Real time in vivo non-invasive optical imaging using near-infrared fluorescent quantum dots

RATIONALE AND OBJECTIVE: Deep-tissue optical imaging is of particular interest, as the equipment costs are lower than for competing technologies such as MRI. For this purpose, the development of novel contrast agents with near-infrared (NIR) fluorescence is especially important. We report on the use of NIR semiconductor nanocrystals in deep-tissue in vivo optical imaging. MATERIALS AND METHODS: Semiconductor nanocrystals of CdMnTe/Hg were grown in aqueous solution and then coated with bovine serum albumin (BSA). The nanocrystals were approximately 5 nm in diameter and have a broad fluorescence peak in the NIR (770 nm). Nanocrystals were injected either subcutaneously or intravenously into athymic NCR NU/NU and C3H/HENCR MTV mice and then excited with a spatially broad 633 nm source; the resulting fluorescence was captured with a sensitive CCD camera. RESULTS: We have demonstrated that the nanocrystals are a useful angiographic contrast agent for vessels surrounding and penetrating a murine squamous cell carcinoma in a C3H mouse. Preliminary assessment of the depth of penetration for excitation and emission was done by imaging a beating mouse heart, both through an intact thorax and after a thoracotomy. The temporal resolution associated with imaging the nanocrystals in circulation has been addressed, and the blood clearance for this contrast agent has also been measured. CONCLUSIONS: There was no significant photobleaching or degradation of the nanocrystals after an hour of continuous excitation. The stability of the nanocrystals together with the time resolution of the optical detection makes them particularly attractive candidates for pharmacokinetic imaging studies.     

无机纳米晶体量子点光学成像应用进展

光学成像是分子成像技术的重要组成部分,主要包括荧光成像、生物发光成像及扩散光学成像。无机纳米晶体量子点（QDs）因具有独特光学性质（如激发光谱宽且连续、发射光谱窄且对称、荧光强度高、抗光漂白能力强等）而成为光学成像的热点研究领域。该文综述了QDs的光学性质、在光学成像中的体内外应用、局限性及目前存在的问题。     

Comparison of subcutaneous and intraperitoneal injection of <Emphasis Type="SmallCaps">d</Emphasis>-luciferin for in vivo bioluminescence imaging

Purpose  We compared subcutaneous (SC) injection and intraperitoneal (IP) injection of d-luciferin for in vivo bioluminescence imaging (BLI) to determine the utility of SC injection. Methods  Mice bearing SC tumours stably expressing firefly luciferase underwent in vivo BLI using SC and IP injection of d-luciferin. BLI studies were repeated at an interval of 3 h using a given injection route to assess repeatability and using different injection routes to assess correlation. In mice bearing both SC and IP tumours, BLI was performed successively using intravenous (IV), SC, and IP injection of d-luciferin. Haematological malignancy model mice underwent BLI using SC and IP injection. Results  In SC tumours, the peak time was slightly shorter and the peak signal was greater using SC injection than using IP injection. The repeatability of determining peak signals was comparable between the two injection routes, and a good correlation was observed between them. In mice bearing both SC and IP tumours, signals from IP tumours relative to those from SC tumours were much greater using IP injection than using IV or SC injection. In the haematological malignancy model, signals from the spleen relative to those from the bone marrow were greater using IP injection than using SC injection. Conclusion  In addition to rare injection failure, the IP injection of d-luciferin led to the overestimation of signals from IP tissues. For BLI, SC injection was shown to be a convenient alternative to IP injection.     

Dual-modality optical and positron emission tomography imaging of vascular endothelial growth factor receptor on tumor vasculature using quantum dots

