Multiview microscopy of single cells through microstructure-based indirect optical manipulation

Abstract

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.

Single-Cell Elasticity Measurement with an Optically Actuated Microrobot

“…The cell stiffness was measured with the tailor-made microtools described above. The microtools were actuated with a holographic optical trap (HOT) system that can create multiple trapping foci and move them in 3D with high precision, as demonstrated in [ 12 ]. In the present experiments, we created 4 trapping foci forming a square of 14 μm side length and moved them with their mutual positions unchanged.…”

“…Furthermore, microtools have been developed to control the flow of the solvent that carries these biological objects [ 6 , 7 ] or to characterize their composition [ 8 ]. The complexity of microrobots spans from simple microspheres [ 1 , 9 ] to complex tailor-made microstructures [ 4 , 10 , 11 , 12 ], and sometimes a group of such structures is needed to perform specific tasks [ 13 , 14 ]. Most often, these microtools are actuated and guided by optical means, but magnetic [ 15 , 16 ] or acoustic [ 17 ] controls are also applied.…”

“…Since the size of these microrobots can range from sub-micrometers to a few hundreds of micrometers, they can be easily optimized for the manipulation of single cells. A broad range of tasks can be performed: cells can be actuated with the tools, which includes their simple translation or rotation either on a hard surface [ 4 , 5 ] or in 3D [ 11 , 18 ]; the tools can enhance imaging of cells [ 12 ]; the internal structure of the cells can be altered by punching holes in them with the tools [ 19 ]; and such microtools have a great potential even in performing cell-to-cell interaction experiments with high precision and selectivity.…”

Applications of Optically Controlled Gold Nanostructures in Biomedical Engineering

“…Later, Vizsnyiczai et al presented a multiview microscopy of single cells based on holographic optical tweezing of microstructures made with two-photon polymerization. The presented tool allows for precise manipulation of the cells in 6 degrees of freedom and solves the axial resolution problem that comes with fluorescence imaging (Vizsnyiczai et al, 2020 ).…”

Universal Light-Driven Micro/Nanoscale Rotors

“…The former often relies on complex optics to create designer laser beams that lead to the strong asymmetric light-rotor interactions and thus the large enough optical torques for the rotation 13,18 . In particular, to rotate homogenous and symmetric particles 19 and live cells 20 , multiple-beam optical tweezers were proposed. Moreover, optical torques are reduced significantly when the rotor size decreases 21 .…”