METHODS AND APPARATUS FOR FULL SPECTRUM FLOW CYTOMETER
In one embodiment, a method of building an optimized color flow cytometry panel is disclosed using a full spectrum flow cytometer with five excitation lasers and five corresponding detection modules. In another embodiment, a graphical user interface is disclosed generated by a server computer from a fluorochrome database and displayed by a client computer to assist in the selection of a set of fluorochromes for use in an assay to analyze biological samples. The GUI can display spectra graphs to visually show how fluorochromes may overlap and can generate similarity indexes for the paired fluorochrome interference and a complexity index for overall many to many interferences generated by a selected group or set of fluorochromes.
1 - 49 . (canceled)
50 - 60 . (canceled)
61 . A method for flow cytometry for a flow cytometer with a plurality of photodetectors, the method comprising:
selecting a plurality of fluorochromes to label biological cells in a sample, wherein the number of the plurality of photodetectors in the flow cytometer is greater than the number of the selected plurality of fluorochromes;
determining similarity indexes for each fluorochrome pair of the plurality of fluorochromes based on a configuration of the flow cytometer having a plurality of excitation lasers and the plurality of photodetectors, wherein each similarity index indicates a measure of fluorochrome interference between two spectra of fluorescence given off by each fluorochrome of each fluorochrome pair;
generating a similarity matrix with the similarity indexes for each fluorochrome pair to visually display interaction between each fluorochrome pair; and
displaying, on a display device to a user, the similarity matrix with each similarity index of each fluorochrome pair to evaluate the selection of the plurality of fluorochromes in the analysis of labeled biological cells in the sample.
62 . The method of claim 61 , wherein the method further comprises:
determining a complexity index for the selected plurality of fluorochromes, wherein the complexity index indicates a measure of overall interference among the plurality of fluorochromes in the sample; and
displaying the complexity index to the user to further evaluate the selection of fluorochromes in the set of the at least one reagent composition.
63 . The method of claim 61 , wherein the configuration of the flow cytometer includes:
three or more detector modules with thirty-two or more photodetectors forming thirty-two or more detector channels; and
three or more lasers respectively associated with the three or more detector modules, wherein the three or more lasers provide three or more different laser excitation wavelengths to excite the selected plurality of fluorochromes for detection by the three or more detector modules with the thirty-two or more photodetectors.
64 . The method of claim 61 , wherein the flow cytometer is a configurable flow cytometer and the method further comprises:
selecting a configuration of the plurality of excitation lasers and a plurality of associated detector modules with the plurality of photodetectors for the configurable flow cytometer.
65 . The method of claim 63 , wherein the selecting of the configuration of the configurable flow cytometer includes:
providing the flow cytometer with three or more detector modules having thirty-two or more photodetectors forming thirty-two or more detector channels; and
providing three or more lasers respectively associated with the three or more detector modules, wherein the three or more lasers provide three or more different laser excitation wavelengths to excite fluorochromes for detection by the three or more detector modules and the thirty-two or more photodetectors.
66 . A method for a spectral flow cytometer having N detector channels, the method comprising:
running single stain reference controls for each of M fluorochromes through the flow cytometer to generate full spectrum signatures for each of the M fluorochromes being used to label particles/cells in a mixed sample, wherein M is less than N;
forming an N by M reference matrix of reference vectors using the plurality of full spectrum signatures over the N detector channels;
labeling the particles/cells in the mixed sample;
running the labeled mixed sample through the flow cytometer to generate mixed signature data in a measured sample event vector for the labeled mixed sample;
with the N by M reference matrix of reference vectors, unmixing the mixed signature data in the measured sample event vector to generate an unmixed sample event vector; and
using the unmixed sample event vector to form one or more dot plots to display counts of the different types of particles/cells in the mixed sample to a user on a display device.
67 . The method of claim 66 , further comprising:
running an unstained reference control through the flow cytometer to generate a full spectrum signature for autofluorescence of the particles/cells.
68 . The method of claim 66 , further comprising:
prior to the unmixing, generating an adjusted spectral spillover matrix with fine adjustments; and
after the unmixing, compensating the unmixed sample event vector into a recompensated event vector that is used to form the dot plots.
69 . The method of claim 66 , wherein
the dot plots further display binned ranges of densities of the particles/cells in the mixed sample using differing colors.
70 . The method of claim 66 , further comprising:
selecting a grouping of dots on a dot plot for analysis with a polygon gate;
setting a positive population gate for the grouping of dots to display a histogram of positive and negative populations; and
displaying an interval gate in a spectrum plot of a full spectrum signature in a detector channel of the N detector channels.