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Imaging In Mice With Fluorescent Proteins: From Macro To Subcellular

View Article: PubMed Central - PubMed

ABSTRACT

Whole-body imaging with fluorescent proteins has been shown to be a powerful technology with many applications in small animals. Brighter, red-shifted proteins can make whole-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 protein with emission at wavelengths longer than 620 nm. This new protein offers potential for noninvasive whole-body macro imaging such as of tumor growth. For subcellular imaging, to observe cytoplasmic and nuclear dynamics in the living mouse, cancer cells were labeled in the nucleus with green fluorescent protein and with red fluorescent protein in the cytoplasm. The nuclear and cytoplasmic behavior of cancer cells in real time in blood vessels was imaged as they trafficked by various means or adhered to the vessel surface in the abdominal skin flap. During extravasation, real-time dual-color imaging showed that cytoplasmic processes of the cancer cells exited the vessels first, with nuclei following along the cytoplasmic projections. Both cytoplasm and nuclei underwent deformation during extravasation. Cancer cells trafficking in lymphatic vessels was also imaged. To noninvasively image cancer cell/stromal cell interaction in the tumor microenvironment as well as drug response at the cellular level in live animals in real time, we developed a new imageable three-color animal model. The model consists of GFP-expressing mice transplanted with the dual-color cancer cells. With the dual-color cancer cells and a highly sensitive small animal imaging system, subcellular dynamics can now be observed in live mice in real 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.

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Time-lapse imaging of nuclear-cytoplasmic dynamics of extravasation of cancer cellsA, 12 hours after injection dual color MMT cells, the skin flap was opened and fixed on a flat stand. Images were acquired every hour for 24 hours with the skin flap open. Two dual color MMT cancer cells are visualized in the process of extravasation 24 hours after injection (arrows). The cancer cells extended fine cytoplasmic projections into the host tissue at the onset of extravasation. One of the cells extended two fine cytoplasmic projections into the host tissue (arrowhead). The nuclei then migrated along the cytoplasmic projection until the whole cell came out of the vessel. Subsequently, the whole cell extravasated. Bar = 20 μm. B, 48 and 72 hours after injection. Cytoplasmic processes were extended along the vessel wall 48 hours after injection (arrows). Cells extravasated in the same direction of the cytoplasmic projections (broken arrows). Images were acquired every 24 hours by opening and closing the skin flap. Bar = 50 μm. C, Invasion and proliferation of MMT cells around a vessel after extravasation. Bar = 50 μm. Images were acquired with the Olympus OV100. Right, schematics of (A), (B), and (C) [21].
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f8-sensors-08-01157: Time-lapse imaging of nuclear-cytoplasmic dynamics of extravasation of cancer cellsA, 12 hours after injection dual color MMT cells, the skin flap was opened and fixed on a flat stand. Images were acquired every hour for 24 hours with the skin flap open. Two dual color MMT cancer cells are visualized in the process of extravasation 24 hours after injection (arrows). The cancer cells extended fine cytoplasmic projections into the host tissue at the onset of extravasation. One of the cells extended two fine cytoplasmic projections into the host tissue (arrowhead). The nuclei then migrated along the cytoplasmic projection until the whole cell came out of the vessel. Subsequently, the whole cell extravasated. Bar = 20 μm. B, 48 and 72 hours after injection. Cytoplasmic processes were extended along the vessel wall 48 hours after injection (arrows). Cells extravasated in the same direction of the cytoplasmic projections (broken arrows). Images were acquired every 24 hours by opening and closing the skin flap. Bar = 50 μm. C, Invasion and proliferation of MMT cells around a vessel after extravasation. Bar = 50 μm. Images were acquired with the Olympus OV100. Right, schematics of (A), (B), and (C) [21].

Mentions: Dual-color cancer cells were injected by a vascular route in an abdominal skin flap in nude mice. The mice were imaged with the Olympus OV100 which has a sensitive CCD camera and five objective lenses, parcentered and parfocal, enabling imaging from macrocellular to subcellular. We observed the nuclear and cytoplasmic behavior of cancer cells in real time in blood vessels as they trafficking by various means or adhered to the vessel surface in the abdominal skin flap (Figure 7). During extravasation, real-time dual-color imaging showed that cytoplasmic processes of the cancer cells exited the vessels first, with nuclei following along the cytoplasmic projections (Figure 8). Both cytoplasm and nuclei underwent deformation during extravasation. Different cancer cell lines seemed to strongly vary in their ability to extravasate. With the dual-color cancer cells and the highly sensitive small animal imaging system described here, the subcellular dynamics of cancer metastasis can now be observed in live mice in real time [21].


