Abstract
Whole-body imaging with fluorescent proteins has been shown to be a powerfultechnology with many applications in small animals. Brighter, red-shifted proteins can makewhole-body imaging even more sensitive due to reduced absorption by tissues and less scatter.For example, a new protein called Katushka has been isolated that is the brightest known proteinwith emission at wavelengths longer than 620 nm. This new protein offers potential for non-invasive whole-body macro imaging such as of tumor growth. For subcellular imaging, toobserve cytoplasmic and nuclear dynamics in the living mouse, cancer cells were labeled in thenucleus with green fluorescent protein and with red fluorescent protein in the cytoplasm. Thenuclear and cytoplasmic behavior of cancer cells in real time in blood vessels was imaged as theytrafficked by various means or adhered to the vessel surface in the abdominal skin flap. Duringextravasation, real-time dual-color imaging showed that cytoplasmic processes of the cancer cellsexited the vessels first, with nuclei following along the cytoplasmic projections. Both cytoplasmand nuclei underwent deformation during extravasation. Cancer cells trafficking in lymphaticvessels was also imaged. To noninvasively image cancer cell/stromal cell interaction in the tumormicroenvironment as well as drug response at the cellular level in live animals in real time, wedeveloped a new imageable three-color animal model. The model consists of GFP-expressingmice transplanted with the dual-color cancer cells. With the dual-color cancer cells and a highlysensitive small animal imaging system, subcellular dynamics can now be observed in live mice inreal time. Fluorescent proteins thus enable both macro and micro imaging technology and thereby provide the basis for the new field of in vivo cell biology.
Highlights
Using the dual-colored cancer cells and a highly sensitive macroimaging/microimaging system, the Olympus OV100, we developed real-time dynamic subcellular imaging of cancer cell trafficking in live mice
Labeling of cells with different color fluorescent proteins in the nucleus and cytoplasm and powerful imaging technologies have enabled the real-time visualization of the nuclear-cytoplasmic dynamics including mitosis and apoptosis
We have been able to visualize the extreme deformation cancer cells undergo in their cytoplasm and to some extent in their nucleus to traffick in narrow vessels and to extravasate
Summary
Green fluorescent protein (GFP) has been shown to be able to be genetically linked with almost any protein providing a permanent and heritable label in live cells to study protein function and location [1]. Many different colors of fluorescent proteins have been produced in the laboratory or found in nature. Many processes can be visualized simultaneously in cells. Cells can be multiply labeled for live imaging of processes that heretofore could be performed only on fixed and stained cells. What previously could only be seen on gels and blots, can be visualized in real-time in living cells expressing fluorescent proteins. Our laboratory pioneered the use of fluorescent proteins for in vivo imaging from macro to subcellular [1,2,3]
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