Micro-CT for Biological and Biomedical Studies: A Comparison of Imaging Techniques

Abstract

Several imaging techniques are used in biological and biomedical studies. Micro-computed tomography (micro-CT) is a non-destructive imaging technique that allows the rapid digitisation of internal and external structures of a sample in three dimensions and with great resolution. In this review, the strengths and weaknesses of some common imaging techniques applied in biological and biomedical fields, such as optical microscopy, confocal laser scanning microscopy, and scanning electron microscopy, are presented and compared with the micro-CT technique through five use cases. Finally, the ability of micro-CT to create non-destructively 3D anatomical and morphological data in sub-micron resolution and the necessity to develop complementary methods with other imaging techniques, in order to overcome limitations caused by each technique, is emphasised.

Keywords: confocal laser scanning microscopy; micro-computed tomography; optical microscopy; scanning electron microscopy.

Figure 1

(a) Egg capsules (×8 magnification) and (b,c) living juveniles of Hexaplex trunculus (×25 magnification) using a stereoscope (Zeiss Discovery.V12).

Figure 2

Volume rendering of an adult Hexaplex trunculus shell acquired using a Skyscan 1172 micro-CT scanner.

Figure 3

Three-dimensional (3D) model of the closed pores (indicated in red) of an adult Hexaplex trunculus shell (shell length 48.33 mm) acquired using a Skyscan 1172 micro-CT scanner.

Figure 4

Colour-coded structure thickness images of (a) whole specimen and (b) cross-section along the specimen of an adult Hexaplex trunculus shell acquired using a Skyscan 1172 micro-CT scanner.

Figure 5

Volume rendering of egg capsules of Hexaplex trunculus acquired using a Skyscan 1172 micro-CT scanner.

Figure 6

Volume rendering of juvenile Hexaplex trunculus acquired using a Skyscan 1172 micro-CT scanner, where internal calcified structures (statoliths, indicated with white arrows) are also visible.

Figure 7

OM images of (a) middle body cross-section of a specimen showing the orientation and the type of chaetae (×40 magnification), (b) detailed view of middle body chaetae (×60 magnification), and (c) body shape of the specimen (×40 magnification).

Figure 8

Micro-CT images of (a) cross-section of the middle body of specimen, with white arrows showing the jaws, (b) body shape of the specimen, and (c) jaws of the specimen.

Figure 9

SEM images of (a) body form of specimen, (b) 3D form of paleae chaete, and (c) shape of paleae chaete and compound chaete of middle body.

Figure 10

(a) Volume rendering of thrombi and (b) 3D color visualisation of a thrombus sample (volume 16 mm³) acquired through the Skyscan 1172 micro-CT scanner: Erythrocyte-rich regions were rendered in red, whereas platelet-rich regions were rendered in white. (b) from [13], reproduced under a CC-BY licence.

Figure 11

OM image (×200 magnification) of a histological examination of a thrombotic specimen revealed a thrombus characterised by fibrin/erythrocytes (occupied 70% of the total area of the thrombus) and leukocytes (30%).

Figure 12

Volume rendering of (a) the whole specimen and (b) the virtually dissected specimen and (c) transaxial, sagittal, and coronal images of the Drosophila melanogaster acquired through the Skyscan 1172 micro-CT scanner. The white arrow and the cross-point of the coloured lines indicate the heart position.

Figure 12

Volume rendering of (a) the whole specimen and (b) the virtually dissected specimen and (c) transaxial, sagittal, and coronal images of the Drosophila melanogaster acquired through the Skyscan 1172 micro-CT scanner. The white arrow and the cross-point of the coloured lines indicate the heart position.

Figure 13

CLSM visualisation (×60 magnification) of different stained Drosophila melanogaster tissues. (a) Drosophila heart tissue following immunofluorescence staining with an antibody against the Atg8a/GABARAP autophagic protein. (b) Different sections of a fly muscle probed with the mitochondrial marker blw/ATP5A (green) in a Parkinson’s disease fly model, showing accumulation of mitochondrial aggregates (arrows) vs. control. In (a,b) nuclei and actin were counterstained with DAPI and phalloidin, respectively. In (a), Z-stacks with a step size of 1 µm were taken using identical settings, and each stack consisted of 26 plane images.

Figure 13

CLSM visualisation (×60 magnification) of different stained Drosophila melanogaster tissues. (a) Drosophila heart tissue following immunofluorescence staining with an antibody against the Atg8a/GABARAP autophagic protein. (b) Different sections of a fly muscle probed with the mitochondrial marker blw/ATP5A (green) in a Parkinson’s disease fly model, showing accumulation of mitochondrial aggregates (arrows) vs. control. In (a,b) nuclei and actin were counterstained with DAPI and phalloidin, respectively. In (a), Z-stacks with a step size of 1 µm were taken using identical settings, and each stack consisted of 26 plane images.

Figure 14

(a) Three-dimensional (3D) volume renderings, (b) transverse section image midway through the right coronal suture, and (c) bone thickness visualisation derived from microCT scans of wild-type and a Erf^loxP/− (CRS-4) animal with multisuture synostosis at P65. Black arrows indicate the fused sutures. Yellow arrows point to the coronal suture position in the transverse sections. White arrows indicate the minimal bone thickness in the coronal suture region in the wild-type animal.

Figure 15

OM image (×1 magnification) of a histological examination of the coronal sutures of P65 mouse calvaria stained with Alizarin Red and Alcian Blue. Black arrows indicate the position of the open suture in wild-type animals and white arrows indicate the position of the ossified suture in the Erf^loxP/− (CRS-4) craniosynostosis animals.

Figure 16

Confocal microscopy (×63 magnification) of coronal sutures transverse cryosections from P15 mouse calvaria stained with BrdU (green) to evaluate cellular proliferation and TOPRO-3 (red) to identify the nuclei. Dotted lines indicate the position of the parietal and frontal bones.