Quantitative analysis of microscopicX‐ray computed tomography imaging: Japanese quail embryonic soft tissues with iodine staining

Abstract

Rapid three-dimensional imaging of embryos to better understand the complex process of morphogenesis has been challenging. Recently introduced iodine staining protocols (I 2 KI and alcoholic iodine stains) combined with microscopic X-ray computed tomography allows visualization of soft tissues in diverse small organisms and tissue specimens. I 2 KI protocols have been developed specifically for small animals, with a limited number of quantitative studies of soft tissue contrasts. To take full advantage of the low X-ray attenuation of ethanol and retain bound iodine while dehydrating the specimen in ethanol, we developed an ethanol I 2 KI protocol. We present comparative microscopic X-ray computed tomography analyses of ethanol I 2 KI and I 2 KI staining protocols to assess the performance of this new protocol to visualize soft tissue anatomy in late stage Japanese quail embryos using quantitative measurements of soft tissue contrasts and sample shrinkage. Both protocols had only 5% shrinkage compared with the original harvested specimen, supporting the use of whole mounts to minimize tissue shrinkage effects. Discrimination within and among the selected organs with each staining protocol and microscopic X-ray computed tomography imaging were comparable to those of a gray scale histological section. Tissue discrimination was assessed using calibrated computed tomography values and a new discrimination index to quantify the degree of computed tomography value overlaps between selected soft tissue regions. Tissue contrasts were dependent on the depth of the tissue within the embryos before the embryos were saturated with each stain solution, and optimal stain saturations for the entire embryo were achieved at 14 and 28 days staining for I 2 KI and ethanol I 2 KI, respectively. Ethanol I 2 KI provided superior soft tissue contrasts by reducing overstaining of fluid-filled spaces and differentially modulating staining of some tissues, such as bronchial and esophageal walls and spinal cord. Delineating the selected soft tissues using optimal threshold ranges derived from the quantitative analyses of the contrast enhancement in optimally stained embryos is possible. The protocols presented here are expected to be applicable to other organisms with modifications to staining time and contribute toward rapid and more efficient segmentation of soft tissues for three-dimensional visualization.

Comparison and Evaluation of the Effectiveness of Two Approaches of Diffusible Iodine‐Based Contrast‐Enhanced Computed Tomography (diceCT) for Avian Cephalic Material

“…We ascribe this to the strong support of bony tissues within the cranial specimens, which restricted the potential shrinkage of soft tissues. Similar results have been reported for mounted whole bird specimens (Tahara and Larsson, ) and our previous results indicate minor shrinkage with longer staining duration on larger cranial specimens (Li et al., ). Less than 10% shrinkage in lineal measurements was observed for the tongue specimens after both I 2 KI‐formaldehyde and I 2 E staining compared to the size before the staining (after fixation).…”

“…As shown in other experiments, it seems to be more efficient to use I 2 E staining, requiring relatively shorter staining durations even though the concentration adopted is usually lower than that of I 2 KI (Metscher, ,b; Degenhardt et al., ; Tahara and Larsson, ). The simulation results also provide a key to interpreting intrinsic differences between the two approaches.…”

Development of the paratympanic pneumatic system of Japanese quail

“…An aqueous PMA staining has been previously used to enhance the soft tissue contrast with micro‐CT (e.g., Golding & Jones, ; Golding, Ponder, & Byrne, ; Pauwels, Van Loo, Cornillie, Brabant, & Van Hoorebeke, ). We modified PMA staining using ethanol solvent as used in Nieminen et al (), to limit background staining (e.g., Tahara & Larsson, ). PMA was made of 2.5 g PMA dissolved in 100 mL of 70% ethanol (2.5% PMA).…”

Diffusible iodine‐based contrast‐enhanced computed tomography (diceCT): an emerging tool for rapid, high‐resolution, 3‐D imaging of metazoan soft tissues

“…; Lautenschlager et al. ; Tahara & Larson, ; Gignac & Kley, ), and mammals (Degenhardt et al. ; Herdina et al.…”