Apparatus and method for cell kill confirmation

Inventors

SAVAGE, Frederick • Appleyard, David • Xia, Zheng • Ebersole, Matthew D. • McAda, Daniel

Assignees

ABS Global Inc

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

The invention relates to producing a sexed semen sample using an optical closed-loop kill-confirmation workflow. A cell is illuminated with a focused beam of electromagnetic radiation, and a first fluorescence is detected to determine a set of characteristics of the cell. Based on the fluorescence, the cell is identified as an undesired cell, a kill laser is set to emit a focused beam of electromagnetic radiation at the cell according to a timing signal, and the kill laser is fired.

After the kill laser firing, a second fluorescence by the cell is detected and used to determine a type of event based on the second fluorescence. The type of event is one of a slice, a kill, or a miss, thereby confirming the outcome of the kill pulse for kill-irradiated cells. The described approach supports automated feedback for operator re-alignment and maintaining kill performance throughout a run.

The document further describes architectures and electronics for capturing kill-confirmation information. Kill acquisition electronics include a wide band transimpedance amplifier connected to a second fluorescence detector, a high-speed comparator with an adjustable VREF and comparator/resistor ladder configuration, a correlated double sampler with a high-speed ADC, or direct high-speed analog-to-digital converter digitization.

The claims also describe a particle orientation-determination method using first and second fluorescence to identify a particle as a first type or a second type and determine orientation from both fluorescence measurements while providing output to the user. The optical and flow arrangement is described in terms of fluorescence behavior under excitation and microfluidic focusing with sheath fluid and optionally inertial flow focusing, to support event-type determination and provide kill-confirmation fluorescence for resolving desired and undesired cell populations.

Claims Coverage

The document includes two independent claims that cover a sexed semen production method with optical kill-confirmation and a particle orientation-determination method. Across the independent claims, two inventive features are centered on sequential fluorescence measurements and event or orientation classification from those measurements.

The independent claims center on closed-loop kill-confirmation by classifying a cell as undesired from first fluorescence, firing a kill laser based on a timing signal, and determining a slice, kill, or miss event type from second fluorescence, together with orientation determination by using first and second fluorescence to classify particle type and determine orientation.

Stated Advantages

Documented Applications