Ultrasound-modulated Fluorescence Research Articles

Newly-Engineered Materials for Bio-Imaging Technology: A Focus on the Hybrid System of Ultrasound and Fluorescence

As an emerging technique, ultrasound-modulated fluorescence (UMF), or ultrasound switchable fluorescence (USF) bioimaging has shown promising features to produce deep-tissue and high-resolution fluorescence imaging for biomedical research and health diagnosis. The success of UMF or USF heavily relies on the design of their contrast agents (CAs). We herein surveyed recent advances in the development of such unique CAs, including configuration, mechanism, stability, sensitivity, and selectivity. Meanwhile, UMF or USF instrumentation has emerged as developmental breakthrough technologies to existing bio-imaging techniques. The best performance of UMF or USF bio-imaging requires an interactive response between CAs and the instrument. In this review, the description of UMF or USF instrumentation are also included for clarification and better understanding. Finally, the UMF and USF's performance in bioimaging is evaluated based on signal-to-noise ratio, resolution, imaging depth and speed, using photoacoustic imaging (PAI) as a standard, a well-developed technique of hybrid bio-imaging. Unlike PAI, UMF or USF is still in its early stage. Although results demonstrated a proof-of-concept landmark being reached, significant efforts are needed to improve the performance of UMF or USF.

Ultrasound-modulated fluorescence based on donor-acceptor-labeled microbubbles.

A fluorescence resonance energy transfer (FRET)-based microbubble contrast agent system was designed to experimentally demonstrate the concept of ultrasound-modulated fluorescence (UMF). Microbubbles were simultaneously labeled with donor and acceptor fluorophores on the surface to minimize self-quenching and maximize FRET. In response to ultrasound, the quenching efficiency was greatly modulated by changing the distance between the donor and acceptor molecules through microbubble size oscillations. Both donors and acceptors exhibited UMF on individual microbubbles. The UMF strength of the donor was more significant compared to that of the acceptor. Furthermore, the UMF of the donor was observed from a microbubble solution in a turbid media. This study exploits the feasibility of donor–acceptor labeled microbubbles as UMF contrast agents.

Ultrasound-modulated fluorescence based on fluorescent microbubbles.

Ultrasound-modulated fluorescence (UMF) imaging has been proposed to provide fluorescent contrast while maintaining ultrasound resolution in an optical-scattering medium (such as biological tissue). The major challenge is to extract the weakly modulated fluorescent signal from a bright and unmodulated background. UMF was experimentally demonstrated based on fluorophore-labeled microbubble contrast agents. These contrast agents were produced by conjugating N-hydroxysuccinimide (NHS)-ester-attached fluorophores on the surface of amine-functionalized microbubbles. The fluorophore surface concentration was controlled so that a significant self-quenching effect occurred when no ultrasound was applied. The intensity of the fluorescent emission was modulated when microbubbles were oscillated by ultrasound pulses, presented as UMF signal. Our results demonstrated that the UMF signals were highly dependent on the microbubbles' oscillation amplitude and the initial surface fluorophore-quenching status. A maximum of ∼42% UMF modulation depth was achieved with a single microbubble under an ultrasound peak-to-peak pressure of 675 kPa. Further, UMF was detected from a 500-μm tube filled with contrast agents in water and scattering media with ultrasound resolution. These results indicate that ultrasound-modulated fluorescent microbubble contrast agents can potentially be used for fluorescence-based molecular imaging with ultrasound resolution in the future.

Effect of object size and acoustic wavelength on pulsed ultrasound modulated fluorescence signals

Detection of ultrasound (US)-modulated fluorescence in turbid media is a challenge because of the low level of fluorescent light and the weak modulation of incoherent light. A very limited number of theoretical and experimental investigations have been performed, and this is, to our knowledge, the first demonstration of pulsed US-modulated fluorescence tomography. Experimental results show that the detected signal depends on the acoustic frequency and the fluorescent target's size along the ultrasonic propagation axis. The modulation depth of the detected signal is greatest when the length of the object along the acoustic axis is an odd number of half wavelengths and is weakest when the object is an integer multiple of an acoustic wavelength. Images of a fluorescent tube embedded within a 22- by 13- by 30 mm scattering gel phantom (μ(s)∼15 cm(-1), g=0.93) with 1-, 1.5-, and 2 MHz frequency US are presented. The modulation depth of the detected signal changes by a factor of 5 depending on the relative size of the object and the frequency. The approach is also verified by some simple experiments in a nonscattering gel and using a theoretical model.