Purpose  To date, the in vivo imaging of quantum dots (QDs) has been mostly qualitative or semiquantitative. The development of a dual-function positron emission tomography (PET)/near-infrared fluorescence (NIRF) probe might allow the accurate assessment of the tumor-targeting efficacy of QDs. Materials and methods  An amine-functionalized QD was conjugated with VEGF protein and DOTA chelator for VEGFR-targeted PET/NIRF imaging after ⁶⁴Cu-labeling. The targeting efficacy of this dual functional probe was evaluated in vitro and in vivo through cell-binding assay, cell staining, in vivo optical/PET imaging, ex vivo optical/PET imaging, and histology. Results  The DOTA–QD–VEGF exhibited VEGFR-specific binding in both cell-binding assay and cell staining experiment. Both NIR fluorescence imaging and microPET showed VEGFR-specific delivery of conjugated DOTA–QD–VEGF nanoparticle and prominent reticuloendothelial system uptake. The U87MG tumor uptake of ⁶⁴Cu-labeled DOTA–QD was less than one percentage injected dose per gram (%ID/g), significantly lower than that of ⁶⁴Cu-labeled DOTA–QD–VEGF (1.52 ± 0.6%ID/g, 2.81 ± 0.3%ID/g, 3.84 ± 0.4%ID/g, and 4.16 ± 0.5%ID/g at 1, 4, 16, and 24 h post injection, respectively; n = 3). Good correlation was also observed between the results measured by ex vivo PET and NIRF organ imaging. Histologic examination revealed that DOTA–QD–VEGF primarily targets the tumor vasculature through a VEGF–VEGFR interaction. Conclusion  We have successfully developed a QD-based nanoprobe for dual PET and NIRF imaging of tumor VEGFR expression. The success of this bifunctional imaging approach may render higher degree of accuracy for the quantitative targeted NIRF imaging in deep tissue.     

In vitro validation of bioluminescent monitoring of disease progression and therapeutic response in leukaemia model animals

Purpose The application of in vivo bioluminescence imaging to non-invasive, quantitative monitoring of tumour models relies on a positive correlation between the intensity of bioluminescence and the tumour burden. We conducted cell culture studies to investigate the relationship between bioluminescent signal intensity and viable cell numbers in murine leukaemia model cells. Methods Interleukin-3 (IL-3)-dependent murine pro-B cell line Ba/F3 was transduced with firefly luciferase to generate cells expressing luciferase stably under the control of a retroviral long terminal repeat. The luciferase-expressing cells were transduced with p190 BCR-ABL to give factor-independent proliferation. The cells were cultured under various conditions, and bioluminescent signal intensity was compared with viable cell numbers and the cell cycle stage. Results The Ba/F3 cells showed autonomous growth as well as stable luciferase expression following transduction with both luciferase and p190 BCR-ABL, and in vivo bioluminescence imaging permitted external detection of these cells implanted into mice. The bioluminescence intensities tended to reflect cell proliferation and responses to imatinib in cell culture studies. However, the luminescence per viable cell was influenced by the IL-3 concentration in factor-dependent cells and by the stage of proliferation and imatinib concentration in factor-independent cells, thereby impairing the proportionality between viable cell number and bioluminescent signal intensity. Luminescence per cell tended to vary in association with the fraction of proliferating cells. Conclusion Although in vivo bioluminescence imaging would allow non-invasive monitoring of leukaemia model animals, environmental factors and therapeutic interventions may cause some discrepancies between tumour burden and bioluminescence intensity.     

Comparison of semiquantitative fluorescence imaging and PET tracer uptake in mesothelioma models as a monitoring system for growth and therapeutic effects

IntroductionVarious techniques are available for in vivo imaging, and precise understanding of their characteristics is essential for effective use of the imaging results. We established human mesothelioma cell lines expressing red fluorescent protein (RFP) and examined their fluorescence intensity and uptake of positron emission tomography (PET) tracer analogs to compare their characteristics and assess their usefulness in the evaluation of therapeutics.MethodA human mesothelioma cell line was stably transfected to express RFP. Fluorescence, cell number and protein amount were measured during cell growth and treatment with cytotoxic reagents. In in vivo experiments, RFP-expressing cells were injected subcutaneously or into the pleural cavity of nude mice, and fluorescence images were taken with or without pemetrexed treatment. The uptake of [³H]3′-deoxy-3′-fluorothymidine ([³H]FLT) and [¹⁴C]2-fluoro-2-deoxy-d-glucose ([¹⁴C]FDG) under treatment with the above reagents in vitro and in vivo were examined.ResultsStrong correlation was observed between fluorescence intensity and total cell number with or without cytotoxic treatment. The uptake of [³H]FLT and [¹⁴C]FDG decreased rapidly after the initiation of treatment with actinomycin D or cycloheximide. When treated with pemetrexed, the uptake of [³H]FLT temporarily increased. The cells formed subcutaneous and orthotopic tumors, with fluorescence intensity correlating with tumor volume. The correlation was sustained under pemetrexed treatment. The uptake of [³H]FLT in vivo increased significantly early after pemetrexed treatment.ConclusionFluorescence imaging could be used to semiquantitatively monitor tumor size, whereas PET could be used to monitor tumor response to therapeutic treatments, and especially, FLT might be a good marker of the response to anti-folate chemotherapeutics.     

