Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.
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 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. 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 load3-4.
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 substrate5-6. 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 models7-9. 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 signal10. 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.
Although following these protocols will usually result in high quality images, it is important to consider a few key issues in order to obtain accurate and consistent data from imaging studies. Luminescence images should be acquired that have counts from 600 to 60,000 to ensure that the signal is above background and the camera is not saturated. If signal obtained is less than 600 the exposure conditions should be adjusted to increase counts. If signal obtained is over 60,000 the camera is saturated in some regions. Wh…
The authors have nothing to disclose.
The authors thank the Cirillo laboratory members for valuable discussions and assistance throughout this study. We thank Dr. Joshua Hill and the laboratory of Dr. James Samuel for assistance with during the filming of this protocol. This work was funded by grant 48523 from the Bill & Melinda Gates Foundation and grant AI47866 from the National Institutes of Health.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Isoflurane | VETONE | 501027 | ||
Ketamine | Butler animal health supply | |||
Xylazine | MP Biomedical | 158307 | ||
Luciferin | GMT | LUCK-100 | ||
Fetal plus solution | VOR tech pharmaceutical | |||
Cathether (22G x 1”) | TERUMO | OX2225CA | ||
Guide wire | Hallowell EMC | 210A3491 | ||
Octocope with speculum | Hallowell EMC | 000A3748 | ||
Xenogen IVIS system | Caliper Life Sciences | |||
XGI-8-gas Anesthsia System | Caliper Life Sciences | |||
Living Imaging Software | Caliper Life Sciences | |||
Transparent nose cones | Caliper Life Sciences | |||
Light baffle divider | Caliper Life Sciences |