Materials
Calcium tungstate middle speed intensifying screen (Yuhuan Medical Equipment Factory, Zhejiang, China) for clinical use (produced under the Sino Food and Drug Administration standard No. YY/T 0095-2004) was chosen in this study. Na99mTcO4 was produced from 99Mo/99mTc generator (Beijing Atom High Tech, China). 18F-fluorodeoxyglucose was produced by a cyclotron (GE Industries Inc., USA) and the FDG reagent kit (ABX, Germany). Na332PO4 solution was purchased from Beijing Atom High Tech.
Basic Features of ISRLI
0.37 MBq (10 µCi) of 18F (γ plus β+), 99mTc (pure γ) or 32P (pure β−) with a final volume of 60 µL was respectively added to two caps of EP tube. Optical imaging was acquired by a luminescence imaging system described in our previous studies, composing an electron multiplying charge-coupled device (EMCCD)camera (iXon3 888, Andor Corp), a standard prime lens (Pentax F/1.8), and a light-tight box, similar to the widely used IVIS optical system. The acquisition protocols of ISRLI and CLI were both EM = 1000, binning = 1, exposure time = 10s, with or without covering the intensifying screen on the tube caps.
The excitation source of Cerenkov luminescence was shielded by inserting a piece of black paper between the intensifying screen and the tube caps. ISRLI with and without shield were respectively acquired and compared. To investigate the spectrum of ISRLI, a fluorescence spectrophotometer (F7000, HITACHI, Japan) was applied to measure the emitted ISRLI spectrum excited by 99mTc. It should be noted that before the acquisition of luminescence images in this study, five background images were acquired to calculate the mean background intensity. By subtracting the background intensity from the luminescence images, the background influence was eliminated.
Signal Quantitative Correlation Study of ISRLI
A series of caps of EP tube containing 0, 0.37, 0.74, 1.11, 1.48 MBq (0, 10, 20, 30, 40 µCi) of 18F or 99mTc with a final volume of 60 µL was respectively prepared. Optical imaging was acquired by the same protocols of CLI and ISRLI described before (EM = 1000, binning = 1, exposure time = 10s), with or without covering the intensifying screen on the tube caps. The optical signal intensity of CLI and ISRLI in each tube cap was quantitatively compared. Further, the PET imaging of 18F was respectively acquired by a micro-PET/CT (NanoPET/CT, Mediso, Hungry), and the scintigraphy of 99mTc was acquired by a clinical used gamma camera (NM670, GE, USA). By drawing the region of interest (ROI), the radioactive concentration in PET imaging and the average counts in scintigraphy of each tube cap was measured. The correlation of the intensity of CLI, ISRLI, and nuclear imaging was then investigated.
Sensitivity Comparison of CLI and ISRLI
Caps of EP tube containing 37, 3.7, 0.37, 0.037 kBq (1, 0.1, 0.01, 0.001 µCi) of 18F and 99mTc with a final volume of 60 µL were prepared. In order to better determine the sensitivity threshold, according to the previous studies on CLI, the optical imaging protocol was adjusted to be EM = 1000, binning = 4, exposure time = 60s. The CLI images of 18F and the ISRLI images of 18F and 99mTc were respectively acquired.
