Particle sorting apparatus and method
View Patent ↗A particle analyzing and/or sorting apparatus and the associated methods. One aspect of the described embodiments relates to an analyzer, or a sorter, having acquisition and sort electronics in the form of a field programmable gate array for processing detected signals. Another aspect relates to a droplet based approach of analyzing and sorting particles and may further include a dynamic element, such a dynamic drop delay. In still another broad aspect, an apparatus and method for dynamically varying other sorting parameters.
1. A method of sorting particles comprising the steps of:
delivering a fluid stream containing particles to an inspection zone and interrogating the particles at the inspection zone with a source of electromagnetic radiation wherein interrogated particles produce emitted or reflected electromagnetic radiation;
detecting the emitted or reflected electromagnetic radiation of the interrogated particles and producing at least one signal containing at least one pulse representative of at least one interrogated particle characteristic;
comparing the at least one signal to a trigger threshold to determine the occurrence of a particle event;
generating an event memory map having event windows, each of the event windows associated with an expected sortable unit of the fluid stream;
coordinating each particle event to one of the event windows in the event memory map;
determining measured pulse parameters for each particle event from the at least one signal;
classifying particles based upon the measured pulse parameters;
determining event parameters for each of the event windows;
applying a sort logic to make a sort decision based on the event parameters and particle classifications associated with each of the event windows;
sorting particles within each of the event windows according to the respective sort decisions; and
collecting at least one population of sorted particles.
2. The method according to claim 1 wherein the step of generating at least one signal containing at least one pulse representative of the at least one interrogated particle characteristic further comprises the steps of:
generating a first signal based upon the emitted or reflected electromagnetic radiation produced by excited particles at the inspection zone in a first direction; and
generating a second signal based upon the emitted or reflected electromagnetic radiation produced by excited particles at the inspection zone in a second direction.
3. The method according to claim 1 wherein the step of coordinating each particle event to one of the event windows in the event memory map further comprises the step of comparing the time stamp of the particle event to a time associated with the event window in the event memory map.
4. The method according to claim 1 wherein an event parameter for each of the event windows corresponds to an amount of time representing sortable units.
5. The method according to claim 1 wherein the sortable units comprise expected droplets.
6. The method according to claim 5 further comprising the steps of:
forming the fluid stream into droplets downstream of the inspection zone;
charging the droplets according to their associated sort decision; and
deflecting charged droplets into collection receptacles.
7. The method according to claim 6 wherein the step of forming droplets further comprises applying a drop drive signal to an oscillator for forming the fluid stream into droplets.
8. The method according to claim 7 wherein the event windows of the event memory map are generated in response to the drop drive signal.
9. The method according claim 1 further comprising the step of recording the sort decision in the event memory map for each of the event windows.
10. The method according to claim 9 further comprising the step of recording the event parameters resulting in a particle in one of the event windows not being sorted.
11. The method according claim 1 wherein the event parameters are selected from the following: droplet number, droplet time stamp, number of particle events in a droplet; sort results, light scatter, light extinction, fluorescence, event duration, event start time, location of a particle in a droplet, particle classifications and combinations thereof.
12. The method according claim 1 wherein the measured pulse parameters are selected from the group consisting of: forward pulse peak, side pulse peak, forward pulse area, side pulse area, forward pulse slope, side pulse slope, pulse duration, and combinations thereof.
13. The method according to claim 1 wherein the particles comprise sperm cells and wherein the particle classification comprises classifications selected from the group consisting of: X-chromosome bearing sperm; Y-chromosome bearing sperm; oriented sperm; unoriented sperm; live sperm; dead sperm, and combinations thereof.
14. The method according to claim 1 wherein the steps of comparing the at least one signal to a trigger threshold to determine the occurrence of a particle event, coordinating each particle event to one of the event windows in the event memory map, determining measured pulse parameters for each particle event, and classifying particles based upon the measured pulse parameters are performed on a field programmable gate array.
15. The method according to claim 1 wherein the step of classifying particles based upon the measured pulse parameters further comprises the step of comparing measured pulse parameters to a bitmap in the form of a look up table.
16. The method according to claim 1 further comprising the step of generating a state record from the event memory map.
17. The method according to claim 16 wherein the state record comprises the information required for making a sort decision.
18. The method according to claim 1 wherein information stored in the event memory map is applied in a sort logic to subsequent particles.
19. The method according to claim 18 further comprising the step of comparing particle classifications within a single one of the event windows.
20. The method according to claim 19 further comprising the step of modifying the sort decision for event windows having more than one classification of particles based upon a projected purity of the collected population of sorted particles.
21. The method according to claim 1 wherein the particles comprise sperm cells.
22. The method according to claim 21 wherein measured pulse parameters are compared against a bitmap look up table to classify a subpopulation of sperm cells as oriented live sperm cells.
23. The method according to claim 22 wherein measured pulse parameters are compared against a bitmap look up table to classify a sperm cells as X chromosome bearing sperm cells or Y chromosome hearing sperm cells.
24. The method according to claim 23 wherein only sperm cells classified as live oriented sperm cells are further classified as X chromosome bearing or Y chromosome bearing sperm cells.
25. The method according to claim 1 further comprising the step of detecting from the event memory expected changes in operational parameters.
26. The method according to claim 25 further comprising the step of applying a compensation when expected changes in operational parameters are detected from information contained in the event memory map.
27. The method according to claim 26 wherein the operational parameters comprises a sort delay and wherein the step of applying a compensation when expected changes in operational parameters are detected further comprises changing the sort delay when a particle is detected at the tail end of an expected sortable unit.
28. The method according to claim 26 wherein the step of applying a compensation further comprises modifying the sort delay for individual expected sortable units.
29. The method according to claim 26 wherein the sort delay is increased for an expected sortable unit with a particle located at tail end of the unit.
30. The method according to claim 26 wherein the sort drop delay is decreased for an expected sortable unit with a particle located at the leading end of the unit.