Planar lightsheet optical tweezer pLOT for 2D trapping and imaging of freely-moving live cells
摘要
We report the realization of the first planar optical trap and imaging system using a sheet of light that allows interrogation of living specimens in a plane. An orthogonal widefield detection is employed to directly visualize the trapping of the target object (dielectric beads/cells) in a plane. The planar trap is realized on an inverted optical stage with illumination from the bottom. The system uses a combination of a cylindrical lens and a high NA objective lens to generate a tightly focused diffraction-limited sheet of light. For trapping objects in a selective XZ-plane, the sample (beads/cells suspended in a solution) is illuminated by a sheet of light (along the Z-axis with coverslip along XZ), whereas the detection is carried out perpendicular to the coverslip (along the Y-axis). Orthogonal detection allows direct visualization of the trapped object in the 2D plane illuminated by the lightsheet. The generated PSF has a dimension of 2073.84 μm2 (along XZ), which defines the active trap region/zone. To estimate the trap stiffness, both variance-based equipartition and video-based object tracking methods are employed. Results (image and video) show real-time trapping of dielectric beads and live cells in the trap zone (2D plane). Prolonged exposure shows objects getting trapped and builds up a 2D layer of beads/cells, demonstrating stable trapping in a selective layer. The technique is furthered by successfully trapping fluorescently labeled live cells in a single plane and simultaneously performing fluorescence imaging on the go with sub-cellular resolution. The potential of the planar trap lies in its ability to confine objects (such as dielectric beads and cells) in a selective 2D plane and allow interrogation, thereby opening up the possibility of new kinds of studies in optical manipulation, fluorescence microscopy and biotechnology.
Statement of Significance: The ability to confine and interrogate living specimens (cells) in a plane is an incredible feat that paves the way for new kinds of studies. Currently, there is no available technique that can trap microscopic living objects in a 2D plane. The successful trapping of live cells in a plane, and high-resolution fluorescence imaging on the go, have applications in the broad field of fluorescence microscopy, optical physics and biotechnology.