Penetration Comparison of CLI and ISRLI
One cap of EP tube containing 0.37 MBq (10 µCi) of 18F-FDG with a final volume of 60 µL was prepared, and the optical signal of CLI or ISRLI was respectively acquired with or without covering a 10mm-thick pork slice. The CLI signal was acquired by the protocol EM = 1000, binning = 1, exposure time = 60s. The ISRLI was acquired by the protocol EM = 1000, binning = 1, exposure time = 10s, with the intensifying screen being fixed with the distance of 10 mm from the cap of EP tube. The reason to prolong the acquisition time of CLI was to precisely determine the existence of CLI signal when the source was covered by biological tissue, which was theoretically very weak in intensity. As the exposure time and binning value of CLI image were larger than that of ISRLI image, the following formula was used to make the optical intensity of CLI image and ISRLI with the same unit:
$${I}_{CLI}^{{\prime }}={I}_{CLI}\text{*}\frac{{T}_{RLISI}}{{T}_{CLI}\text{*}{2}^{BV}}$$
1
where \({I}_{CLI}\) was the optical intensity of CLI; \({T}_{RLISI}\) and \({T}_{CLI}\) were the exposure time of ISRLI and CLI; \(BV\) was the binning value of EMCCD camera. In this experiment, \({T}_{RLISI}=10s\), \({T}_{CLI}=60s\), and \(BV=4\), resulting the unit to be \(\text{a}\text{r}\text{b}.\text{u}\text{n}\text{i}\text{t}\text{s}/10\text{s}/\text{b}\text{i}\text{n}\text{n}\text{i}\text{n}\text{g}1\).
Resolution study of ISRLI
0.37 MBq (10 µCi) of 18F was used as a signal source. ISRLI was acquired by the protocol EM = 1000, binning = 1, and exposure time = 10s, respectively, with a distance of 0, 0.5, 1.0, 1.5, 2.0 cm between the intensifying screen and the radionuclide. The resolution was determined by the full width at the half maximum (FWHM) measured on images. Additionally, before the imaging experiment, a ruler was used to establish the relationship between the pixel on the image and the actual length, and the result showed that 25 pixels equaled 1 mm.
ISRLI-based tumor imaging on tumor xenografted mice
Mice models bearing HT-1080 human fibrosarcoma cells were established. The mice models were injected with 100 µL 14.8 MBq of 18F-FDG via the tail vein. PET and CLI images were respectively acquired at1 h post-injection (p.i.). ISRLI was acquired by placing the intensifying screen closely on the surface of the tumor flank, which pointed at the EMCCD camera. CLI was acquired by the adjusted protocol EM = 1000, binning = 4, and exposure time = 120s to compromise the weak intensity of CLI. ISRLI was acquired by the previous protocol (EM = 1000, binning = 1, exposure time = 10s). The comparison of the intensity between CLI and ISRLI images were in the uniformed unit photon yield rate (arb. units/10s/binning1). The final CLI and ISRLI were presented by fusing the CLI or ISRLI and the white-light view without the intensifying screen of each mouse.
ISRLI-based lymphography on rabbits
37 MBq (1 mCi) of 99mTc-DX was intradermally injected in the right foot of rabbits, SPECT/CT was acquired at 15 min post-injection. According to the location of positive subdermal popliteal lymph nodes shown on SPECT/CT imaging, ISRLI was then acquired by placing the intensifying screen close to the skin projection of popliteal lymph nodes. Surgical resection of the popliteal lymph nodes was then performed. The intraoperative ISRLI of the exposed and the resected lymph nodes were acquired, respectively. The acquisition protocol of ISRLI was adjusted to EM = 1000, binning = 4, and exposure time = 10s according to the imaging quality of the preliminary experiment. The final ISRLI was presented by fusing the ISRLI and the white-light view without the intensifying screen of each rabbit. Anesthesia was maintained throughout the entire procedure, followed by CO2 asphyxiation immediately after the surgery.
Image processing method
Because a small part of the high-energy rays produced during the decay of radionuclide may travel through the intensified screen and hit the EMCCD camera, there would be some impulse noise on the luminescence images. In order to reduce the image noise and improve the image quality, the median filter method was used. All luminescence images were colored luminesce to the pseudo-color look-up table, and some pseudo-colored luminescence images were then overlapped on the corresponding white-light images. All the above processes were completed by using Matlab R2016a (Mathworks, Inc, USA).
Statistics Analysis
All of the above procedures were repeated for 5 times. All data were analyzed by GraphPad Prism 5.0 and presented as mean ± S.D. Differences between the 2 groups were determined by Student’s t-test, and multiple comparisons were performed by one-way ANOVA and completed by Bonferroni’s multiple comparison test. A P-value less than 0.05 was considered statistically significant.