An improved protocol for optical projection tomography imaging reveals lobular heterogeneities in pancreatic islet and β-cell mass distribution

Abstract

Optical projection tomography (OPT) imaging is a powerful tool for three-dimensional imaging of gene and protein distribution patterns in biomedical specimens. We have previously demonstrated the possibility, by this technique, to extract information of the spatial and quantitative distribution of the islets of Langerhans in the intact mouse pancreas. In order to further increase the sensitivity of OPT imaging for this type of assessment, we have developed a protocol implementing a computational statistical approach: contrast limited adaptive histogram equalization (CLAHE). We demonstrate that this protocol significantly increases the sensitivity of OPT imaging for islet detection, helps preserve islet morphology and diminish subjectivity in thresholding for tomographic reconstruction. When applied to studies of the pancreas from healthy C57BL/6 mice, our data reveal that, at least in this strain, the pancreas harbors substantially more islets than has previously been reported. Further, we provide evidence that the gastric, duodenal and splenic lobes of the pancreas display dramatic differences in total and relative islet and β-cell mass distribution. This includes a 75% higher islet density in the gastric lobe as compared to the splenic lobe and a higher relative volume of insulin producing cells in the duodenal lobe as compared to the other lobes. Altogether, our data show that CLAHE substantially improves OPT based assessments of the islets of Langerhans and that lobular origin must be taken into careful consideration in quantitative and spatial assessments of the pancreas.

Figure 1

CLAHE facilitates OPT-based assessments of islets and BCM. (A–H) Illustration of the effect of CLAHE (E–H) as compared to non-normalized tomographic projection data (A–D). (A and B) Original projection image. (E and F) Images showing effect of CLAHE when applied to (A). (C and G) Intensity histograms of the patches shown in (B and F) respectively. (D and H) Segmentation of the patches shown in (B and F) respectively. Scale bar in (E) is 2 mm in (A and E). Scale bar in (F) is 1 mm in (B and F).

Figure 2

Adaptive contrast normalization facilitates detection of islets by OPT imaging. (A–J) OPT images of splenic pancreas from eight-week C57Bl/6 mice labelled for insulin showing non-normalized images (A–C) and CLAHE processed images (E–G) of OPT projection data (A and E), resultant volume-renderings (B and F) and iso-surface reconstructions (C and G). (D) Overlay of the non-normalized data in ((C), pseudo colored green) and the CLAHE processed data ((G), pseudo colored red). CLAHE processing enables detection of a larger number of insulin labelled islets (red only). (H) high magnification image corresponding to inset in (D). (I and J) Representative high magnification iso-surface reconstructions (of high intensity islets) illustrating the effect on islet morphology with non-normalized (I, green) versus CLAHE processed data (J, red). Scale bar in (D) is 1,000 µm in (A–G). Scale bar in (H) is 500 µm. Scale bar in (I) is 500 µm in (I and J).

Figure 3

The lobular compartments of the pancreas display significant differences in relative β-cell mass and islet densities. (A) Photomicrograph of a gut segment including the stomach, duodenum, spleen and pancreas from a C57Bl/6 mouse at eight weeks. For comparative OPT assessments, the lobular compartments were separated based on morphologic features and the relationship to embryological origin (indicated by broken lines). (B–D) Representative iso-surface reconstructions of the islet β-cell volumes in the splenic (B, red), gastric (C, yellow) and duodenal (D, green) lobes of the pancreas from C57Bl/6 mice at eight weeks. In (B–D) the exocrine parenchyma (grey) is reconstructed based on the signal from endogenous tissue autofluorescence. (E) Graph showing the total lobular distribution of pancreatic tissue, insulin⁺ islets and β-cell volumes. (F) Graph showing the lobular distribution of insulin⁺ islets/mm³ of pancreatic tissue. (G) Graph showing lobular β-cell volume/total lobe volume. In (E–G), n = 5, values are given ±SEM. Significance levels indicated correspond to **p < 0.01, ***p < 0.001. CLAHE processing was implemented for all display items in (B–G). Scale bar in (A) is 2 mm. Scale bar in (D) is 1 mm in (B–D). Abbreviations: GL, gastric lobe; DL, duodenal lobe; SL, splenic lobe; St, stomach; Spl, spleen; Duo, duodenum.