不同粒径的石墨烯量子点在PC12细胞中的成像效应及细胞毒性

 目的 探究不同粒径的石墨烯量子点(graphene quantum dots,GQDs)在PC12细胞中的细胞成像效应及细胞毒性。方法 通过动态光散射法(DLS)对GQDs的水合粒径和zeta电位进行表征,采用CCK-8试剂盒检测GQDs对PC12细胞的毒理效应,使用激光共聚焦显微镜比较不同粒径的GQDs在PC12细胞中的荧光成像效应。结果 GQDs对PC12细胞的毒性效应是呈尺寸依赖性的,15 nm的GQDs比50 nm GQDs对PC12细胞的细胞毒性低,500 μg/ml的15 nm GQDs孵育48 h后,细胞活力仍保持在80%以上。对15 nm的GQDs更容易被PC12细胞摄取,80%以上的细胞能成功显影,表现优异的细胞成像效应。结论 GQDs细胞毒性低,细胞成像效果好,是一种适合神经系统成像的纳米材料。     

脑卒中细胞凋亡的活体成像进展

脑卒中是全球第二大常见死因,是我国第一大死亡和致残原因.缺血脑卒中急性期及迟发性神经元死亡期均存在神经元细胞的凋亡,凋亡决定了脑卒中最终的梗死体积.应用活体成像技术能够无创地检测脑内的细胞凋亡信息,为脑卒中的早期诊断、病程及治疗效果监测提供了新的方向.     

Near-infrared fluorescence imaging of HER-2 protein over-expression in tumour cells

The aim of this study was to evaluate in vitro and in vivo imaging of HER-2-over-expressing tumours using near-infrared optical imaging. A fluorochrome probe was designed by coupling Cy5.5 to anti-HER-2 antibodies. Cells over-expressing (SK-BR-3 cells) or normally expressing (PE/CA-PJ34 cells) the HER-2 protein were incubated with the probe. After removing unbound probe molecules, fluorescence intensities were determined (a.u.: arbitrary units). Cells were additionally investigated using FACS and laser scanning microscopy. The probe was also injected intravenously into tumours bearing SK-BR-3 (n=3) or PE/CA-PJ34 (n=3). Whole-body fluorescence images were generated and analysed. The incubation of SK-BR-3 cells with the probe led to higher fluorescence intensities [2,133 (±143) a.u.] compared to controls [975 (±95) a.u.]. The results from FACS and immunocytochemical analysis were in agreement with these findings. A distinct dependency between the fluorescence intensity and the cell number used in the incubations was detected. In vivo, the relative fluorescence intensities in SK-BR-3 tumours were higher than in PE/CA-PJ34 tumours at 16–24 h after probe application. HER-2-over-expressing tumours were depictable in their original size. Labelling of HER-2 with Cy5.5 is suitable for in vitro and in vivo detection of HER-2-over-expressing tumour cells.     

荧光成像技术在神经定位中的研究进展

术中神经定位困难常可增大神经损伤的风险,从而导致患者神经功能障碍。因此,如何在涉及神经的手术(如复发性腮腺肿瘤、前列腺等手术)中准确定位神经成为手术成功的关键因素之一。术中实时定位神经的方法众多,其中荧光成像技术具有高灵敏、易使用、低成本、无辐射的独特优势,因此受到越来越多研究者的关注。该文就荧光成像技术在神经定位中的相关研究成果进行综述。     

