Toward a Fully Automated High-Throughput Phototransfection System

Abstract

W e have designed and implemented a framework for creating a fully automated high-throughput phototransfection system. Integrated image processing, laser target position calculation, and stage movements show a throughput increase of O23Â over the current manual phototransfection method although the potential for even greater throughput improvements (O110Â) is described. A software tool for automated off-line singlecell morphological measurements, as well as real-time image segmentation analysis, has also been constructed and shown to be able to quantify changes in the cell before and after the process, successfully characterizing them, using metrics such as cell perimeter, area, major and minor axis length, and eccentricity values. ( JALA 2010;15:329-41) DJC is a recipient of a 2009 ALA Young Scientist Award from the IEEE Robotics and Automation Society (www.ieee-ras.org).

Software-aided automatic laser optoporation and transfection of cells

“…It would be possible to apply a more elaborate cell-recognition algorithm that, for example, determines the entire cell shape and marks precisely the desired number of target positions on that cell as described in Ref. 20 . However, concerning how different samples should be treated, there can be numerous variables and inconsistences as a result of different cell types, shapes as well as confluence and illumination conditions; making such an algorithm inherently complex and time consuming.…”

“…This process is tedious, time-consuming and while enabling to address individual cells which make single-cell studies possible, only a small number of cells can actually be targeted per procedure. The number of addressable cells for manual laser optoporation reported with different setups varies approximately between 20 and about 1000 cells per hour 9 17 20 . Another hurdle posed for automating the procedure is the necessary optimization of several parameters such as exposure time, laser power and optimal location of irradiation for specific cell lines or experimental setups which can be time consuming as well and may have to be repeated between experiments 15 .…”

“…An automation based on a sophisticated analysis of the cell morphology as described in Ref. 20 can by itself be very time consuming due to the many possible eventualities that may occur with different cell types, individual cell variations as well as varying illumination conditions and may ultimately offer little to no real net time benefit compared to a manual approach. Therefore, a simple yet rapid cell-detection algorithm based on the image analysis of contrast variations was implemented to identify cell positions suitable for transient laser illumination and combined with microscope hardware control.…”

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].…”

Frapid: achieving full automation of FRAP for chemical probe validation

“…In the arena of custom-built systems, the open source Micropilot [10] has indeed given scientists the ability to run automated FRAP assays using training sets to study complex biological questions. Other systems have also capitalized on homebuilt and programmable microscopes to process cells for other advanced optical experiments such as optoporation [11,12]. Although these systems are allowing scientists to run more advanced experiments without supervision, they lack the ability to deliver small molecules or proteins at predefined timepoints.…”