An ICCD camera-based time-domain ultrasound-switchable fluorescence imaging system

Fluorescence imaging in centimeter-deep tissues with high resolution is highly desirable for many biomedical applications. Recently, we have developed a new imaging modality, ultrasound-switchable fluorescence (USF) imaging, for achieving this goal. In our previous work, we successfully achieved USF imaging with several types of USF contrast agents and imaging systems. In this study, we introduced a new USF imaging system: an intensified charge-coupled device (ICCD) camera-based, time-domain USF imaging system. We demonstrated the principle of time-domain USF imaging by using two USF contrast agents. With a series of USF imaging experiments, we demonstrated the tradeoffs among different experimental parameters (i.e., data acquisition time, including CCD camera recording time and intensifier gate delay; focused ultrasound (FU) power; and imaging depth) and the image qualities (i.e., signal-to-noise ratio, spatial resolution, and temporal resolution). In this study, we also discussed several imaging strategies for achieving a high-quality USF image via this time-domain system.

2) ZnttbPc in 5% F98 nanocapsules. Fig. S2 (a) shows the normalized fluorescence intensity and lifetime change of ADP(OH) 2 -Bottom in 5% F98 nanocapsules over a temperature rise. Likewise, Fig. S2 (b) shows the same information for ZnttbPc in 5% F98 nanocapsules. The result is similar to that from the ICCD camera imaging system measurements. ADP(OH) 2 -Bottom in 5% F98 nanocapsule has a I on /I off ratio (I on /I off = ~ 24 folds), and its fluorescence lifetime slightly changes as a function of temperature ( on / off = ~ 1.07, on = 1.48 ns); its T th = ~ 31°C. ZnttbPc in 5% F98 nanocapsule has both significant intensity and lifetime changes (I on /I off = ~ 4200 folds, on / off = ~ 11.23 folds, on = 2.97 ns); its T th = ~ 28°C.
Different lifetime of fluorescence and laser pulse on ICCD images. In this work it shows that the laser pulse can be much shorter (usually its lifetime < 500 ps) than some fluorescence pulses that have long fluorescence lifetimes (> 1.0 ns). In this section we demonstrated that this ICCD camera imaging system was capable of measuring different lifetimes on the same FOV. Fig. S3 (a) left shows the experiment sample setup. A silicone phantom (μ s ' = 3.5 cm -1 , μ a = 0.03 cm -1 , total thickness Z = 3 mm) imbedded with a silicone tube (I.D. = 760 μm, Z1 = Z2 = 1.5 mm) was placed on a plastic Petri dish, and they were placed on the microscope stage for imaging (with a 4× objective lens). When the laser beam was focused on the bottom surface of the phantom, near the tube, the plastic Petri dish gave laser reflection and generated autofluorescence on the pathway. CCD images captured the corresponding signal spot (the round red spot in Fig. S3 (a) left). This signal should have a short lifetime. Meanwhile, the silicone tube was filled with ZnttbPc in 5% F98 nanocapsule. The Petri dish was filled with hot water at 40 °C so that the phantom was heated and the contrast agent inside the tube was switched on and presented a long lifetime. CCD images also captured the scattered fluorescence signal from the tube (the green line in Fig. S3 (a) left). Fig. S3 (a) right shows one real example image from the experiment. Fig.   S3 (b) shows a series of CCD images captured at different gate delay t; t was counted from the start time of the gate, and the gate width was fixed at t w = 300 ps as previously (seen in Fig. 1 (c)). Fig. S3 (b) clearly shows that the round beam spot appeared first but disappeared fast and that the fluorescence in the tube appeared later but disappeared much more slowly because of its long lifetime. Fig. S3 (c) shows the different lifetimes measured when different ROIs were selected. ROI 1 was selected on the round beam spot, so it showed a relatively short lifetime of laser leakage plus plastic autofluorescence. Its decay time (down to 37%, without deconvolution from impulse response function (IRF), same below) was ~ 0.49 ns. On the other hand, ROI 2 was selected on the tube so it showed a long lifetime of fluorescence signal. Its decay time was ~ 3.87 ns.
USF signal lifetime measurement. In this study it shows that it is feasible to select a proper time-gated window of signal acquisition to improve SNR in USF imaging. This is based on the assumption that the USF signal has a longer lifetime than that of the background. Although it was shown that both ADP(OH) 2 -Bottom nanocapsule and ZnttbPc nanocapsule demonstrated a long lifetime when the samples were tested in the cuvette in a high temperature environment (i.e., T > T th ), a question remained whether the USF signal itself had a long lifetime. In the previous section, we assumed that the USF signal came from the contrast agents that were thermally "switched-on" due to a temperature rise by FU heating. In this experiment, we measured the fluorescence lifetime of a USF signal, in order to demonstrate whether the "ultrasonically switched-on" fluorescence had a lifetime similar to that of the "thermally switched-on" fluorescence. Fig. S4 (a) shows the sample configuration and the experimental setup. In this experiment, we tested the USF signal from ADP(OH) 2 -Bottom in 5% F98 nanocapsule. The silicone tube was filled with ADP(OH) 2 -Bottom in 5% F98 nanocapsule, and the cuvette was filled with ZnttbPc in 5% nanocapsule. Thus, the background had a strong-intensity but shortlifetime fluorescence. The FU transducer was focused on the silicone tube for acquiring the USF signal. We adopted a long exposure time (3 s) to get the USF signal. The experimental setup was basically the same as that in Fig. 6 (a), Case 2, except that the FU transducer did not scan across the tube but acquired the USF signals from the tube at different time-gated windows.
This experiment followed three steps. In step 1, the ICCD camera measured the IRF of the system (when the cuvette and tube were first filled with water). Fig. S4 (b) presents the measurement principle, which is the same as that in Fig. 1 (c). Here, the gate width t w = 400 ps, the delay time interval ∆ = 200 ps, and the measured gate time range t = 0 -10 ns. In Fig. S4 (c), the solid blue line represents the normalized IRF of the system (i.e., the measured excitation laser pulse). In step 2, the system measured the strong-intensity background fluorescence (BF) pulse from the cuvette filled with ZnttbPc in 5% nanocapsule, which is the solid red line (normalized) in Fig. S4 (c). Its calculated lifetime is 0.26 ns (function y1, dash red line in Fig. S4 (c)).
Step 3 repeated the measurement in step 2, with the difference that a FU signal (Vpp = 130 mV, FU exposure time = 400 ms) came in. The FU signal was synchronized with each camera frame (camera exposure time = 3 s) that captured the fluorescence pulse within the designed time-gated window as in step 2. Thus, in step 3, the measured fluorescence pulse represents the BF pulse (from the cuvette) plus the USF pulse (from the tube). Then, this measured pulse in step 3 was subtracted from the BF pulse in step 2; the remainder was only the USF pulse from the tube. In Fig. S4 (c), the solid green line represents the normalized USF pulse after subtraction. Its calculated lifetime is 1.10 ns (function y2, dash green line in Fig. S4 (c)). The result shows that the USF signal has a relatively long lifetime compared with that of the background (τ = 1.10 ns > 0.26 ns). It also shows the USF signal lifetime of ADP(OH) 2 -Bottom nanocapsule is similar to the lifetime of the nanocapsule (τ = 1.02 ns as seen in Fig. 1 (f)) in a high-temperature (i.e., T = 40 °C > T th = 31 °C) water-bath environment.