Apparatus and method for cell kill confirmation
Inventors
Savage, Frederick Hershel • Appleyard, David • Xia, Zheng • Ebersole, Matthew • McAda, Daniel
Assignees
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Publication Number
US-11427804-B2
Publication Date
2022-08-30
Expiration Date
Abstract
A method and related apparatus for confirming whether a kill laser successfully destroys an undesired population of cells includes introducing fluorescent dye into cells, exciting the cells with a detection laser or a light emitting diode to cause the cell to fluoresce for a first time, measuring the amount of fluorescence in the cells with a detector capable of emitting a detection pulse, classifying the cells via embedded processing as undesired or desired cells based on the amount of fluorescence, firing a kill beam with a kill laser at any undesired cells, measuring the amount of fluorescence in the cells a second time to determine whether a fluorescent event was generated from the kill beam striking the cells, and providing feedback to an operator of the kill laser as to whether any fluorescent events were generated from the kill beam striking the cells.
Core Innovation
A closed-loop feedback method provides for a system that destroys an undesired cell by detecting a first fluorescence of a cell with a detector and classifying the cell as an undesired cell or a desired cell based on the detected first fluorescence. The method destroys the undesired cell if classified as undesired and confirms whether the undesired cell was destroyed by detecting a second fluorescence of the cell as part of the same feedback loop.
The method further excites the cell with a detection laser or a light emitting diode to cause the first fluorescence, with the detection laser utilizing beam conditioning to aid in achieving a correct spot size at the cell. The method also includes aligning a kill laser, linking the illumination geometry used for classification and the kill action geometry used for destroying undesired cells.
In the sexed semen system context, sperm cells are excited by a detection laser and their sex is determined from the output of a first fluorescence detector using a computer processing unit. A kill laser destroys undesired sperm cells, and closed-loop feedback confirms whether undesired sperm cells are destroyed by using a second fluorescence detector output.
Claims Coverage
The independent claims cover feedback-based cell destruction with fluorescence-based confirmation, and separate formulations for a sexed semen system including detector-based sex determination, kill laser actuation, and closed-loop confirmation, with additional independent coverage for correlated double sampling and specific kill acquisition electronics architectures. Across the independent claims, the inventive features cluster into five main features.
Fluorescence-based confirmation feedback for destroying undesired cells
Detecting a first fluorescence of a cell with a detector; classifying the cell as an undesired cell or a desired cell based on detection of the first fluorescence; destroying the undesired cell if the cell is classified as an undesired cell; confirming whether the undesired cell was destroyed by detecting a second fluorescence of the cell.
Detection laser beam conditioning and kill laser alignment in the feedback loop
Exciting the cell with a detection laser or a light emitting diode to cause the first fluorescence, wherein the detection laser utilizes beam conditioning to aid in achieving a correct spot size at the cell; and aligning a kill laser.
Purity percentage determination using stored destruction-confirmation output
Storing an output related to whether the undesired cell was destroyed; using the output to determine whether to repeat any one or more of the detecting, classifying, destroying, confirming, and storing steps; and calculating a purity percentage associated with a population of cells.
Sexed semen system with first and second fluorescence detectors and closed-loop kill confirmation
A detection laser capable of exciting a sperm cell; a first fluorescence detector; a computer processing unit for determining the sex of the sperm cell based on an output of the first fluorescence detector; a kill laser; a second fluorescence detector; closed-loop feedback confirming whether undesired sperm cells are destroyed by the kill laser based on an output from the second fluorescence detector.
Kill acquisition electronics with voltage adjustment and bias supply for a second fluorescence detector
Kill acquisition electronics including at least a wide band transimpedance amplifier operatively connected to the second detector and readout circuitry; and a USB to serial converter or an analog signal input that allows a voltage at the second detector to be adjusted and a high voltage custom power bias supply which generates the voltage at the second fluorescence detector.
Correlated double sampler for kill acquisition electronics
Kill acquisition electronics comprising a correlated double sampler.
Kill/confirmation architecture for a sexed semen system with two detection laser lens assemblies
A detection laser capable of exciting a sperm cell; a fluorescence detector; a computer processing unit for determining the sex of the sperm cell based on an output of the fluorescence detector; a first detection laser lens assembly and a second detection laser lens assembly; a kill laser; and closed-loop feedback confirming whether undesired sperm cells are destroyed by the kill laser based on an output from the fluorescence detector.
The independent claims emphasize a feedback method and corresponding sexed semen systems that detect first fluorescence to classify cells as desired or undesired, destroy undesired cells using a kill laser, and confirm destruction using fluorescence detection. Additional independent coverage includes purity determination from stored confirmation output and sexed semen system integration with kill acquisition electronics, voltage-adjustable biasing, a correlated double sampler, and lens assemblies.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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