Microscopic dual-energy CT (microDECT): a flexible tool for multichannel ex vivo 3D imaging of biological specimens

Dual-energy computed tomography (DECT) uses two different x-ray energy spectra in order to differentiate between tissues, materials or elements in a single sample or patient. DECT is becoming increasingly popular in clinical imaging and preclinical in vivo imaging of small animal models, but there have been only very few reports on ex vivo DECT of biological samples at microscopic resolutions. The present study has three main aims. First, we explore the potential of microscopic DECT (microDECT) for delivering isotropic multichannel 3D images of fixed biological samples with standard commercial laboratory-based microCT setups at spatial resolutions reaching below 10 m. Second, we aim for retaining the maximum image resolution and quality during the material decomposition. Third, we want to test the suitability for microDECT imaging of different contrast agents currently used for ex vivo staining of biological samples. To address these aims, we used microCT scans of four different samples stained with x-ray dense contrast agents. MicroDECT scans were acquired with five different commercial microCT scanners from four companies. We present a detailed description of the microDECT workflow, including sample preparation, image acquisition, image processing and postreconstruction material decomposition, which may serve as practical guide for applying microDECT. The MATLAB script (The Mathworks Inc., Natick, MA, USA) used for material decomposition (including a graphical user interface) is provided as a supplement to this paper (). In general, the presented microDECT workflow yielded satisfactory results for all tested specimens. Original scan resolutions have been mostly retained in the separate material fractions after basis material decomposition. In addition to decomposition of mineralized tissues (inherent sample contrast) and stained soft tissues, we present a case of double labelling of different soft tissues with subsequent material decomposition. We conclude that, in contrast to in vivo DECT examinations, small ex vivo specimens offer some clear advantages regarding technical parameters of the microCT setup and the use of contrast agents. These include a higher flexibility in source peak voltages and x-ray filters, a lower degree of beam hardening due to small sample size, the lack of restriction to nontoxic contrast agents and the lack of a limit in exposure time and radiation dose. We argue that microDECT, because of its flexibility combined with already established contrast agents and the vast number of currently unexploited stains, will in future represent an important technique for various applications in biological research. Lay description Conventional x-ray computed tomography (CT) is usually restricted to producing greyscale image volumes from patients or samples. This is because standard detectors used for x-ray imaging do not collect spectral information. Dual-energy computed tomography (DECT) circumvents this problem by acquiring two tomographic scans based on two different x-ray energy spectra. A fusion of the two scans displays spectral information and creates a colour CT image volume. Based on this colour image volume, densities of different materials can be calculated during a process called basis material decomposition. Recently, DECT has become increasingly popular in clinical and preclinical imaging, but to date there have been only very few reports on high-resolution ex vivo DECT of biological samples. The present study aims to establish DECT for imaging small biological samples at micron resolutions. We explore the potential of microscopic DECT (microDECT) for delivering colour 3d images of fixed biological samples with commercial microCT setups. In addition, we test the suitability of different contrast agents currently used for ex vivo staining of biological samples for microDECT imaging protocols. We present a detailed description of the microDECT workflow including sample preparation, image acquisition, image processing, and post-reconstruction material decomposition that may serve as practical guide for applying microDECT. The presented workflow yielded satisfactory results for all tested specimens. Original scan resolutions have been mostly retained in material fractions after basis material decomposition. We present examples for two-colour discrimination of mineralized tissues (inherent sample contrast) and stained soft tissues, and one case of double labelling of different soft tissue fractions with subsequent material decomposition. We conclude that microDECT will in future represent an important technique for various applications in biomedical research.