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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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Imaging nuclear-cytoplasmic dynamics of intravascular trafficking of cancer cellsA, Schematic diagram of the skin flap model in live mice for imaging intravascular trafficking and extravasation. An arc-shaped incision was made in the abdominal skin, and then the skin flap was spread and fixed on a flat stand. HT-1080-GFP-RFP cells were injected into the epigastric cranialis vein through a catheter. Immediately after injection, the inside surface of the skin flap was directly observed. B, HT-1080-GFP-RFP cell crawls smoothly along the vessel wall without rolling in a capillary (arrow). The nucleus and cytoplasm are slightly stretched. The nucleus is in the front of the cell while the cell is crawling. When the cell advanced into a part of the capillary where the diameter is smaller than of deformation limit of the cell, the cell could not advance any further. Bar = 100 μm. C, HT-1080-GFP-RFP cell, trafficking at low velocity, advanced between other cells and the vessel wall. The moving cancer cell contacted the other cells (arrow). The cell deformed slightly and continued to move without adhesion. Bar = 100 μm. Right, schematics of (B) and (C). D, One cancer cell migrating in the post capillary with slow velocity. The cytoplasm is at the head of the cell while the cell is moving in a large vein, but the nucleus is at the head in a small vein. The velocity of the cells in (A) and (B) is an average of 24.2 μm/s. The average velocity in cells in (D) and (E), however, is only 6.1 μm/s because the cells are in a narrower vein. Images were taken every 3.30 seconds. Bar = 50 μm. E, Multicellular aggregate collided with another aggregate that was already attached to the vessel wall. The two aggregates attached and formed a larger aggregation. Some cells (arrow) escaped from the aggregate because of weak adhesion and recommenced movement. Images were taken every 1.04 seconds. Bar = 100 μm. Images were acquired in real time with the Olympus OV100. Right, schematics of (B) and (C) [21].
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f7-sensors-08-01157: Imaging nuclear-cytoplasmic dynamics of intravascular trafficking of cancer cellsA, Schematic diagram of the skin flap model in live mice for imaging intravascular trafficking and extravasation. An arc-shaped incision was made in the abdominal skin, and then the skin flap was spread and fixed on a flat stand. HT-1080-GFP-RFP cells were injected into the epigastric cranialis vein through a catheter. Immediately after injection, the inside surface of the skin flap was directly observed. B, HT-1080-GFP-RFP cell crawls smoothly along the vessel wall without rolling in a capillary (arrow). The nucleus and cytoplasm are slightly stretched. The nucleus is in the front of the cell while the cell is crawling. When the cell advanced into a part of the capillary where the diameter is smaller than of deformation limit of the cell, the cell could not advance any further. Bar = 100 μm. C, HT-1080-GFP-RFP cell, trafficking at low velocity, advanced between other cells and the vessel wall. The moving cancer cell contacted the other cells (arrow). The cell deformed slightly and continued to move without adhesion. Bar = 100 μm. Right, schematics of (B) and (C). D, One cancer cell migrating in the post capillary with slow velocity. The cytoplasm is at the head of the cell while the cell is moving in a large vein, but the nucleus is at the head in a small vein. The velocity of the cells in (A) and (B) is an average of 24.2 μm/s. The average velocity in cells in (D) and (E), however, is only 6.1 μm/s because the cells are in a narrower vein. Images were taken every 3.30 seconds. Bar = 50 μm. E, Multicellular aggregate collided with another aggregate that was already attached to the vessel wall. The two aggregates attached and formed a larger aggregation. Some cells (arrow) escaped from the aggregate because of weak adhesion and recommenced movement. Images were taken every 1.04 seconds. Bar = 100 μm. Images were acquired in real time with the Olympus OV100. Right, schematics of (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
Imaging nuclear-cytoplasmic dynamics of intravascular trafficking of cancer cellsA, Schematic diagram of the skin flap model in live mice for imaging intravascular trafficking and extravasation. An arc-shaped incision was made in the abdominal skin, and then the skin flap was spread and fixed on a flat stand. HT-1080-GFP-RFP cells were injected into the epigastric cranialis vein through a catheter. Immediately after injection, the inside surface of the skin flap was directly observed. B, HT-1080-GFP-RFP cell crawls smoothly along the vessel wall without rolling in a capillary (arrow). The nucleus and cytoplasm are slightly stretched. The nucleus is in the front of the cell while the cell is crawling. When the cell advanced into a part of the capillary where the diameter is smaller than of deformation limit of the cell, the cell could not advance any further. Bar = 100 μm. C, HT-1080-GFP-RFP cell, trafficking at low velocity, advanced between other cells and the vessel wall. The moving cancer cell contacted the other cells (arrow). The cell deformed slightly and continued to move without adhesion. Bar = 100 μm. Right, schematics of (B) and (C). D, One cancer cell migrating in the post capillary with slow velocity. The cytoplasm is at the head of the cell while the cell is moving in a large vein, but the nucleus is at the head in a small vein. The velocity of the cells in (A) and (B) is an average of 24.2 μm/s. The average velocity in cells in (D) and (E), however, is only 6.1 μm/s because the cells are in a narrower vein. Images were taken every 3.30 seconds. Bar = 50 μm. E, Multicellular aggregate collided with another aggregate that was already attached to the vessel wall. The two aggregates attached and formed a larger aggregation. Some cells (arrow) escaped from the aggregate because of weak adhesion and recommenced movement. Images were taken every 1.04 seconds. Bar = 100 μm. Images were acquired in real time with the Olympus OV100. Right, schematics of (B) and (C) [21].
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Related In: Results  -  Collection

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f7-sensors-08-01157: Imaging nuclear-cytoplasmic dynamics of intravascular trafficking of cancer cellsA, Schematic diagram of the skin flap model in live mice for imaging intravascular trafficking and extravasation. An arc-shaped incision was made in the abdominal skin, and then the skin flap was spread and fixed on a flat stand. HT-1080-GFP-RFP cells were injected into the epigastric cranialis vein through a catheter. Immediately after injection, the inside surface of the skin flap was directly observed. B, HT-1080-GFP-RFP cell crawls smoothly along the vessel wall without rolling in a capillary (arrow). The nucleus and cytoplasm are slightly stretched. The nucleus is in the front of the cell while the cell is crawling. When the cell advanced into a part of the capillary where the diameter is smaller than of deformation limit of the cell, the cell could not advance any further. Bar = 100 μm. C, HT-1080-GFP-RFP cell, trafficking at low velocity, advanced between other cells and the vessel wall. The moving cancer cell contacted the other cells (arrow). The cell deformed slightly and continued to move without adhesion. Bar = 100 μm. Right, schematics of (B) and (C). D, One cancer cell migrating in the post capillary with slow velocity. The cytoplasm is at the head of the cell while the cell is moving in a large vein, but the nucleus is at the head in a small vein. The velocity of the cells in (A) and (B) is an average of 24.2 μm/s. The average velocity in cells in (D) and (E), however, is only 6.1 μm/s because the cells are in a narrower vein. Images were taken every 3.30 seconds. Bar = 50 μm. E, Multicellular aggregate collided with another aggregate that was already attached to the vessel wall. The two aggregates attached and formed a larger aggregation. Some cells (arrow) escaped from the aggregate because of weak adhesion and recommenced movement. Images were taken every 1.04 seconds. Bar = 100 μm. Images were acquired in real time with the Olympus OV100. Right, schematics of (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