Multiple-Beam Laser Guidance-Based Microscope for Patterning Adult Cardiomyocytes

Abstract

Traditional cell-culture techniques lack the spatial control of single cells that is necessary to recreate the cell-cell contact arrangement found in tissue. Since Ashkins developed optical force-based cellmanipulation techniques [1], the laser microbeam has been used in a microscopic system to explore various biological interactions at molecular and cellular levels. These explorations of a single cell or subcellular organelle involve trapping (using a laser-tweezers microscope [2]) and guidance (using a laser-guided direct writing microscope [3,4]). When laser tweezers are used, a high numerical aperture (NA) microscope objective generates a strongly focused laser beam, which 3D traps a particle, such as a biological cell, in the beam's focal point. In laser-guided direct writing, a low NA microscope objective generates a weakly focused laser beam, which traps a particle in the beam axis and guides it to move along the direction of beam propagation.Based on these techniques, we have developed a laser cell-micropatterning system in which the laser beam is focused in a transition state between generating an optical trap and optical guidance [5]. Using this system, a single biological cell can be trapped in a typical (e.g., 30mm) cell culture dish and patterned onto a designated cell culture niche with very high spatial and temporal resolution [6][7][8].However, this and the other currently available laser-based single-cell-manipulation techniques can be used to pattern only spherical cells, not irregularly shaped cells such as rod-shape cardiomyocytes. The work stems from the use of a spatial light modulator (SLM) loaded with a computer generated phase map to shape a single laser beam into multiple microbeams analogous to techniques used in holographic optical tweezers (HOT) [9]. In contrast to the optical configuration in HOT, our system uses a low NA objective to produce multiple weakly focused laser microbeams in our multiple beam laser guidance system. Based on the cell image acquired during laser cell patterning, the multiple beams can be distributed around the outer contour of an irregularly shaped cell to achieve accurate cell patterning. In addition to describing the principle and practice of the system design, here we present what is to our knowledge the first achievement of patterning large, irregularly (rod) shaped adult rat cardiomyocytes (ACMs) in an end-to-end connected alignment to replicate the in vivo heart-muscle structure without use of substrate surface modifications that may interfere with in vivo-like cell-cell and cell-extracellular matrix interactions.The basic structure of the multiple-beam laser cell-patterning system is schematically shown in Figure 1. The cell suspension was loaded into the microfluidics-based cell feeding system built in the celldeposition chamber, with a microfluidic channel width of 200 µm. The guidance region (also the imaging region of the objective) was initially focused onto the outlet of the cell-feeding microfluidic channel by controlling the movement of the ce...

Application of optical tweezers in cardiovascular research: More than just a measuring tool

“…However, this early version of the patterning system is able to pattern only spherical cells and not irregularly shaped cells. By using a computer-mapped spatial light modulator to shape a single microbeam into multiple microbeams, a later version of the micropatterning system is able to place beams around the contour of large and irregularly shaped cells to achieve precise patterning, such as end-to-end tandem alignment of rat cardiomyocytes, without adding surface modifiers ( Schmidt et al, 2016 ). It is thus clear that optical tweezers are not just a nanophysical measuring tool but have versatile potential to be explored in biomedicine.…”