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Once LEAP™ identifies the cells selected for laser-based manipulation, the laser(s) are fired at >1 kHz at the identified cells. Laser beam diameters, power density, pulse frequency, etc. are all controllable and enable optimization for a wide range of experimental objectives. By applying different laser wavelengths, cells can be eliminated in situ by non-destructive means or completely obliterated. Alternatively, by reducing laser power cells be transiently permeabilized to uptake a wide variety of experimentally important molecules, a process called “optoinjection.”
Cell purification
LEAP™’s unique ability for in situ cell identification and laser-based elimination enables both high yield and high purity of the desired cells. This outcome is supported by LEAP™’s closed-loop processing capability, during which LEAP™ will re-image a processed well and eliminate remaining undesired cells by conducting one or more iterations until a threshold outcome is achieved. To Cyntellect’s knowledge, LEAP™ is the only system that can enable this capability with minimal impact on cell yield because desirable cells are simply sitting in the well during all processing and elimination of undesirable cells.
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| LEAP™ Delivers High Purity and High Yield. A sample B cell population (green) contaminated with ~40% T cells tagged with a phycoerythrin-tagged T cell specific antibody (red) is imaged and analyzed by LEAP™. Following laser processing of this same sample by LEAP™, both resulting purity and yield exceeded 99%. |
The laser parameters can be adjusted to achieve cell elimination through one of three mechanisms:
- photothermal (results in cell coagulation without lysis);
- photochemical (results in apoptosis induction over 24 hours); and
- photomechanical (results in instant cell lysis)
Cell optoinjection
Laser-based manipulation of cells can be performed under sub-lethal conditions which enables efficient uptake of biomolecules by cells, a process called “optoinjection”, with relative low cell toxicity. Optoinjection has been demonstrated to deliver a wide variety of molecules from low molecular weight ions to higher molecular weight proteins. Optoinjection has even been demonstrated to deliver certain biomaterials, such as fluorescent semiconductor nanocrystals, which are greater than five nanometers in diameter.
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| Optoinjection of Living Cells. LEAP™ employs targeted lasers to transiently permeabilize cells allowing uptake of a wide variety of molecules including certain: (a) ions, (b) small molecules, (c) dextran, (d) proteins, (e) fluorescent biosensors, and (f) QDots™ quantum dots. |
Most importantly, optoinjection can be utilized to deliver certain biomolecules into cells that are typically resistant to other delivery techniques – for example, primary cells.
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