量子点的表面修饰、表征及其活细胞成像研究

目的 通过对疏水性量子点表面进行亲水性改造并偶联单克隆抗体,制备免疫荧光探针用于细胞荧光标记示踪.方法 将牛血清白蛋白(BSA)作为乳化剂分子对疏水性量子点进行表面亲水性改造,检测亲水性改造后的量子点物理特性,采用MTT法测定其对细胞活力的影响.将亲水性改造后的量子点与trastuzumab偶联,对HER-2阳性乳腺癌细胞进行实时荧光成像检测.结果 经亲水性改造后的量子点粒径约70nm,分布较均一;在不同离子强度和pH环境中,仍保持良好的发光性能和胶体稳定性;生物相容性佳,未检测到明显细胞毒作用;经表面偶联肿瘤特异性单克隆抗体后,成功地对HER-2阳性乳腺癌细胞进行了长时间的活细胞跟踪成像研究.结论 亲水性量子点可通过选择性偶联抗体获得免疫量子点荧光探针,从而对细胞、组织等进行特异性荧光成像和检测.     

Tumor tissue characterization evaluating the luciferase activity under the control of a hsp70 promoter and MR imaging in three tumor cell lines

We investigated the luciferase activity under the control of a hsp70 promoter and MR imaging for three tumor cell lines. Three tumor cell lines, SCCVII, NIH3T3 and M21 were transfected with a plasmid containing the hsp70 promoter fragment and the luciferase reporter gene and grown in mice. Bioluminescence imaging of the tumors was performed every other day. MR imaging, pre- and post-contrast T1-wt SE, T2-wt FSE, Diffusion-wt STEAM-sequence, T2-time determination were obtained on a 1.5-T GE MRI scanner at a tumor size of 600–800 mm³ and 1400–1600 mm³. Comparing the different tumor sizes the luciferase activity of the M21 tumors increased about 149.3%, for the NIH3T3 tumors about 47.4% and for the SCCVII tumors about 155.8%. Luciferase activity of the M21 tumors (r = 0.82, p < 0.01) and the SCCVII tumors (r = 0.62, p = 0.03) correlated significant with the diffusion coefficient. In the NIH3T3 tumors the best correlation between the luciferase activity and the MRI parameter was seen for the SNR (T2) values (r = 0.78, p < 0.01). The luciferase activity per mm³ tumor tissue correlated moderate with the contrast medium uptake (r = 0.55, p = 0.01) in the M21 tumors. In the NIH3T3 and SCCVII tumors a negative correlation (r = −0.78, p < 0.01, respectively, r = −0.49, p = 0.02) was found with the T2 time. Different tissue types have different luciferase activity under the control of the same hsp70 promoter. The combination of MR imaging with bioluminescence imaging improves the characterization of tumor tissue giving better information of this tissue on the molecular level.     

Dynamic bioluminescence imaging for quantitative tumour burden assessment using IV or IP administration of <Emphasis Type="SmallCaps">d</Emphasis>-luciferin: effect on intensity,time kinetics and repeatability of photon emission

Introduction In vivo bioluminescence imaging (BLI) is a promising technique for non-invasive tumour imaging. d-luciferin can be administrated intraperitonealy or intravenously. This will influence its availability and, therefore, the bioluminescent signal. The aim of this study is to compare the repeatability of BLI measurement after IV versus IP administration of d-luciferin and assess the correlation between photon emission and histological cell count both in vitro and in vivo. Materials and methods Fluc-positive R1M cells were subcutaneously inoculated in nu/nu mice. Dynamic BLI was performed after IV or IP administration of d-luciferin. Maximal photon emission (PE_max) was calculated. For repeatability assessment, every acquisition was repeated after 4 h and analysed using Bland–Altman method. A second group of animals was serially imaged, alternating IV and IP administration up to 21 days. When mice were killed, PE_max after IV administration was correlated with histological cell number. Results The coefficients of repeatability were 80.2% (IV) versus 95.0% (IP). Time-to-peak is shorter, and its variance lower for IV (p < 0.0001). PE_max was 5.6 times higher for IV. A trend was observed towards lower photon emission per cell in larger tumours. Conclusion IV administration offers better repeatability and better sensitivity when compared to IP. In larger tumours, multiple factors may contribute to underestimation of tumour burden. It might, therefore, be beneficial to test novel therapeutics on small tumours to enable an accurate evaluation of tumour burden. Marleen Keyaerts is a Ph. D. fellow of the Research Foundation—Flanders (Belgium; FWO).     

