Chang Mi Hee, Cirillo Suat L G, Cirillo Jeffrey D
Microbial & Molecular Pathogenesis, Texas A&M Health Science Center.
J Vis Exp. 2011 Feb 18(48):2547. doi: 10.3791/2547.
Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load. This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.
成像技术是一种可用于监测生物过程的重要技术。特别是，癌细胞、干细胞、特定免疫细胞类型、病毒病原体、寄生虫和细菌的存在可以在活体动物体内实时追踪。与动物模型中感染分析的传统策略相比，生物发光成像技术在病原体研究中的应用具有诸多优势。随着时间推移，感染情况可在个体动物体内可视化，无需实施安乐死来确定病原体的位置和数量。光学成像能够对所有组织和器官进行全面检查，而不是对先前已知感染的部位进行采样。此外，在继续进行整个实验中未成功接种的动物实验之前，可以直接确定将病原体接种到特定组织中的准确性。由于成像允许对每只动物进行单独跟踪，因此可以控制动物之间的变异性。成像技术有可能大幅减少所需动物数量，因为它能够从多个时间点获取数据，而无需对组织进行采样来确定病原体载量。本方案描述了使用生物发光成像技术对表达荧光素酶的重组细菌菌株在活体动物中可视化感染的方法。叩甲荧光素酶（CBRLuc）和萤火虫荧光素酶（FFluc）都以荧光素作为底物。CBRLuc和FFluc产生的光波长范围较宽，从500纳米到7纳米，这使得这些荧光素酶成为活体动物模型光学成像的优秀报告基因。这主要是因为需要大于600纳米的光波长来避免被血红蛋白吸收，从而能够有效地穿透哺乳动物组织。荧光素酶通过基因手段导入细菌以产生光信号。小鼠通过气管内接种生物发光细菌，以便实时监测感染情况。注射荧光素后，使用IVIS成像系统采集图像。在成像过程中，使用XGI - 8气体麻醉系统用异氟烷对小鼠进行麻醉。图像可以进行分析，以定位和量化信号源，信号源分别代表细菌感染部位和数量。成像后，对匀浆组织进行菌落形成单位（CFU）测定，以确认细菌的存在。使用几种不同剂量的细菌来关联细菌数量与发光情况。成像技术可应用于发病机制研究以及抗菌化合物和疫苗疗效评估。
