In the lab, autopsies of research animals have helped researchers make great strides in understanding the various pathways and biological processes that take place in mammalian cell biology. But that’s after the animal has been euthanized and those biological processes have ceased.
Now, intravital imaging is allowing researchers to capture images of those same biological processes in live animals. It’s providing answers about how cells behave in resting tissue, how they interact with one another, exchange information, respond to pathological stimuli, and mediate functions. In addition, the technique is providing insight into disease processes that take place in cells by defining the impact of specific disturbances in real-time, allowing researchers to test concepts gathered in vitro while providing instructive observations not readily captured by the histological evaluation of tissue.
At the Albert Einstein College of Medicine, Jeffrey Pollard is the director of the Center for the Study of Reproductive Biology and Women's Health and deputy director of the Albert Einstein Cancer Center. One of his goals is to develop intravital imaging technologies that will make imaging-based cell collection and gene expression analysis of tissue cells routine.
“The use of high-resolution imaging as a front end in the collection and interpretation of DNA microarray data is a unique combination of advanced technologies and potentially a vast improvement over existing methods of immunocytochemistry and laser capture microdissection as front ends for DNA chip analyses,” says Pollard.
One main advantage of intravital imaging is the ability to observe cell-to-cell interactions, thus proving they occur in vivo. Using photoswitchable fluorescent proteins such as the green-to-red transitioning Dendra2, researchers can track individual cells as they travel through blood vessels within the body.
But there are limitations. For one thing, the technology is reliant upon fluorescently labeled cells. In addition, even using infrared lazers, researchers can only image to a depth of 300-400 µm. And it’s still quite expensive. In his recent study published in Nature Protocols(1), the two-laser multiphoton microscope used by his team cost around $1 million.
In that article, Pollard and colleagues used the microscope for multichannel intravital fluorescence imaging of a mouse model of breast cancer. The instrument extended the wavelength range for excitation, thereby expanding the number of simultaneously usable fluorophores, and markedly increased single-to-noise via overclocking of detection. Overall, Pollard notes that the procedure can be completed in less than 24 hours.
At the Department of Oncology in the University of Alberta, Canada, associate professor John Lewis is studying the tumor microenvironment to functionally elucidate the molecular switches of tumorigenesis. Namely, he’s looking at tumor neoangiogenesis and the acquisition of tumor cell motility. He is also investigating novel nanoparticles that are being developed for the early detection of prostate cancer, drug delivery, and the in vivo study of tumor cell invasion and metastasis. All of these projects are facilitated by long-term time-lapse intravital imaging of human cancer progression.
Most intravital imaging studies have been in mice, which Lewis believes limits the data that are being collected. His solution is chicken embryos. “You can generate embryos faster and in greater quantity than you can mice. You can also see what is going on for days in chicken embryos compared to 6-8 hours in mice. The data from the embryos allows for more confidence statistically,” says Lewis.
In an article in the Journal of Visualized Experiments (2), Lewis demonstrated that chicken embryos can be a useful model in assessing the vascular dynamics and the pharmacokinetics of xenografted human tumors. “The structure and position of the chorioallantoic membrane (CAM) allows high-quality image acquisition and accommodates many kinds of cancer xenografts without invasive surgical procedures. Moreover, cancer tumor xenografts implanted into the chorioallantoic membrane become vascularized within seven days, offering a rapid, inexpensive and semi-high-throughput means to assess the accumulation of nanoparticles in tumor tissue,” says Lewis.
Lewis also noted that the cancer xenografts implanted in the CAM of the chicken embryos were “accessible to the high-resolution optics of an upright epifluorescence or confocal microscope; contextual and temporal information regarding nanoparticle uptake in the tumor vasculature can be readily obtained.”
In the end, the use of chicken embryos and other models might increase with the continued interest in intravital imaging. The cancer xenografts tend to grow laterally across the CAM, which results in tumors that are large but less than 200 µm in depth. At this depth, a standard epifluorescence microscope can image the entire tumor. “In contrast, tumors implanted in either superficial or orthotopic sites within the mouse proliferate in three dimensions, making it difficult to accurately localize nanoparticles deep within these tumors by non-invasive techniques,” says Lewis.
Claudio Vinegoni is an instructor at Harvard Medical School and also director of the In Vivo Microscopy Program in the Center for Systems Biology at Massachusetts General Hospital. His research focuses on developing novel in vivo imaging methods. His work in fluorescence molecular tomography has increased the resolution possible for 3-D imaging in living mice.
Currently, he’s developing novel optical mesoscopic molecular imaging techniques to generate in vivo 3-D data in optically diffusive non-transparent living organisms up to a few millimeters in size such as Drosophila melanogaster and zebrafish. These techniques provide both in vivo anatomical and functional imaging. Other research activities in his lab involve in vivo near infrared fluorescence imaging of protease activity in rabbit models of atherosclerosis, the development of novel fiber-based imaging systems and combined optical and opto-acoustic multispectral tomographic imaging for in-vivo imaging applications.
“The great advantage of intravital imaging is that it allows you to have direct access to mouse models and is a well-established technique. You can do it for hours, days, and weeks,” says Vinegoni. “You don't have to cut open the mouse repeatedly to see what is going on."
However, some organs are difficult to get a look at. "Imaging the heart is a challenge. It beats a lot. The average mouse has a heartbeat of 350 beats per minute. It makes imaging difficult," says Vinegoni.
Despite these challenges, intravital imaging is providing researchers with a window into the cellular processes in living animals and continuing to grow in popularity, especially as it becomes more affordable.
- Entenberg, D., J. Wyckoff, B. Gligorijevic, E. T. Roussos, V. V. Verkhusha, J. W. Pollard, and J. Condeelis. 2011. Setup and use of a two-laser multiphoton microscope for multichannel intravital fluorescence imaging. Nat. Protocols 6(10):1500-1520.
- Cho, C.-F. F., A. Ablack, H.-S. S. Leong, A. Zijlstra, and J. Lewis. 2011. Evaluation of nanoparticle uptake in tumors in real time using intravital imaging. Journal of visualized experiments : JoVE 52.