Near Infrared Optical Projection Tomography for Assessments of β-cell Mass Distribution in Diabetes Research

Abstract

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen's (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR) 2 ; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction 2 and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations 3. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.

Let’s shine a light on fungal infections: A noninvasive imaging toolbox

“…Moreover, as OPT and SPIM offer the possibility to multiplex different fluorescent signals, it can be developed to look both at labeled pathogens and host cells. OPT and SPIM are successfully used in embryology and diabetes research [24,25] and are currently being optimized for fungal infections as well (Fig 1F and 1G).…”

Spatial and quantitative datasets of the pancreatic β-cell mass distribution in lean and obese mice

“…By the implementation of a custom built specimen mount 6 and by alignment of the (overlapping) first and last projection image recorded in each scan, we could determine that any potential movements of the specimen during scanning were negligible. Highly experienced animal caretakers with long previous experience of the models performed phenotyping of the utilized animals.…”

Sleeve gastrectomy, but not duodenojejunostomy, preserves total beta-cell mass in Goto-Kakizaki rats evaluated by three-dimensional optical projection tomography

“…The pancreas was moved to a second Petri dish of ice-cold PBS where superficial connective tissue and fat were removed from the pancreatic surface. Subsequent steps for OPT analysis were performed according to a previous protocol with the modification that the freeze-thaw steps were performed prior to incubation in modified Dent's bleach and subsequent washes [27]. Briefly, the pancreas was fixated for 2 h in fresh 4 % paraformaldehyde (Fisher Scientific, Loughborough, UK), washed twice in PBS for 15 min and dehydrated for 15 min each in 33, 66 and 100 % methanol (Scharlau, Sentmenat, Spain).…”

“…The technique yields a volume reconstruction of tissue that has been fluorescently stained in toto ('whole mount'). This method has been thoroughly validated and refined for the assessment of BCM distribution (individual islet volumes and corresponding centroid coordinates) throughout the volume of intact pancreata [25][26][27].…”