In vivo optical molecular imaging and analysis in mice using dorsal window chamber models applied to hypoxia, vasculature and fluorescent reporters - PubMed (original) (raw)

In vivo optical molecular imaging and analysis in mice using dorsal window chamber models applied to hypoxia, vasculature and fluorescent reporters

Gregory M Palmer et al. Nat Protoc. 2011.

Abstract

Optical techniques for functional imaging in mice have a number of key advantages over other common imaging modalities such as magnetic resonance imaging, positron emission tomography or computed tomography, including high resolution, low cost and an extensive library of available contrast agents and reporter genes. A major challenge to such work is the limited penetration depth imposed by tissue turbidity. We describe a window chamber technique by which these limitations can be avoided. This facilitates the study of a wide range of processes, with potential endpoints including longitudinal gene expression, vascular remodeling and angiogenesis, and tumor growth and invasion. We further describe several quantitative imaging and analysis techniques for characterizing in vivo fluorescence properties and functional endpoints, including vascular morphology and oxygenation. The procedure takes ∼2 h to complete, plus up to several weeks for tumor growth and treatment procedures.

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Figures

Figure 1

Window chamber. The window chamber, retaining nuts and window glass are shown. Scale bar, 1 cm. Reproduced with permission (G.M.P., A.N.F., S.S. and M.W.D., unpublished data).

Figure 2

Equipment and surgical tools. Some of the equipment and surgical tools described in this protocol are shown here with descriptions. Scale bar, 10 cm.

Figure 3

Imaging mount. Imaging mount used to fix the window chamber onto a microscope stage. Large central hole is at least 12 mm in diameter to permit imaging, and the three smaller holes are positioned to allow the bolts on the window chamber to pass through. The mount can be custom designed for a given microscope to permit secure attachment to the stage. Scale bar, 1 cm.

Figure 4

Surgical platform preparation. With the heated paraffin pad beneath the surgical stage and the viewing stage attached with binder clips, the sterile field is draped over the entire assembly. A rectangular section is cut from the sterile field so that the viewing stage is exposed. Reproduced with permission (G.M.P., A.N.F., S.S. and M.W.D., unpublished data).

Figure 5

Preparing the mouse for surgery. The line drawn along the mouse’s spine will facilitate symmetrical placement of the window chamber. The dots will be used to ensure that the window chamber is sutured at the proper height and lateral position on the torso.

Figure 6

Securing the dorsal skin fold. Four sutures along the marked line attach the dorsal skin to the C-holder. This provides the working space for window chamber attachment.

Figure 7

Tumor cell inoculation. Approximately 20 μl of the cell suspension is injected between the dermis and the superficial fascia. Proper positioning of the needle tip will cause the injected cell suspension to form a small bubble.

Figure 8

Functional imaging. Magnified (×2.5) raw and processed images of a 4T1 tumor expressing GFP concurrently with HIF-1α. (a) Bright-field transmission image of the central tumor and surrounding normal tissue. (b) Grayscale image of raw fluorescence intensity. (c) Mapping of relative variations in total hemoglobin. (d) Simultaneous visualization of inverse total hemoglobin absorption and hemoglobin saturation. Increased pixel brightness represents an increased hemoglobin component at that pixel location. Hemoglobin saturation is mapped on a blue-red color gradient, with the deoxygenated hemoglobin component represented in the blue color channel, and the oxygenated hemoglobin component represented in the red color channel. (e) Simultaneous visualization of total hemoglobin absorption (brightness of red-blue signal), hemoglobin saturation (red-blue color scale) and GFP expression (green channel). Scale bars, 1 mm.

Figure 9

Spectral decomposition. Decomposition of overlapping spectra using PARAFAC. (a) Bright-field image in which the tumor can be seen in the left-hand side of the image. (b) Fluorescence intensity averaged across the entire wavelength range. (c) PARAFAC-derived spectral components corresponding to fluorescence and phosphorescence emission. (d–f) The factor scores are shown for the fluorescence (d) and phosphorescence (e) channels, as well as the ratio of fluorescence/phosphorescence (f) (scale bars are 1 mm). Portions of this figure are reprinted with permission from Palmer et al.

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In vivo optical molecular imaging and analysis in mice using dorsal window chamber models applied to hypoxia, vasculature and fluorescent reporters - PubMed (original) (raw)