Imaging In Mice With Fluorescent Proteins: From Macro To Subcellular
Time-lapse imaging of nuclear-cytoplasmic dynamics of extravasation of cancer cellsA, 12 hours after injection dual color MMT cells, the skin flap was opened and fixed on a flat stand. Images were acquired every hour for 24 hours with the skin flap open. Two dual color MMT cancer cells are visualized in the process of extravasation 24 hours after injection (arrows). The cancer cells extended fine cytoplasmic projections into the host tissue at the onset of extravasation. One of the cells extended two fine cytoplasmic projections into the host tissue (arrowhead). The nuclei then migrated along the cytoplasmic projection until the whole cell came out of the vessel. Subsequently, the whole cell extravasated. Bar = 20 μm. B, 48 and 72 hours after injection. Cytoplasmic processes were extended along the vessel wall 48 hours after injection (arrows). Cells extravasated in the same direction of the cytoplasmic projections (broken arrows). Images were acquired every 24 hours by opening and closing the skin flap. Bar = 50 μm. C, Invasion and proliferation of MMT cells around a vessel after extravasation. Bar = 50 μm. Images were acquired with the Olympus OV100. Right, schematics of (A), (B), and (C) [21].
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f8-sensors-08-01157: Time-lapse imaging of nuclear-cytoplasmic dynamics of extravasation of cancer cellsA, 12 hours after injection dual color MMT cells, the skin flap was opened and fixed on a flat stand. Images were acquired every hour for 24 hours with the skin flap open. Two dual color MMT cancer cells are visualized in the process of extravasation 24 hours after injection (arrows). The cancer cells extended fine cytoplasmic projections into the host tissue at the onset of extravasation. One of the cells extended two fine cytoplasmic projections into the host tissue (arrowhead). The nuclei then migrated along the cytoplasmic projection until the whole cell came out of the vessel. Subsequently, the whole cell extravasated. Bar = 20 μm. B, 48 and 72 hours after injection. Cytoplasmic processes were extended along the vessel wall 48 hours after injection (arrows). Cells extravasated in the same direction of the cytoplasmic projections (broken arrows). Images were acquired every 24 hours by opening and closing the skin flap. Bar = 50 μm. C, Invasion and proliferation of MMT cells around a vessel after extravasation. Bar = 50 μm. Images were acquired with the Olympus OV100. Right, schematics of (A), (B), and (C) [21].
Mentions: Dual-color cancer cells were injected by a vascular route in an abdominal skin flap in nude mice. The mice were imaged with the Olympus OV100 which has a sensitive CCD camera and five objective lenses, parcentered and parfocal, enabling imaging from macrocellular to subcellular. We observed the nuclear and cytoplasmic behavior of cancer cells in real time in blood vessels as they trafficking by various means or adhered to the vessel surface in the abdominal skin flap (Figure 7). During extravasation, real-time dual-color imaging showed that cytoplasmic processes of the cancer cells exited the vessels first, with nuclei following along the cytoplasmic projections (Figure 8). Both cytoplasm and nuclei underwent deformation during extravasation. Different cancer cell lines seemed to strongly vary in their ability to extravasate. With the dual-color cancer cells and the highly sensitive small animal imaging system described here, the subcellular dynamics of cancer metastasis can now be observed in live mice in real time [21].

View Article: PubMed Central - PubMed

ABSTRACT

Whole-body imaging with fluorescent proteins has been shown to be a powerful technology with many applications in small animals. Brighter, red-shifted proteins can make whole-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 protein with emission at wavelengths longer than 620 nm. This new protein offers potential for noninvasive whole-body macro imaging such as of tumor growth. For subcellular imaging, to observe cytoplasmic and nuclear dynamics in the living mouse, cancer cells were labeled in the nucleus with green fluorescent protein and with red fluorescent protein in the cytoplasm. The nuclear and cytoplasmic behavior of cancer cells in real time in blood vessels was imaged as they trafficked by various means or adhered to the vessel surface in the abdominal skin flap. During extravasation, real-time dual-color imaging showed that cytoplasmic processes of the cancer cells exited the vessels first, with nuclei following along the cytoplasmic projections. Both cytoplasm and nuclei underwent deformation during extravasation. Cancer cells trafficking in lymphatic vessels was also imaged. To noninvasively image cancer cell/stromal cell interaction in the tumor microenvironment as well as drug response at the cellular level in live animals in real time, we developed a new imageable three-color animal model. The model consists of GFP-expressing mice transplanted with the dual-color cancer cells. With the dual-color cancer cells and a highly sensitive small animal imaging system, subcellular dynamics can now be observed in live mice in real 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.

No MeSH data available.


Related in: MedlinePlus