Noninvasive high-resolution in vivo imaging of cell biology in the anterior chamber of the mouse eye

Abstract

There is clearly a demand for an experimental platform that enables cell biology to be studied in intact vascularized and innervated tissue in vivo. This platform should allow observations of cells noninvasively and longitudinally at single-cell resolution. For this purpose, we use the anterior chamber of the mouse eye in combination with laser scanning microscopy (LSM). Tissue transplanted to the anterior chamber of the eye is rapidly vascularized, innervated and regains function. After transplantation, LSM through the cornea allows repetitive and noninvasive in vivo imaging at cellular resolution. Morphology, vascularization, cell function and cell survival are monitored longitudinally using fluorescent proteins and dyes. We have used this system to study pancreatic islets, but the platform can easily be adapted for studying a variety of tissues and additional biological parameters. Transplantation to the anterior chamber of the eye takes 25 min, and in vivo imaging 1-5 h, depending on the features monitored.

Figure 1

Imaging setup for noninvasive in vivo imaging in the anterior chamber of the eye. Photograph shows an upright microscope with custom-built stage (1) for incorporation of the holding equipment, including metal plate (2), head holder (3), universal joint (4) and heating pad (5).

Figure 2

Noninvasive in vivo imaging in the anterior chamber of the eye. (a) The head of the mouse is restrained in the head holder with the eye containing the transplanted pancreatic islets facing upward. Anesthesia is applied via the gas mask. The eye lids of the graft-bearing eye are held back by a polyethylene tubing loop between the tips of a forceps to expose and stabilize the eye. Viscotears is applied as immersion liquid between the objective APO 10 × 0.3 W and the eye. (b) Head holder equipped with nose piece for fixation. (c) Head holder equipped with gas mask for fixation and simultaneous application of gas anesthesia.

Figure 3

Custom-built stabilizer of the mouse eye for noninvasive in vivo imaging. A forceps is attached to a Universal Joint (black) via a metal bar and the tips of the forceps covered with a loop of polyethylene tube. At the front part of the forceps a screw enables adjustment of the loop size. The universal joint is attached to a metal plate (silver), which is covered by a heating pad (white). The head holder was removed for this picture.

Figure 4

Perfusion of the anterior chamber of the mouse eye. Two micropipettes are penetrating the cornea at opposite sides of the eye and allow flow of a fluorescent indicator from the inflow pipette (right) through the anterior chamber into the outflow pipette (left).

Figure 5

Morphological characterization of a pancreatic islet graft by imaging reflection and GFP. Islets of mice expressing GFP under the insulin promoter (green β cells) were transplanted to mice expressing GFP under the Tie2 promoter (green endothelial cells). (a) GFP was excited with 488 nm at 35% laser power and emission measured between 495 and 530 nm. (b) Reflection was imaged by exciting with 633 nm at 35% laser power and measuring emission between 632 and 639 nm. (c) Overlay of a and b. Objective APO 10 × 0.3 W. Pinhole: 1 airy unit. Zoom factor: 1.7. Image bit depth: 8 bit. Resolution xyz: 1.38 × 1.38 × 5 μm. Scale bar: 100 μm.

Figure 6

Imaging of tissue vascularization. Panels show the same graft as in Figure 5. Blood vessels were visualized by an intravenous injection of a 70 kDa Texas Red labeled dextran. GFP (a) and Texas Red (b) were excited with a two-photon laser at 890 nm at minimal necessary laser power required and emission collected onto nondescanned detectors using a dichroic mirror (RSP 560) and emission filters (BP 525/50 and BP 640/20). (c) Overlay of a and b. Objective APO 10 × 0.3 W. Pinhole: 1 airy unit. Zoom factor: 1.7. Image bit depth: 8 bit. Resolution xyz: 1.38 × 1.38 ×5 μm. Scale bar: 100 μm.

Figure 7

Imaging of cell death. Panels show the same graft as in Figures 5 and 6. β cell death was induced by intravenous injection of alloxan. Apoptotic and dead cells were visualized by intravenous injection of annexin V-APC. (a) Reflection was imaged by exciting with 543 nm and emission measured between 539 and 547 nm at 35% laser power. (b) GFP was excited at 488 nm and emission measured between 495 and 530 nm at 35% laser power. (c) Allophycocyanin (APC) was excited at 633 nm with collection of emission light between 645 and 680 nm at 75% laser power. (d) Overlay of a,b and c. Objective APO 10 × 0.3 W. Pinhole: 1 airy unit. Zoom factor: 1.7. Image bit depth: 8 bit. Resolution xyz: 1.38 × 1.38 × 10 μm. Scale bar: 100 μm.

Figure 8

Loading of cells within the anterior chamber of the eye with calcium indicators. For loading, the anterior chamber was perfused with Fluo-4 and Fura-Red. Fluo-4 (a) and Fura-Red (b) were excited with 488 nm at 25% laser power and emission measured for Fluo-4 between 495 and 535 nm and for Fura-Red between 600 and 700 nm. Reflection was imaged by exciting with 543 nm and emission measured between 539 and 547 nm at 15% laser power. (c) Overlay of a and b. Objective APO 20 × 0.5 W. Pinhole: 1 airy unit. Zoom factor: 1.7. Image bit depth: 12 bit. Resolution xyz: 0.7 × 0.7 × 3.3 μm. Scale bar: 100 μm.