Noninvasive in vivo imaging of pancreatic islet cell biology

Abstract

Advanced imaging techniques have become a valuable tool in the study of complex biological processes at the cellular level in biomedical research. Here, we introduce a new technical platform for noninvasive in vivo fluorescence imaging of pancreatic islets using the anterior chamber of the eye as a natural body window. Islets transplanted into the mouse eye engrafted on the iris, became vascularized, retained cellular composition, responded to stimulation and reverted diabetes. Laser-scanning microscopy allowed repetitive in vivo imaging of islet vascularization, beta cell function and death at cellular resolution. Our results thus establish the basis for noninvasive in vivo investigations of complex cellular processes, like beta cell stimulus-response coupling, which can be performed longitudinally under both physiological and pathological conditions.

Figure 1

Pancreatic islet transplantation into the anterior chamber of the eye. (a,b) Illustration of islet transplantation into the anterior chamber of the eye (a) and noninvasive in vivo imaging (b). (c) Photograph of islets engrafted on the iris. Scale bar, 2 mm. (d) Islet graft sections stained for insulin (red) and glucagon (green) at indicated time points after transplantation. Scale bar, 50 μm. (e) Ratio of insulin⁺ to glucagon⁺ cells in islets in the pancreas and in the anterior chamber of the eye at the indicated time points after transplantation (differences are not significant; ANOVA, P = 0.38, n = 4 sections per pancreas, n = 3–4 mice per time point).

Figure 2

Noninvasive imaging of RIP-GFP islet engraftment and vascularization. (a) Maximum projections of image stacks (110-μm thick) of an islet graft in the anterior chamber of the eye captured at indicated time points after transplantation. Top row, GFP fluorescence (beta cells); middle row, Texas Red fluorescence (blood vessels); bottom row, overlay of GFP and Texas Red. Two-photon excitation, 890 nm; objective: 10× 0.3 W; zoom factor, 2–2.3. Scale bar, 100 μm. (b,c) Quantification of vessel density (b) and vessel diameter (c) in islet grafts at the indicated time points after transplantation (n = 5 for each time point).

Figure 3

Pancreatic islets engrafted in the anterior chamber of the eye maintain glucose homeostasis. (a) Nonfasting glycemia in streptozotocin-induced diabetic mice after transplantation of islets (~300 islet equivalents) to the anterior chamber of the eye (n = 4; ▲, ◊, ◆ and Δ each correspond to one mouse). Removal of the islet graft–bearing eye is indicated by the arrow. Dashed line indicates upper limit for normoglycemia. (b) Plasma glucose evels during intraperitoneal glucose tolerance tests performed 6 weeks after transplantation of islets in the anterior chamber of the eye (□, n = 4), in normal controls (●, n = 9), and in streptozotocin-induced diabetic mice (○, n = 7).

Figure 4

Noninvasive in vivo imaging of beta cell [Ca²⁺]_i handling. (a) Fluorescence images of Fluo-4 and Fura-Red at indicated time points after systemic application of glibenclamide (1 mg/kg) at 180 s, 3 months after transplantation. Excitation, 488 nm; objective, 20× W 0.5; zoom factor, 2.3; pinhole, 1 airy unit. (b) Whole-frame Fluo-4/Fura-Red ratio change in response to glibenclamide stimulus (arrow indicates stimulation start). (c) Ratio change in individual cells within the imaging plane as indicated in panel a. ROI, region of interest. (d) Ratiometric maximum projections of Fluo-4/Fura-Red of a whole islet before (left) and after (right) glibenclamide stimulation. Excitation, 488 nm; objective, 20× W 0.5; zoom factor, 1.7, pinhole, 1 airy unit. (e) A representative Fluo-4/Fura-Red ratio change in response to a glibenclamide stimulus, start as indicated by arrows, recorded from the same islet graft at days 34 (left) and 37 (right) after transplantation. Scale bars, 50 μm.

Figure 5

Noninvasive in vivo imaging of beta cell death. (a–h) Maximum projections of image stacks (260-μm thick) captured of an individua RIP-GFP islet graft in the anterior chamber of the eye 2 months after transplantation. Excitation, 488, 543 and 633 nm; objective, 20× W 0.5 (a–d) and 40× W 0.8 (e–h); zoom factor, 1.7; pinhole: 1 airy unit. (a–d) GFP fluorescence (beta cells, a), annexin V–APC labeling (b), reflected light of the islet graft (c) and overlay of a– c(d) with reflected light in blue under normoglycemic conditions (top row) and under hyperglycemic conditions 24 h after alloxan treatment (bottom row). (e–h) High magnification images of a RIP-GFP islet graft area strongly labeled with annexin V–APC after alloxan-nduced beta cell death. (e) GFP fluorescence (beta cells). (f) Annexin V-APC fluorescence. (g) Overlay of e and f. (h) Overlay of g and reflected light in blue. Scale bars, 100 μm.