Abstract:We introduce a method that combines two-photon polymerization (TPP) and surface functionalization to enable the indirect optical manipulation of live cells. TPP-made 3D microstructures were coated specifically with a multilayer of the protein streptavidin and nonspecifically with IgG antibody using polyethylene glycol diamine as a linker molecule. Protein density on their surfaces was quantified for various coating methods. The streptavidin-coated structures were shown to attach to biotinated cells reproducibly. We performed basic indirect optical micromanipulation tasks with attached structure-cell couples using complex structures and a multi-focus optical trap. The use of such extended manipulators for indirect optical trapping ensures to keep a safe distance between the trapping beams and the sensitive cell and enables their 6 degrees of freedom actuation.
Fluorescent observation of cells generally suffers from the limited axial resolution due to the elongated point spread function of the microscope optics. Consequently, three-dimensional imaging results in axial resolution being several times worse than the transversal. The optical solutions to this problem usually require complicated optics and extreme spatial stability. A straightforward way to eliminate anisotropic resolution is to fuse images recorded from multiple viewing directions achieved mostly by the mechanical rotation of the entire sample. In the presented approach, multiview imaging of single cells is implemented by rotating them around an axis perpendicular to the optical axis by means of holographic optical tweezers. For this, the cells are indirectly trapped and manipulated with special microtools made with two-photon polymerization. The cell is firmly attached to the microtool and is precisely manipulated with 6 degrees of freedom. The total control over the cells position allows for its multiview fluorescence imaging from arbitrarily selected directions. The image stacks obtained this way are combined into one 3D image array with a special image processing algorithm resulting in isotropic optical resolution. The presented tool and manipulation scheme can be readily applied in various microscope platforms.
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