In vivo measurement of partial oxygen pressure in large vessels and in the reticuloendothelial system using fast 19F-MRI

Quantitative in vivo 19F-MRI was performed in a rat model to monitor partial oxygen pressure (pO₂) using a perfluorocarbon (PFC) emulsion as contrast agent. On Days 1, 4, and 8 postin-jection of the PFC emulsion, transaxial T₁ and pO₂ maps were acquired of the abdomen of rats that were consecutively ventilated with pure oxygen, air, and a mixture of 10% oxygen and 90% nitrogen. The images had a resolution of 0.75 mm x 0.75 mm x 2 mm and a total acquisition time of 24 min. In these images it was possible to distinguish between different vessels and hepatic and splenic tissue in the selected imaging plane. Serial 19F-MRI measurements on the different days postinjection of the PFC allowed to determine separately the pO₂ of arterial and venous blood and the intracellular pO₂ in macrophages of the liver and spleen.     

In vivo observation of anisotropic motion of brain water using 2H double quantum filtered NMR spectroscopy

The ²H DQF NMR spectra of deuterated water molecules were measured for the first time in in vivo rat brain. The observation of the DQF signal indicates that there is a water population that exhibits anisotropic motion. The characteristics of the DQF spectra premortem and postmortem are very similar (lineshape and relaxation times). In the 1st h there is a 10–15% decrease in the signal intensity of the DQF spectra followed by a gradual but a much slower decrease in the DQF signal intensity that reaches 65–70% of its initial value after only 12 h. When the brains were kept at 4°C, a 40% decrease in the DQ signal intensity was observed only after 7 days. Mechanical chopping of the brain tissues causes an immediate loss of more that 97% of the DQ signals. The slow, temperature-sensitive decay of the signal, and its sensitivity to mechanical treatment point out that these signals originate from water molecules that interact with structural components in the brain. The characteristics of the DQF spectra depend on the amount of bulk water as exemplified by increased residual quadrupolar interaction and relaxation rates obtained when dehydrating the brain tissue.     

4D血流MR成像评估颅内动脉血流动力学状态的实验研究

目的旨在应用4D血流MR成像研究正常志愿者及大脑中动脉(MCA)狭窄病人的颅内动脉血流动力学状态,并比较正常和狭窄颅内动脉的双侧血流动力学参数差异。方法纳入正常志愿者5名和MCA狭窄病人3例。应用3 T MR设备进行检查,采用3D时间飞跃法(TOF)MRA及高分辨3D-T1WI-SPACE序列显示及分析Willis环结构特点。应用4D血流MR成像获取血流数据,分析双侧颈内动脉(ICA)颅内段、MCA及大脑前动脉(ACA)的血流动力学状态,计算心动周期不同时相的平均血管面积、平均及最大血流速度、平均瞬时血流率,生成血流矢量图、流线图及粒子追踪图。分析颅内Willis环的结构特征及血流动力学状态。采用独立样本t检验比较正常志愿者及MCA狭窄病人双侧动脉血流动力学参数的差异。结果正常志愿者中,4名显示对称Willis环结构,1名右侧胚胎型大脑后动脉。双侧ICA、MCA和ACA的血流动力学参数包括平均血管面积、平均血流速度、最大血流速度和平均瞬时血流率之间差异均无统计学意义(均P0.05)。血流矢量图、流线图及粒子追踪图显示,ICA可见高速血流,收缩晚期及舒张期显示血流速度下降,血流分布欠均匀。1名右侧胚胎型大脑后动脉志愿者双侧血流动力学参数明显不对称,除右侧ACA最大血流速度低于左侧,其余颅内血管段的平均血管面积、平均和最大血流速度以及平均瞬时血流率右侧均明显高于左侧。3例MCA狭窄病人的ICA、MCA及ACA双侧血流动力学参数之间的差异未见统计学意义(P0.05)。3例狭窄侧平均瞬时血流率均低于对侧。2例局部平均血流速度及相对压力梯度较对侧降低;另1例则明显较对侧增大。血流矢量图、流线图及粒子追踪图显示狭窄局部血流矢量明显偏离血管主轴方向,血流分布明显不规则。结论应用4D血流MR成像可以提供对颅内动脉复杂血流动力学状态的综合评估,包括对血流参数进行定量评价。颅内Willis环的结构与其血流动力学状态及分布密切相关。     

