Abstract
For many years, investigators have sought after high-resolution fluorescence imaging in centimeter-deep tissue because many interesting in vivo phenomena—such as the presence of immune system cells, tumor angiogenesis, and metastasis—may be located deep in tissue. Previously, we developed a new imaging technique to achieve high spatial resolution in sub-centimeter deep tissue phantoms named continuous-wave ultrasound-switchable fluorescence (CW-USF). The principle is to use a focused ultrasound wave to externally and locally switch on and off the fluorophore emission from a small volume (close to ultrasound focal volume). By making improvements in three aspects of this technique: excellent near-infrared USF contrast agents, a sensitive frequency-domain USF imaging system, and an effective signal processing algorithm, for the first time this study has achieved high spatial resolution (~ 900 μm) in 3-centimeter-deep tissue phantoms with high signal-to-noise ratio (SNR) and high sensitivity (3.4 picomoles of fluorophore in a volume of 68 nanoliters can be detected). We have achieved these results in both tissue-mimic phantoms and porcine muscle tissues. We have also demonstrated multi-color USF to image and distinguish two fluorophores with different wavelengths, which might be very useful for simultaneously imaging of multiple targets and observing their interactions in the future. This work has opened the door for future studies of high-resolution centimeter-deep tissue fluorescence imaging.
Highlights
Fluorescence microscopy has broken many physical limits to allow imaging of biological samples and live tissues with unprecedented resolution, depth, contrast, sensitivity, and specificity [1]
High-resolution deep-tissue fluorescence imaging is highly desirable for at least the following reasons [3–17]: (1) Much interesting in vivo micro-information may deeply locate in tissue, including microcirculation, micro-angiogenesis, micro-lymphangiogenesis, tumor micro-metastasis, and vascularization of implanted tissue scaffolds, etc; (2) Far-red or near-infrared (NIR) light can penetrate centimeter-deep tissue via scattering, the micro-information is completely blurred; (3) Compared with other imaging modalities such as ultrasound (US), computed tomography (CT), magnetic resonance imaging (MRI), or positron emission tomography (PET), fluorescence imaging stands out in the following respects: high sensitivity, low cost, usage of non-ionizing radiation, feasibility of multi-wavelength imaging, and feasibility of tissue structural, functional, and molecular imaging
We achieved high-resolution fluorescence imaging in centimeter-deep tissue phantoms with high signal-to-noise ratio (SNR) and picomole sensitivity by using a series of newly developed technologies
Summary
Fluorescence microscopy has broken many physical limits to allow imaging of biological samples and live tissues with unprecedented resolution, depth, contrast, sensitivity, and specificity [1]. Previously our group developed a technique named continuous-wave ultrasoundswitchable 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. Despite these successes, these technologies face a fundamental barrier: the quick degradation of SNR and sensitivity to an unacceptable level with the increase of the imaging depth, the spatial resolution, or both. High-resolution centimeter-deep tissue fluorescence imaging with high SNR and high sensitivity still remains unachievable
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