Method and apparatus for calibrating a flow cytometer
View Patent ↗A method of continuously verifying proper sort calibration in a droplet sorting flow cytometer by selecting a fraction of droplets estimated to have substantially zero probability of containing a particle; applying one charge of a set of charges to the selected droplets in order to form a test stream out of the selected droplets; illuminating the droplets in the test stream; and detecting any light emitted or scattered by any particles in the selected droplets.
1. A method of continuously verifying proper sort calibration in a droplet sorting flow cytometer that selectively applies or does not apply one of a set of one or more electrical charges to a plurality of droplets as they form from a continuous fluid stream at a droplet break-off location and electrostatically sorts the droplets into two or more separate droplet streams, the selection of the electrical charge for each droplet being dependent on expected particles in the droplet based on a drop delay setting representing an estimate of the time elapsing between the moment a particle contained in the continuous fluid stream is detected at an interrogation location of the flow cytometer and the arrival of that particle at the droplet break-off location, the method comprising: selecting a fraction of droplets in one of said separate droplet streams estimated to have substantially zero probability of containing a particle; applying one charge of said set of charges to the fraction of selected droplets in order to form a calibration test stream out of the selected droplets; illuminating the droplets in the calibration test stream; and detecting any light emitted or scattered by any particles in the selected droplets.
2. The method of claim 1 , further comprising adjusting the drop delay setting if light detecting in the detecting step exceeds a threshold level.
3. The method of claim 2 , wherein the adjusting step is performed automatically.
4. The method of claim 1 , wherein the charge applied in the charging step is a neutral charge.
5. The method of claim 1 , wherein the illuminating and detecting steps are performed using an epi-illumination sensor.
6. A method of continuously verifying proper sort calibration in a droplet sorting flow cytometer that selectively applies or does not apply one of a set of one or more electrical charges to a plurality of droplets as they form from a continuous fluid stream at a droplet break-off location and electrostatically sorts the droplets into two or more separate droplet streams, the selection of the electrical charge for each droplet being dependent on expected particles in the droplet based on a drop delay setting representing an estimate of the time elapsing between the moment a particle contained in the continuous fluid stream is detected at an interrogation location of the flow cytometer and the arrival of that particle at the droplet break-off location, the method comprising:
(a) selecting a fraction of droplets in one of said separate droplet streams estimated to have substantially zero probability of containing a particle and applying one charge of said set of charges to the fraction of selected droplets in order to form a calibration test stream out of the selected droplets;
(b) illuminating the selected droplets in said calibration test stream by directing an illumination beam along a beam axis in a forward direction through a lens system to cause emission of fluorescent light by any particles contained in the droplets;
(c) using said lens system to collect some of said fluorescent light and direct it in a rearward direction along the beam axis;
(d) detecting at least some of the collected fluorescent light and generating an output signal representative of the detected light; and
(e) analyzing the output signal and, based on the analysis, automatically adjusting at least one of: (1) the drop delay setting; and (2) the amplitude of a charge in said set of charges.
7. The method of claim 6 , wherein the adjusting step comprises adjusting the drop delay setting as a function of the difference between the detected fluorescence emissions and the fluorescence emissions that would have been produced by droplets having the expected particles.
8. The method of claim 6 , wherein the adjusting step comprises adjusting the amplitude of a charge in said set of charges as a function of variation in an average peak intensity of fluorescence emissions detected for droplets containing fluorescent particles.
9. The method of claim 6 , wherein the illuminating step comprises using a fiber optic cable to guide light from a light source to a position adjacent said sorted droplet streams.
10. The method of claim 6 , wherein an electrical field is generated by a pair of electrically charged deflector plates, and wherein the illuminating step comprises illuminating the droplets as they move between the deflector plates.
11. The method of claim 10 , wherein at least one of the deflector plates is held by an electrically insulated support, and wherein the illuminating step comprises illuminating the droplets through a hole in the support as the droplets move through the electric field.
12. The method of claim 6 , further comprising using a light source to illuminate the particles at the interrogation location and in the separate droplet streams.
13. The method of claim 6 , further comprising performing steps (a)-(d) for each of the remainder of said two or more droplet streams, and wherein step (e) further comprises analyzing each of the respective output signals and adjusting at least one of: (1) the drop delay setting as a function of the difference between the detected fluorescence emissions and the fluorescence emissions that would have been produced by droplets having expected particles; and (2) the amplitude of a charge in said set of charges as a function of variations in the average peak intensity of fluorescence emissions detected for droplets containing fluorescent particles in at least one of said droplet streams.
14. The method of claim 13 , wherein an electrical field is generated by a pair of electrically charged deflector plates, and wherein the illuminating step comprises illuminating the droplets as they move between the deflector plates.
15. The method of claim 14 , wherein at least one of the deflector plates is held by an electrically insulated support, and wherein the illuminating step comprises illuminating the droplets through holes in the support as the droplets move through the electric field.
16. An apparatus for continuously and automatically calibrating a droplet sorting flow cytometer that selectively applies or does not apply one of a set of one or more electrical charges to a plurality of droplets as they break off of a continuous fluid stream at a droplet break-off location and electrostatically sorts the droplets into two or more separate droplet streams, the selection of the electrical charge for each droplet being dependent on expected particles in the droplet based on a drop delay setting representing an estimate of the time elapsing between the moment a particle contained in the continuous fluid stream is detected at an interrogation location of the flow cytometer and the arrival of that particle at the droplet break-off location, the apparatus comprising:
(a) a sort calibration system for selecting a fraction of droplets in one of said separate droplet streams estimated to have substantially zero probability of containing a particle and for applying one charge of said set of charges to the fraction of selected droplets in order to form a calibration test stream out of the selected droplets;
(b) an epi-illumination sensor downstream of the interrogation location for illuminating said calibration test stream, detecting any fluorescence emissions emitted by particles contained in the droplets of the calibration test stream, and generating an output signal representative of the detected fluorescence emissions; and
(c) a control operable to analyze the output signal and to automatically adjust at least one of: (1) the drop delay setting; and (2) the amplitude of a charge in said set of charges.
17. The apparatus of claim 16 , wherein the control is operable to adjust the drop delay setting as a function of the difference between the detected fluorescence emissions and the fluorescence emissions that would have been produced by droplets having the expected particles.
18. The apparatus of claim 16 , wherein the control is operable to adjust the amplitude of a charge in said set of charges as a function of variation in an average peak intensity of fluorescence emissions detected for droplets containing fluorescent particles.
19. The apparatus of claim 16 , wherein said epi-illumination sensor comprises a light source, a dichroic filter, a lens system, and a photodetector.
20. The apparatus of claim 19 , wherein the light source is operable to illuminate particles at the interrogation location.
21. The apparatus of claim 16 , wherein the control is operable to automatically maintain a phase relationship between droplet formation and droplet charging to within about 10% of its optimal phase of droplet formation.