Mr. Sheng-Hong Chen Profile

Sheng-Hong Chen

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Proceedings Article | 27 February 2019 Paper

Functional calcium imaging using optical-resolution photoacoustic microscopy in a 3D tumor cell culture

Wei-Wen Liu, Sheng-Hong Chen, Pai-Chi Li

Proceedings Volume 10878, 108782G (2019) https://doi.org/10.1117/12.2510936

KEYWORDS: Calcium, Tumors, Imaging systems, Signal detection, 3D image processing, Cancer, Photoacoustic imaging, Photoacoustic spectroscopy, Photoacoustic microscopy, Pulsed laser operation

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Imaging intracellular calcium dynamics to evaluate the cellular activity is crucial for an understanding of the related cell behavior and signal transduction. Identifying spatial-temporal activity patterns in three-dimensional (3D) tumor cell culture can provide novel insights into calcium-associated tumorigenesis and lead to optimizing the tumor-killing strategies. However, the information cannot be obtained without adequate penetration in 3D cell culture. Herein, we develop optical-resolution photoacoustic microscopy (OR-PAM) by tuning the laser wavelength to 627 nm to investigate calcium waves in 3D tumor cell culture using a novel photoacoustic contrast agent, Chlorophosphonazo-III (CPZ). CPZ has a peak optical absorbance at 650 nm. Not like another Bisazo derivative that can also be used for PA calcium imaging, Arsenazo-III, non-toxic and the high survival rate is observed even when cells are treated with 150 μM of CPZ (<97%). Moreover, CPZ can differentiate calcium changes using single wavelength like some conventional single wavelength calcium indicators used in optical imaging. Phantom results show that the photoacoustic (PA) signal intensity is highly correlated with calcium concentration using 100 μM or 150 μM CPZ, where R2 values are 0.94 and 0.97, respectively. To investigate the feasibility of live-cell PA calcium imaging in 3D cell culture, we evoke the intracellular calcium puffs of tumorspheres using thapsigargin and high concentration of extracellular calcium. A 2-fold enhancement of PA calcium intensity is observed after stimulating tumorspheres with thapsigargin or extracellular calcium. It demonstrates that the functional calcium imaging in 3D tumor cell culture can be detected by our OR-PAM system and CPZ can serve as a functional PA contrast agent.

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