Light scattering as an intrinsic indicator for pancreatic islet cell mass and secretion

Abstract

The pancreatic islet of Langerhans is composed of endocrine cells producing and releasing hormones from secretory granules in response to various stimuli for maintenance of blood glucose homeostasis. In order to adapt to a variation in functional demands, these islets are capable of modulating their hormone secretion by increasing the number of endocrine cells as well as the functional response of individual cells. A failure in adaptive mechanisms will lead to inadequate blood glucose regulation and thereby to the development of diabetes. It is therefore necessary to develop tools for the assessment of both pancreatic islet mass and function, with the aim of understanding cellular regulatory mechanisms and factors guiding islet plasticity. Although most of the existing techniques rely on the use of artificial indicators, we present an imaging methodology based on intrinsic optical properties originating from mature insulin secretory granules within endocrine cells that reveals both pancreatic islet mass and function. We demonstrate the advantage of using this imaging strategy by monitoring in vivo scattering signal from pancreatic islets engrafted into the anterior chamber of the mouse eye, and how this versatile and noninvasive methodology permits the characterization of islet morphology and plasticity as well as hormone secretory status.

Figure 1. Pancreatic islets can be visualized using their intrinsic backscattering properties.

(A) Intrinsic scattering properties of pancreatic islets from various species allow their visualization in vitro by imaging side scattering of light by optical microscopy and backwards scattering of a laser beam scanning by confocal microscopy. This latter imaging technique not only offers high-resolution imaging but also provides three-dimensional information by the imaging of optical slices. (B) Different laser illumination wavelengths can be used to acquire backscatter images, the maximum signal intensity being collected at the incident light wavelength. (C) Confocal imaging of MIP-GFP islets at higher magnification reveals a punctuated pattern of backscatter signal, excluded from cell nuclei. The backscatter signal is present non-exclusively in each GFP-positive cell. Confocal images are presented as maximum intensity projections (A, B) or single optical planes (C). Size bars = 100 μm (A, B), 10 μm (C).

Figure 2. Backscatter signal can be used as an intrinsic indicator for islet volume.

(A) Photography of a transplanted eye shows a number of individual islets of various dimensions. (B) The islets were analyzed individually by computational analysis after in vivo imaging (islets represented as maximum intensity projection on XZ; white is backscatter signal; red line is equatorial area). (C) Mice were then sacrificed and their eyes collected, processed using an anti-insulin antibody for staining, and scanned by optical projection tomography (OPT) to determine individual islet volumes. (D) Comparison of individual islet volumes as quantified from images obtained in vivo by confocal microscopy and from images obtained ex vivo by OPT. (E) The ratios between individual islet volumes from islet grafts in three different mice are plotted and shown to be independent of the imaging technique in use. Size bar = 50 μm.

Figure 3. Quantification and characterization of islet growth in vivo by backscatter imaging.

(A) A schematic representation of dimensional information obtained by in vivo islet backscatter imaging is overlaid on an islet graft section stained by immunohistochemistry (green = insulin; blue = DAPI; black = pigmented iris). In vivo imaging permits longitudinal analysis of the islet equatorial volume and projected area. (B) Equatorial volume and projected area from individual islets imaged in vivo and (C) calculated diameter based on a spherical model. (D) In vivo imaging of the same islets engrafted into the anterior chamber of the eye at two different time points allows the longitudinal assessment of islet growth in the ob/ob mouse. (E) Growth of 27 islets was assessed over a period from 1 to 3 months in 7 ob/ob mice. The average X-, Y-, or Z-growth based either on the equatorial volume or on the projected area shows no preference for directional growth in the ob/ob mouse model. Size bars = 50 μm.

Figure 4. Backscatter intensity in beta cells originates from dense core secretory granules and provides information on islet secretory status.

(A) Electron microscopy imaging shows that untreated isolated pancreatic islets display typical dense core secretory granules containing zinc-insulin crystals. (D) The chelation of Zn²⁺ by treatment with TPEN impedes with normal crystallization, resulting in pale granules. (B, E) Dithizone staining in treated versus non-treated islets demonstrates a clear loss of Zn crystals after TPEN treatment. (C, F, G) The reduction in the number of dense core granules results in a decreased backscatter signal intensity. (H) Islets were isolated from mice having either free access to food or been food-deprived for 12 h, and their backscatter signal was acquired by confocal microscopy. (I) Both backscatter signal intensity and insulin content were increased when mice were fasted overnight. (J) In vivo imaging of an islet engrafted into the anterior chamber of the ob/ob mouse eye on two consecutive days shows differences in backscatter signal intensities depending on whether the mouse had free access to food or had been food-deprived for 12 h prior to imaging. Note that the backscatter signal originating from the strongly reflective pigmented iris is similar in both images, illustrating in this case that under identical imaging conditions the variations observed in signal intensities are islet-specific. (K) Quantification of backscatter signal intensities in vivo shows an increase under fasting conditions, correlating with lower blood glucose levels resulting in decreased insulin secretion from islets (n = 19 islets in 3 mice). Values are average ± SEM. ^*P < 0.05; ^**P < 0.01; ^****P < 0.0001. Size bars = 100 μm.