High resolution imaging beyond the acoustic diffraction limit in deep tissue via ultrasound-switchable NIR fluorescence

Abstract

Fluorescence imaging in deep tissue with high spatial resolution is highly desirable because it can provide details about tissue's structural, functional, and molecular information. Unfortunately, current fluorescence imaging techniques are limited either in penetration depth (microscopy) or spatial resolution (diffuse light based imaging) as a result of strong light scattering in deep tissue. To overcome this limitation, we developed an ultrasound-switchable fluorescence (USF) imaging technique whereby ultrasound was used to switch on/off the emission of near infrared (NIR) fluorophores. We synthesized and characterized unique NIR USF contrast agents. The excellent switching properties of these agents, combined with the sensitive USF imaging system developed in this study, enabled us to image fluorescent targets in deep tissue with spatial resolution beyond the acoustic diffraction limit.

High-Resolution Ultrasound-Switchable Fluorescence Imaging in Centimeter-Deep Tissue Phantoms with High Signal-To-Noise Ratio and High Sensitivity via Novel Contrast Agents

“…The FWHM of the transducer’s lateral acoustic profile was defined by the lateral focal size of the HIFU transducer (with a central frequency of 2.5 MHz and f-number of 0.83), which was measured ~0.42 mm. The estimated FWHM of the PSF (~0.9 mm) is ~2 times larger, larger also than what we have observed in our previous continuous wave (CW) mode system (0.29 mm) [13]. Current USF resolution is ~2 times worse than the lateral acoustic focal size of the HIFU transducer and ~3 times worse than the resolution our previous study reported, where we had adopted a CW mode system.…”

“…Fig 1(f) shows how their fluorescence intensity switches according to temperature; S1 Table provides a summary of those contrast agents. Impressively, all the intensity ratios (I ON /I OFF ) are >200, while previously developed agents have I ON /I OFF <10 [13],[15],[16],[20]. Fig 1(g) and 1(h) show that the switching property from ADP(CA) 2 -encapsulated Pluronic-F127 nanocapsules has excellent stability and repeatability.…”

“…We used the two edged micro-tubes (the first and fifth tubes) for image co-registration and filled them with the corresponding contrast agent of each modality: air for US imaging, ICG-based agent for color-1 USF imaging, ADP(CA) 2 -based agent for color-2 USF imaging, and ADP(CA) 2 -based agent for DF imaging. Our procedures for DF imaging and ultrasound imaging were similar to those we adopted in our previous paper [13]. …”

“…Using this method, high-resolution (from microns to hundreds of microns) fluorescence images have been demonstrated at millimeters depth in tissue phantoms [8–10],[12–14]. For example, previously our group developed a technique named continuous-wave ultrasound-switchable fluorescence (CW-USF) [13]. This technique has been demonstrated to achieve high spatial resolution of 290 μm in 8 mm-thick tissue phantoms by using indocyanine green (ICG)-encapsulated poly(N-isopropylacrylamide) (PNIPAM) nanoparticles (NPs) and a continuous-wave USF imaging system.…”

“…In this study, we developed an NIR frequency-domain ultrasound-switchable fluorescence (FD-USF) imaging technique to break these limitations. First, we discovered and synthesized unique USF contrast agents whose fluorescence emission can be switched on or off by a focused ultrasound wave [13],[14]. The emission intensity on-to-off ratio (I ON /I OFF ) can reach >200, which is >100 times higher than that of the current agent with a similar structure (~1.8) [15],[16].…”