Software-aided automatic laser optoporation and transfection of cells

Abstract

Optoporation, the permeabilization of a cell membrane by laser pulses, has emerged as a powerful non-invasive and highly efficient technique to induce transfection of cells. However, the usual tedious manual targeting of individual cells significantly limits the addressable cell number. To overcome this limitation, we present an experimental setup with custom-made software control, for computer-automated cell optoporation. The software evaluates the image contrast of cell contours, automatically designates cell locations for laser illumination, centres those locations in the laser focus, and executes the illumination. By software-controlled meandering of the sample stage, in principle all cells in a typical cell culture dish can be targeted without further user interaction. The automation allows for a significant increase in the number of treatable cells compared to a manual approach. For a laser illumination duration of 100 ms, 7-8 positions on different cells can be targeted every second inside the area of the microscope field of view. The experimental capabilities of the setup are illustrated in experiments with Chinese hamster ovary cells. Furthermore, the influence of laser power is discussed, with mention on post-treatment cell survival and optoporation-efficiency rates.

Multiphoton imaging with a novel compact diode‐pumped Ti:sapphire oscillator

“…The non-linear nature of the excitation process provides intrinsic 3D-imaging capability by minimizing out-of-focus excitation. A similar reasoning applies to harmonic generation microscopy (Sun, 2005) and other nonlinear interactions between light and cells/ tissue like laser-assisted cell transfection (Breunig et al, 2015). Under ideal conditions, when the incident pulses are only minimally absorbed and/or scattered, high resolution imaging at depths in the mm range inside tissue can be achieved in vivo (Kobat et al, 2011).…”

Plasmonic Intracellular Delivery

“…For instance, the delivery of gene-editing tools could be used to manipulate cells and tissues for regenerative medicine or engineer cells for personalized cell therapies [4][5][6][7]. Intracellular delivery methods include biological vectors such as viruses, chemical modifications of delivery cargoes such as lipofection, and physical techniques such as microinjection, electroporation, and optoporation [11][12][13][14][15][16][17][18][19][20][21][22][23][24]. While research efforts have led to a continuous increase in efficiency and sophistication, each of the currently available approaches has its own advantages and disadvantages.…”

“…Optoporation, a physical delivery technique, utilizes a tightly focused laser beam to create a transient pore in the cell membrane [18,19,33,34]. This technique offers high delivery efficiency, high cell viability and is versatile with respect to cargo and cell type.…”

Laser-assisted photoporation: fundamentals, technological advances and applications

“…With image processing, the location of the individual cells can be determined. In combination with an auto-focus system to control the axial position of the photoporation laser beam, this allows cells to be photoporated in an automated fashion [107,108]. In one example, it was demonstrated that ~300 cells/min can be photoporated that way [107].…”

“…In combination with an auto-focus system to control the axial position of the photoporation laser beam, this allows cells to be photoporated in an automated fashion [107,108]. In one example, it was demonstrated that ~300 cells/min can be photoporated that way [107]. Finally, a microfluidic approach has also developed in an attempt to increase photoporation throughput, with cells flowing one by one through the focused photoporation laser beam.…”