third dye;
• a detector configured to measure emissions from the sample;
• a first emission spectral element characterized by a first average emission wavelength;
• a second emission spectral element characterized by a second average emission wavelength that is different than the first average emission wavelength;
• at least one processor comprising at least one non-transitory memory encoded with instructions that are executed by the processor to:
• illuminate the sample with the first radiant source and, in response, (1) measure emissions from the sample using the detector and the first emission spectral element and (2) measure emissions from the sample using the detector and the second emission spectral element;
• illuminate the sample with the second radiant source and, in response, measure emissions from the sample using the detector and the second emission spectral element;
• wherein (1) the first dye comprises a first absorption spectrum comprising a first maximum absorption wavelength and the second dye comprises a second absorption spectrum comprising a second maximum absorption wavelength that is equal to or substantially equal to the first maximum absorption wavelength;
• and (2) the second dye comprises a second emission spectrum comprising a second maximum emission wavelength and the third dye comprises a third emission spectrum comprising a third maximum emission wavelength that is equal to or substantially equal to the second maximum emission wavelength-;
• wherein at least one of the first dye, the second dye, and the third dye is an off-axis dye, an off-axis dye being a dye having a maximum absorption or excitation wavelength that is an absolute maximum over an entire spectrum of the dye and having a maximum absorption or excitation wavelength that is a local maximum and is separated from the absolute maximum by at least 60 nanometers;
• and
• wherein each radiant source is characterized by electromagnetic radiation within the visible light range, near infrared range, infrared range, or ultraviolet range.