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 . A system for analyzing biological fluids, the system comprising:
a full spectrum flow cytometer having five (5) or more differing lasers each emitting a different wavelength of laser light and four (4) or more associated detector modules providing N individual photo detectors to capture a full spectrum of visible light, wherein N is forty-eight (48) or more and each individual photo detector is a photodiode;
a single labeled biological sample to run through the full spectrum flow cytometer, the single labeled biological sample having over thirty (30) different markers respectively labeled with over thirty (30) and less than N different fluorochrome-conjugated antibodies to generate over thirty (30) and less than N different colors when excited by the five (5) or more lasers of the flow cytometer that can be detected by the four (4) or more associated detector modules with the N individual photo detectors; and
a computer system coupled in communication with the flow cytometer, the computer system having a display device, a storage device, and a processor to execute instructions, wherein the storage device stores data samples from the flow cytometer in response to running the single labeled biological sample through the flow cytometer, and the processor executes instructions to process the stored data samples to generate full spectrum signatures of the fluorescence given off by each marked biological cells in the single labeled biological sample with a single run through the flow cytometer, wherein each full spectrum signature is used to determine the type and quantity of the plurality of biological cells in the single labeled biological sample with a single run through the flow cytometer, wherein the display device displays the full spectrum signatures to a user.
2 . The system of claim 1 , wherein
each of the N individual photo detectors is an avalanche photodiode.
3 . The system of claim 1 , wherein
each of the N individual photo detectors has a bandpass filter in the optical path to pass a predetermined wavelength range of light for each individual photo detector.
4 . The system of claim 1 , wherein
each of the N individual photo detectors is coupled to a gain amplifier with an independently adjustable gain to increase or decrease light intensity for each individual photo detector.
5 . The system of claim 1 , wherein
a red detector module of the four or more associated detector modules detects fluorescent light over a wavelength range from 625 nanometers (nm) to 828.5 nm for fluorescent particles excited by a red laser of the four or more lasers;
a yellow green detector module of the four or more associated detector modules detects fluorescent light over a wavelength range from 567 nm to 828.5 nm for fluorescent particles excited by a yellow green laser of the four or more lasers;
a blue detector module of the four or more associated detector modules detects fluorescent light over a wavelength range from 498 nm to 828.5 nm for fluorescent particles excited by a blue laser of the four or more lasers; and
a violet detector module of the four or more associated detector modules detects fluorescent light over a wavelength range from 420 nm to 828.5 nm for fluorescent particles excited by a violet laser of the four or more lasers.
6 . The system of claim 5 , wherein
an ultra violet detector module of the four or more associated detector modules detects fluorescent light over a wavelength range from 365 nm to 828.5 nm for fluorescent particles excited by an ultra violet laser of the four or more lasers.
7 . The system of claim 6 , wherein
the ultraviolet detector module has at least sixteen individual photo detectors dividing up the wavelength range of the ultraviolet detector module;
the violet detector module has at least sixteen individual photo detectors dividing up the wavelength range of the violet detector module;
the blue detector module has at least fourteen individual photo detectors dividing up the wavelength range of the blue detector module;
the yellow green detector module has ten individual photo detectors dividing up the wavelength range of the yellow green detector module; and
the red detector module has at least eight individual photo detectors dividing up the wavelength range of the red detector module.
8 . The system of claim 1 , wherein
the full spectrum flow cytometer has six (6) or more lasers each emitting a different wavelength of laser light.
9 . The system of claim 1 , wherein
the full spectrum flow cytometer captures a full spectrum of visible light and a portion of the spectrum of ultraviolet light.
10 . The system of claim 9 , wherein
N is sixty-four (64) or more, and
the associated detector modules provide sixty-four (64) or more individual photo detectors to capture the full spectrum of visible light and the portion of the spectrum of ultraviolet light.
11 . The system of claim 10 , wherein
the associated detector modules provide at least sixteen individual photo detectors for ultraviolet light spectrum;
the associated detector modules provide at least sixteen individual photo detectors for violet light spectrum;
the associated detector modules provide at least fourteen individual photo detectors for blue light spectrum;
the associated detector modules provide at least ten individual photo detectors for yellow-green light spectrum; and
the associated detector modules provide at least eight individual photo detectors for red light spectrum.
12 . The system of claim 10 , wherein
each of the sixty-four (64) or more individual photo detectors is an avalanche photodiode.
13 . The system of claim 1 , wherein
each individual photo detector is in a detector channel with a focusing lens.
14 . The system of claim 13 , wherein
each individual photo detector is packaged in a thin outline (TO) can package.
15 . The system of claim 1 , wherein
the single labeled biological sample is in a test tube.
16 . The system of claim 1 , wherein
the full spectrum flow cytometer further has four (4) or more optical fibers to collect light and direct it into the four (4) or more associated detector modules.
17 . A system for analyzing biological fluids in a labeled biological sample, the system comprising:
a flow cytometer having five (5) or more lasers each emitting a different wavelength of laser light and five (5) or more associated detector modules providing sixty-four (64) or more individual photo detectors to capture visible and ultraviolet light spectrum, wherein each individual photo detector is a photodiode;
wherein the labeled biological sample has over thirty (30) different markers respectively labeled with over thirty (30) different fluorochrome-conjugated antibodies to generate over thirty (30) different colors when excited by the five or more lasers of the flow cytometer that can be detected by the five (5) or more associated detector modules with the sixty-four (64) or more individual photo detectors; and
a computer system coupled in communication with the flow cytometer, the computer system having a display device, a storage device, and a processor to execute instructions, wherein the storage device stores data samples from the flow cytometer in response to running the labeled biological sample through the flow cytometer, and the processor executes instructions to process the stored data samples to generate thirty (30) or more dot plots of color assay results of the fluorescence given off by marked biological cells in the single labeled biological sample with a single run through the flow cytometer, wherein the display device displays the thirty (30) or more dot plots of the color assay results to a user.
18 . The system of claim 17 , wherein
the full spectrum flow cytometer further has five (5) or more optical fibers to collect light and direct it into the five (5) or more associated detector modules.
19 . The system of claim 17 , wherein
each of the N individual photo detectors has a bandpass filter in the optical path to pass a predetermined wavelength range of light for each individual photo detector.
20 . The system of claim 17 , wherein
one of the five (5) or more associated detector modules is an ultra violet detector module that detects fluorescent light over a wavelength range from 365 nanometers (nm) to 828.5 nanometers (nm) for fluorescent particles excited by an ultra violet laser of the five (5) or more lasers.