Mr. Felix S. Alfonso Profile

Felix S. Alfonso

at Stanford Univ

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Proceedings Article | 4 March 2019 Presentation

Quantitative phase imaging of action potentials in mammalian cell culture (Conference Presentation)

Tong Ling, Kevin Boyle, Georges Goetz, Peng Zhou, Yi Quan, Felix Alfonso, Tiffany Huang, Daniel Palanker

Proceedings Volume 10887, 1088715 (2019) https://doi.org/10.1117/12.2508813

KEYWORDS: Action potentials, Phase imaging, Microscopy, Atomic force microscopy, Interferometry, Refractive index, Ions

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Movements of the cell membrane accompanying action potentials have been detected by various methods, including reflection of a laser beam, atomic force microscopy and even bright-field microscopy. However, imaging of the entire cell dynamics during action potential has not been achieved, and the mechanism behind this phenomenon is still actively debated. Here we report full-field interferometric imaging of cellular movements during action potential by simultaneous quantitative phase microscopy (QPM) and multi-electrode array (MEA) recordings. Using spike-triggered averaging of the movies synchronized to electrical recording, we demonstrate deformations of up to 3 nm (0.9 mrad) during the action potential in spiking HEK-293 cells, with a rise time of 4 ms. The time course of the optically-recorded action potential is very similar to intracellular potential recorded with a whole-cell patch clamp, while the time derivative of the rising edge of the optical spike matches the timing and duration of the extracellular electrical recording on MEA. In some cells, phase increases at the center and decreases along the cell boundaries, while in others it increases on one side and decreases on the other. These findings suggest that optical phase changes during an action potential are due to cellular deformation, likely associated with changes in the membrane tension, rather than refractive index change due to ion influx or cell swelling. High-speed QPM may enable all-optical, label-free, full-field imaging of electrical activity in mammalian cells.

Proceedings Article | 4 March 2019 Presentation

Automated detection of action potentials in interferometric imaging of spiking cells (Conference Presentation)

Kevin Boyle, Tong Ling, Georges Goetz, Peng Zhou, Yi Quan, Felix Alfonso, Tiffany Huang, Daniel Palanker

Proceedings Volume 10890, 1089025 (2019) https://doi.org/10.1117/12.2508871

KEYWORDS: Action potentials, Interferometry, Microscopy, Imaging systems, Iterative methods, Temporal resolution, Data analysis

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Wide-field interferometric imaging systems can detect mechanical deformations of a cell during an action potential (AP), such as in quantitative phase microscopy, which is highly sensitive to the changing optical path length. This enables non-invasive optophysiology of spiking cells without exogeneous markers, but high-fidelity imaging of such deformations requires averaging of a large number of spikes synchronized by electrical recordings. We have developed new iterative methods for detecting single APs from quantitative phase microscopy of spiking cells, enabling an all-optical detection system with high accuracy and good temporal resolution. We demonstrate performance of the method across multiple preparations of spiking HEK-293 cells and compare the outcomes of the all-optical measurements with the ground truth detected on a multi-electrode array. We initially use a spike-triggered average, synchronized to an electrical recording, to measure deformations during the AP in spiking cells, which reach up to 3 nm (0.9 mrad) with a rise time of 4 ms and fall time of about 120 ms. Based on this knowledge of the AP dynamics, optical data analysis can provide reliable spike detection, within a standard deviation of 11.6 ms (9.7% of the length of the action potential) with an 8.5% false negative detection rate. The method is robust to natural variations between cells and can be modified to function without any prior knowledge of the AP dynamics. Such a system could achieve high-throughput measurements of network activity in culture and help identify the mechanisms linking cell deformations to the changes of transmembrane potential.

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