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Updated: Nov 15, 2025
Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy
Published on: January 7, 2019
Improving sensitivity and imaging depth of ultrasound-switchable fluorescence via an EMCCD-gain-controlled system and
Tingfeng Yao1,2, Yang Liu1,2, Liqin Ren1,2
1Ultrasound and Optical Imaging Laboratory, Department of Bioengineering, The University of Texas at Arlington, Arlington, TX, USA.
Background:
The ultrasound-switchable fluorescence (USF) technique was recently developed to achieve high-resolution fluorescence imaging in centimeters-deep tissue. This study introduced strategies to significantly improve imaging sensitivity and depth using an electron multiplying charge-coupled device (EMCCD) camera-based USF imaging system and a newly developed USF contrast agent of indocyanine green (ICG)-encapsulated liposomes. For a quantitative study, a phantom of a sub-millimeter silicone tube embedded in centimeter-thick chicken breast tissue was adopted in this study as a model.
Methods:
The synthesized ICG-liposome was characterized and compared with the previously reported ICG-nanogel. The exposure of the EMCCD camera was controlled via the MATLAB (The MathWorks, Inc. USA), instead of an external hardware trigger. The stability of the electron multiplying (EM) gain of the EMCCD camera was compared between two trigger modes: the MATLAB trigger mode and the external hardware trigger mode. The signal-to-noise ratio (SNR) of the USF imaging with different EM gain in various thick tissue was studied.
Results:
The hydrodynamic size of the ICG-liposome was ~181 nm. The ICG-liposome had a sharper temperature switching curve and a better USF performance than the previously reported ICG-nanogel. The EM gain was more stable in MATLAB trigger mode than the external hardware trigger mode. Although, as usual, the SNR decreased quickly with the increase of the tissue thickness, the proposed strategies improved the SNR and the imaging depth significantly by adopting the novel contrast agent and controlling the EM gain.
Conclusions:
We successfully imaged the sub-millimeter silicone tube with an inner diameter of 0.76 mm and an outer diameter of 1.65 mm in 5.5 cm-thick chicken breast tissue using 808 nm excitation light with a low intensity of 28.35 mW/cm2, the improved EMCCD camera-based USF imaging system and the novel ICG-liposomes.
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