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

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.

Fig 1. Characterization of ADP(CA)₂ and ADP(CA)₂-based USF contrast agents.

(a) A scheme displays the principle of USF contrast agents (LCST: lower critical solution temperature). (b) and (c) show the chemical structure, absorption and emission spectra (in dichloromethane) of the environment-sensitive dye, ADP(CA)₂. (d) and (e) show the fluorescence intensity of ADP(CA)₂ as function of polarity and viscosity of the solvent, respectively. Five solvents with different polarity index were employed, which are water (62.8), dimethyl sulfoxide (45.1), 1,2-dichloroethane (41.3), 1,4-dioxane (36) and toluene (33.9). A small polarity index represents low polarity. Viscosity was adjusted by varying the volume ratio of glycerol/(ethylene glycol). A high ratio means a high viscosity. (f) The switching relationship between the fluorescence intensity of these ADP(CA)₂-based USF contrast agents and the temperature: Pluronic-F127 (stars); Pluronic-F98 (squares); Pluronic-F98~PEG20k (diamonds); Pluronic-F98~PEG30k (triangles); Pluronic-F98~PEG40k (solid circles). (g) shows the two switching curves measured at different time from the adopted contrast agent, ADP(CA)₂-encapsulated Pluronic-F127 nanocapsules. The blue line represents the data acquired three days after the agent was synthesized. The red dashed line represents the data acquired ten months after the agent was synthesized. (h) Repeatable switching of the fluorescence intensity measured from ADP(CA)₂-encapsulated Pluronic-F127 nanocapsules at low (15°C) and high (35°C) temperatures.

Fig 2. The FD-USF imaging system.

(a) The schematic diagram of the USF imaging system. PDG: pulse delay generator; FG: function generator; RF-Amp: radio-frequency power amplifier; MNW: matching network; HIFU: high intensity focused ultrasound; PMT: photomultiplier tube; F1-F5: emission filters; F6: excitation filter; DAC: data acquisition card; PC: personal computer. (b) The schematic diagram of time sequences of six different events in USF imaging, including HIFU transducer gating pulse, temperature change at HIFU focus, modulated laser output, USF signal, lock-in reference and lock-in output. (c) The sample configuration, including the sample, the excitation and emission fiber bundles, and the HIFU transducer.

Fig 3. Typical USF signals and the correlation method.

(a) and (b) show the typical USF signal and background noise acquired from ADP(CA)₂-encapsulated Pluronic-F127 nanocapsules in the 8-mm thick tissue USF experiment, respectively. (c) The normalized USF signals (from ADP(CA)₂-based agents) with different signal strengths (strength 1–7: 171, 239, 290, 310, 325, 306, and 270 mV, respectively) to show that the shape is independent of the signal strength. Different signal strengths were generated by varying the HIFU driving voltage. (d) The normalized USF signals from ADP(CA)₂-encapsulated Pluronic-F127 nanocapsules (blue solid line) and ICG-encapsulated PNIPAM NPs (red dash line) agents to show that the shape is dependent on the type of the agents. (e) and (f) are the normalized USF profiles of the micro-tube (filled with the ADP(CA)₂-based agent) before (SNR: 88) and after (SNR: 300) correlation analysis, respectively. The experiment conditions are presented in Methods.

Fig 4. Images of micro-tubes in a tissue-mimic phantom.

(a) The photographs and schematic diagram of the tissue-mimic silicone phantom (on x-y plane): front view (left photo), back view (right photo) and the cross section of the micro-tube structure on the x-y plane (the bottom figure in (a)). (b) The image acquired by directly detecting fluorescence (DF) without ultrasound. (c) and (d) show the USF images acquired from ICG-encapsulated PNIPAM NPs (color-1) and ADP(CA)₂-encapsulated Pluronic-F127 nanocapsules (color-2), respectively. (e) and (f) are the overlapped images between USF images ((c) and (d)) and direct fluorescence image (b). (g) Ultrasound image of the micro-tubes acquired from the same ultrasound transducer (i.e. a C-mode ultrasound image). The three non-edge tubes (the second, third, and fourth tubes) were filled with water (as background control), ICG-based agent (color-1) and ADP(CA)₂-based agent (color-2), respectively. The two edged micro-tubes (the first and fifth tubes) were used for image co-registration and were filled with the corresponding contrast agent of each modality (see Methods). (h) The normalized profiles along x-axis acquired from different imaging modalities (i.e. the cross section of the five micro-tubes in the silicone phantom). Due to the significant overlap of the tube profiles in the DF image, the two dotted brown lines are used to show how FWHM of the central tube in the DF image (the dashed black line) is estimated. The horizontal dashed brown line with double arrows represents ½ of the FWHM that is ~2.5 mm and therefore the estimated FWHM is ~5 mm.

Fig 5. USF images of micro-tubes in porcine muscle tissue samples.

(a)-(d) The USF images of the micro-tubes (before correlation) that were embedded into pork muscle tissue samples with thicknesses of 0.8, 1.2, 2.2 and 3.1 cm, respectively. The I.D./O.D. of the tube is 0.31/0.64 mm; (e)-(h) The corresponding USF images processed by the correlation algorithm. (i) The relationship between the SNR and the thickness of the sample before (triangles) and after (diamonds) the correlation processing (error bar: mean±standard deviation). (j) The relationship between the SNR and the LIA time constant before (triangles) and after (diamonds) the correlation processing (error bar: mean±standard deviation).