In vivo MR tractography of thigh muscles using diffusion imaging: initial results

The aims of this preliminary study were (1) to demonstrate the feasibility of providing in vivo 3D architecture of human thigh muscles using tractography on a 1.5T magnet, and (2) to assess the value of tractography images to obtain averaged microstructural parameters, i.e., the fractional anisotropy (FA) and the mean apparent diffusion coefficient (ADC), over the whole thigh. Five healthy volunteers were included in this study. Their right thighs were imaged using diffusion tensor imaging and gradient-echo T2* sequences. Muscular tractography was performed on each muscle. MR tractography provided a good approach of the muscle shape and of the orientation of the muscle fibers. There was no aberration in the color-encoding scheme nor in the luminosity assigned to each fiber. In contrast, tendons were not drawn in any of the muscles studied. FA values ranged from 0.27 to 0.38. Mean ADC values ranged from 0.76 to 0.96 × 10⁻³ mm²/s. Our study demonstrated the feasibility of providing in vivo 3D architecture of human thigh muscles using tractography on a 1.5T magnet, and of determining muscular microstructural parameters (FA and ADC). Musculoskeletal radiologists should be aware of these new developments that may provide complementary information on muscles to the usual sequences.     

In vivo measurement and imaging of tumor oxygenation using coembedded paramagnetic particulates.

Tumor tissue oxygenation is an important parameter that is positively correlated to the chemo- or radiation treatment outcome of certain tumors. Hence, methods to accurately and noninvasively determine the concentration of oxygen (pO2) in tumors will be valuable. In this study, electron paramagnetic resonance (EPR) spectroscopy, utilizing microcrystalline particulates of lithium phthalocyanine (LiPc), was used to perform repeated measurements of pO2 as a function of tumor growth. We permanently embedded the particulates in the tumor by coimplanting them with RIF-1 tumor cells during inoculation in mice. This procedure enabled repeated measurements of oxygen concentration in the tumor to be obtained for >2 weeks during its growth phase. The particulates were stable and nontoxic to the tumor cells. Both an in vitro clonogenic assay and an in vivo tumor growth rate examination in C3H mice showed no apparent effect on cell proliferation or tumor growth rate. The measurements indicated that the pO2 of the tumor decreased exponentially with tumor growth and reached hypoxic levels ( approximately 4 mmHg) within 4 days after inoculation of the tumor cells. Spatial EPR imaging revealed a nonuniform distribution of the embedded particulates, which were localized mainly in the middle of the tumor volume. Oxygen mapping of the tumor, obtained by spectroscopic EPR imaging, showed significant variation of pO2 within the tumor. In summary, EPR spectroscopy and imaging with an embedded oximetry probe enabled accurate and repeated measurements of pO2 to be obtained in growing tumors under nonperturbing conditions.     

In vivo imaging of the spinal cord cholinergic system with PET

PURPOSE: Our goal was to demonstrate the feasibility of an in vivo noninvasive method for imaging spinal cord cholinergic terminals using (+)-4-[18F]fluorobenzyltrozamicol ([18F]FBT) and PET. METHOD: In vitro and in vivo experiments in rats were conducted to demonstrate the specific binding characteristics, localization, and time course of [3H]FBT binding in the spinal cord. PET imaging was then performed on seven rhesus monkeys. RESULTS: The rat studies demonstrate high specific binding in the spinal cord with a distribution coinciding with the known distribution of cholinergic terminals. In vivo tracer concentrations in the spinal cord and basal ganglia were of the same magnitude. With use of [18F]FBT and PET in the rhesus monkey, the spinal cord was clearly visualized, with tracer concentration in the spinal cord being approximately one-fourth of that seen in the basal ganglia. CONCLUSION: This work demonstrates the feasibility of imaging cholinergic terminals in vivo in the spinal cord using [18F]FBT and PET.