Effect of Fluorescent Particle Size on the Modulation Efficiency of Ultrasound-Modulated Fluorescence

To investigate whether the size of fluorescent particles affects the modulation efficiency of ultrasound-modulated fluorescence (UMF), we measured UMF and DC (direct current) signals of the fluorescence emission from four different sized fluorescent particles: (1) three carboxylate-modified fluorescent microspheres (FM) with diameters of 20 nm, 200 nm, and 1.0 µm and (2) streptavidin-conjugated Alexa Fluor 647 with a diameter of approximately 5 nm. The UMF and DC signals were simultaneously measured using a broadband lock-in amplifier and a narrowband amplifier, respectively. The ratio of the UMF strength to the DC signal strength is defined as the modulation efficiency. This modulation efficiency was then used to evaluate the effects of fluorophore size and concentration. Results show that the modulation efficiency was improved by approximately a factor of two when the size of the fluorescent particles is increased from 5 nm to 1 µm. In addition, the linear relationship between the UMF strength and ultrasound pressure (observed in our previous study) were maintained regardless of the fluorescent particle sizes.

Ultrasound-modulated fluorescence from rhodamine B aqueous solution

We report experimentally observed ultrasound-modulated fluorescence (UMF) from a submillimeter tube filled with rhodamine B aqueous solution. The tube was submerged in water and a scattering medium. Based on the measured data, we find that the UMF signals might be generated from three mechanisms: modulation of the excitation light, modulation of the emission light, and modulation of the properties of fluorophore. In addition, a linear relationship between the UMF and the drive voltage applied to the ultrasound transducer is found.

Microbubble-enhanced ultrasound-modulated fluorescence in a turbid medium

The feasibility of using ultrasound to modulate fluorescence in a turbid medium is still in debate due to the difficulty of detecting the modulated signal. We have demonstrated a system that could detect the weak signals of ultrasound-modulated fluorescence (UMF) by using a broadband lock-in amplifier and microbubbles as enhancement agents. By detecting the microbubble-enhanced UMF signal, a sub-millimeter fluorescent tube submerged in a turbid medium with a depth of 2 cm has been clearly observed with an ultrasonic spatial resolution. The modulation efficiency was significantly improved by using microbubbles, and was found to linearly increase with the drive voltage applied to the ultrasound transducer and the fluorophore concentration within the range adopted in this study. Possible modulation mechanisms are discussed.

Ultrasound-modulated fluorescence based on a fluorophore-quencher-labeled microbubble system

Ultrasound-modulated fluorescence from a fluorophore-quencher-labeled microbubble system driven by a single ultrasound pulse was theoretically quantified by solving a modified Herring equation (for bubble oscillation), a two-energy-level rate equation (for fluorophore excitation), and a diffusion equation (for light propagation in tissue). The efficiency of quenching caused by fluorescence resonance energy transfer (FRET) between the fluorophore and the quencher was modulated when the microbubble oscillates in size driven by the ultrasound pulse. Both intensity- and lifetime-based imaging methods are discussed in three different illumination modes of the excitation light: continuous wave (DC), frequency domain (FD), and time domain (TD). Results show that microbubble expansion opens a time period during which the quenching efficiency is dramatically reduced so that the emitted fluorescence strength and fluorophore lifetime are significantly increased. The modulation efficiency may even reach 100%. In addition, an important finding in this study is that in TD illumination mode, the modulated fluorescence photons may be temporally separated from the unmodulated photons, which makes the modulation efficiency limited only by thermal noise of the measurement system.

Quadrature Detection of Ultrasound-Modulated Photons with a Gain-Modulated, Image-Intensified, CCD Camera

Acousto-optic imaging promises to provide in vivo images of optical contrast but with the superior spatial reso- lution of ultrasound imaging. Here we present novel quadrature detection of ultrasound-modulated photons with a gain- modulated, image-intensified, CCD camera. The additional detection of ultrasound-modulated fluorescence photons dem- onstrates potential for in vivo acousto-optic molecular imaging.