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.
KEYWORDS: Tumors, 3D modeling, Gold, Absorption, Photoacoustic spectroscopy, 3D image processing, In vitro testing, 3D photoacoustic microscopy, Tissue optics, Nanoparticles
Three-dimensional (3D) in vitro models bridge the gap between typical two-dimensional cultures and in vivo conditions. However, conventional optical imaging methods such as confocal microscopy and two-photon microscopy cannot accurately depict cellular processing in 3D models due to limited penetration of photons. We developed a dualwavelength optical-resolution photoacoustic microscopy (OR-PAM), which provides sufficient penetration depth and spatial resolution, for studying CD8+ cytotoxic T lymphocytes (CTLs) trafficking in an in vitro 3D tumor microenvironment. CTLs play a cardinal role in host defense against tumor. Efficient trafficking of CTLs to the tumor microenvironment is a critical step for cancer immunotherapy. For the proposed system, gold nanospheres and indocyanine green (ICG) have been remarkable choices for contrast agents for photoacoustic signals due to their excellent biocompatibility and high optical absorption. With distinct absorption spectrums, targeted cells with gold nanospheres and ICG respectively can be identified by switching 523-nm and 800-nm laser irradiation. Moreover, we use an x–y galvanometer scanner to obtain high scanning rate. In the developed system, lateral and axial resolutions were designed at 1.6 μm and 5 μm, respectively. We successfully showed that dual-spectral OR-PAM can map either the distribution of CTLs with gold nanospheres at a visible wavelength of 523 nm or the 3D structure of tumor spheres with ICG in an in vitro 3D microenvironment. Our OR-PAM can provide better biological relevant information in cellular interaction and is potential for preclinical screening of anti-cancer drugs.
A method for concurrent photoacoustic (PA) and ultrasound (US) imaging with single laser pulses was previously demonstrated. An optical-absorbing multilayer film that can generate a US pulse based on the thermoelastic effect is used. With such a film, the generated US can be adjusted so that it does not overlap with the spectrum of the PA signal generated by the light transmitting through the layer. Thus, the US signal and the PA signal can be generated and separated by using a single laser pulse with spectral filtering. In this study, we continue with the same concurrent imaging approach and propose a cost-effective and portable design. The design consists of a pulsed laser diode with the repetition rate up to 25 kHz and energy of 2 μJ/pulse. A multilayer film is employed to generate narrow band US signals under laser excitation for US imaging. With simple spectral filtering, the PA signals and the US signals can be separated. With optical resolution, the system has a theoretical lateral resolution of 2 μm in PA imaging and 200 μm in US imaging. One of the applications of the proposed microscope is for tumor biology, where angiogenesis is an essential topic for understanding tumor growth and tumor metastasis. We will demonstrate performance of the proposed system by imaging vasculature networks.
Acoustic droplet vaporization has been proposed for sonoporation. In this study, we hypothesize that, by using gold nanodroplets (AuNDs), vaporization can be triggered with external application of laser irradiation. In addition, the vaporization assisted sonoporation can enhance delivery of gold nanoparticles (AuNPs) into the cells, thus potentially enhancing effects of plasmonic photothermal therapy. To test our hypothesis, in vitro studies were conducted. The delivery efficiency of AuNDs was also compared to that of AuNPs encapsulated in ultrasound microbubbles (AuMBs). The inertial cavitation dose (ICD), and optical density (OD) value of AuNPs were all measured under the applications of ultrasound only, laser only, and both ultrasound and laser. Results show that the cavitational effects and microbubble destruction were the highest with both ultrasound and laser being applied. In addition, destruction ratio of AuNDs was around 43%, compared to 35% microbubble destruction of AuMBs. Likewise, the OD value of AuNDs is 1.3 times higher than that of AuMBs under the same conditions, indicating that cavitation resulting from microbubble destruction did have the capability to assist the delivery of AuNPs into the cells. After the delivery, laser heating resulted in cell death. The cell viability with AuNDs was 45% left in the in vitro studies. Synergistic effects were also evident when combing laser with ultrasound.
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