Methods for spectrally resolving fluorophores of a sample by generalized least squares and systems for same

A method comprising:

• (a) receiving first excitation measurements of a first sample consisting of a first fluorophore, wherein the first excitation measurements are measured by a light detection system;
• (b) spectrally unmixing the first excitation measurements based on a spectral matrix with the fluorescence spectra of the first fluorophore and a second fluorophore to obtain unmixed data;
• (c) determining a median intensity level for the first fluorophore and the second fluorophore in the unmixed data;
• (d) determining a ground truth unmixed variance for the first fluorophore and the second fluorophore in the unmixed data;
• (e) adjusting one or more coefficients of a noise model until a variance estimated by the model for the first fluorophore and the second fluorophore in the spectral matrix is equal to the ground truth unmixed variance;
• (f) receiving second excitation measurements of a second sample comprising a second fluorophore, wherein the second excitation measurements are measured by the light detection system;
• and
• (g) classifying a particle based at least in part on the second excitation measurements and the noise model.

A system comprising:

• a light source;
• a light detection system;
• and
• a processor comprising memory operably coupled to the processor wherein the memory comprises instructions stored thereon, which when executed by the processor, cause the processor to:
• (h) receive first excitation measurements of a first sample consisting of a first fluorophore, wherein the first excitation measurements are measured by a light detection system;
• (i) spectrally unmix the first excitation measurements based on a spectral matrix with the fluorescence spectra of the first fluorophore and a second fluorophore to obtain unmixed data;
• (i) determine a median intensity level for the first fluorophore and the second fluorophore in the unmixed data;
• (k) determine a ground truth unmixed variance for the first fluorophore and the second fluorophore in the unmixed data;
• (l) adjust one or more coefficients of a noise model until a variance estimated by the model for the first fluorophore and the second fluorophore in the spectral matrix is equal to the ground truth unmixed variance;
• (m) receive second excitation measurements of a second sample comprising a second fluorophore, wherein the second excitation measurements are measured by the light detection system;
• and
• (n) classify a particle based at least in part on the second excitation measurements and the noise model.

A non-transitory computer readable storage medium comprising instructions stored thereon for performing a method comprising:

• (a) receiving first excitation measurements of a first sample consisting of a first fluorophore, wherein the first excitation measurements are measured by a light detection system;
• (b) spectrally unmixing the first excitation measurements based on a spectral matrix with the fluorescence spectra of the first fluorophore and a second fluorophore to obtain unmixed data;
• (c) determining a median intensity level for the first fluorophore and the second fluorophore in the unmixed data;
• (d) determining a ground truth unmixed variance for the first fluorophore and the second fluorophore in the unmixed data;
• (e) adjusting one or more coefficients of a noise model until a variance estimated by the model for the first fluorophore and the second fluorophore in the spectral matrix is equal to the ground truth unmixed variance;
• (f) receiving second excitation measurements of a second sample comprising a second fluorophore, wherein the second excitation measurements are measured by the light detection system;
• and
• (g) classifying a particle based at least in part on the second excitation measurements and the noise model.