1Department of Molecular Genetics, Weizmann Institute of Science, 2Department of Biological Regulation, Weizmann Institute of Science, 3Department of Chemical Infrastructure, Weizmann Institute of Science
Sharir, A., Ramniceanu, G., Brumfeld, V. High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT. J. Vis. Exp. (52), e2688, doi:10.3791/2688 (2011).
Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (μCT).
High resolution μCT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the μCT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, μCT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in μCT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180° or 360°between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.
1. Sample preparation
2. Sample immobilization
At high resolution, it is important to avoid any change in the sample position during the measurement. For this, the sample is tightly fixed into a plastic recipient that fits its size. Polystyrene pipette tips, plastic Pasteur pipettes or specially built plastic holders are used in this respect. According to the experimental requirements, the sample can be examined in air or immersed in ethanol or buffer solutions. Typical immobilization and final positioning of the leg of the mouse embryo in the instrument is shown in Fig 1.
Figure 1. Final positioning of the embryonic mouse leg in the micro CT instrument.
3. Setting acquisition parameters: x-ray voltage and current, CCD exposure time
4. Sample positioning
The whole field to be viewed in 3D should be present in the projection image at all angles. One should check this by rotating the sample at different angles and by bringing the sample as close as possible to the rotation axis. For this, one should follow the following steps:
5. High resolution tomogram
Figure 2. Projection images of the lung of rat at 0°(A), 45° (B) and 90° (C) rotation angle.
6. Image scale calibration
The pixel level (value) in a reconstructed image is unique for that image. In order to compare two different images, a unique intensity scale has to be imposed on each image. For this
7. Image processing and analysis
After obtaining high resolution images, one has to extract relevant information by using image analysis software. The software package to be used has to be designed to work with very large files (up to 20Gb).
8. Representative Results
A representation of a femur from a C57/Bl6 mouse at embryonic day 18.5 (E18.5) - four days after the initiation of the mineralization process is shown in Fig 3. The mineral layers are clearly visible (white), while soft tissues are not visible in this preparation. We took 1000 projection images with a linear magnification of 4x. The final resolution is 8 microns. A careful analysis of the volume rendering shown in Fig.1, shows that the bone volume fraction (the fraction of the bone volume that is occupied by mineralized tissue) is 0.18, and the bone mineral density is 723 mg/cm3. Those values allow us to compare this structure with bones in other stages of development.
Figure 3. Different representations of a 3D image of a mouse femur embryo. The transversal (cross section) (A), the sagital (medio-lateral) section (B) and a snapshot of the volume rendering (C) are shown.
Figure 4 shows a 3D image of the lungs of a female nude rat (RNU), 12 weeks old, implanted orthotopically with non small cell lung carcinoma (NSCLC) NCI-H460. 2500 projection images were taken with a linear magnification of 0.5x, ensuring a final resolution of 16 microns. The image shows the Microfil stained blood vessels (down to 20 micron diameter).The image analysis shows that 4 weeks after implantation, multiple cancer nodules are formed. They are covering a significant fraction of the lung volume (17%). Most of the pulmonary staining was found in the peripheral areas of the tumors. Significantly, as shown in the fig 4B, several blood vessels are present also inside the nodules, covering, according to preliminary analysis some 3% of their volume.
Figure 4. 3D image of cancer nodules growing in a rat lung. A snapshot of the volume rendering (A) and a section through the volume (B) are shown. The cancer nodules are marked with arrows.
Movie 1. Volume rendering of the mouse femur in Figure 1. Click here to watch the movie.
Movie 2. Volume rendering of the rat lungs in Figure 2. Click here to watch the movie.
Movie 3. Serial sections through the lungs. The nodules appear as grey areas in most of the slices. Click here to watch the movie.
C57/Bl6 mouse at embryonic day 18.5 (E18.5) is four days after the initiation of the mineralization process. At this stage of development, the future bone is made of many layers of mineralized osteoids, clearly seen in Fig 3. At this point, one should stress that mineralized tissues can be visualized at lower resolution with different instruments which require less sample handling. The present protocol (and the micro CT instrument used in it) besides providing higher resolution, offers the highest flexibility for the user to choose the best geometrical parameters for the measurement.
Results in Fig 4 show that in the orthotopic lung cancer animal models, human non small cell lung cancer can induce recruitment of blood vessels and neovascularization. We consider that the lung tissue was neither moved, nor has its shape changed during the measurement. The user should take special precautions to avoid such changes during a tomography. For some samples, especially for the softer tissue, one has to build special holding devices that immobilize perfectly the sample during the measurement. Unfortunately the presence of high leakages of contrast agent in the surroundings of the tumors prevented reliable quantification of the peripheral blood vessels. As a result the images are tainted by some staining agent especially at the edges, which is clearly present in movies 2 and 3. We could not prevent this spill, but the useful information about the cancer nodules, including their size, shape and the presence of inner blood vessels was not affected. We could clearly conclude that at least for the bronchial circulation which was studied here, peripheral blood supply participates in tumor perfusion, with some perfusion present also inside the tumor.
No conflicts of interest declared.
The studies were conducted at the Irving and Cherna Moskowitz Center for Nano and Bio-Nano Imaging at the Weizmann Institute of Science.
We are grateful to Orna Yeger for her help in designing and running this protocol.
|For image acquisition we have used a MICRO XCT-400 microfocussed X-ray tomographic system produced by Xradia, Concord, USA.|
|Images were processed and analyzed using ImageJ (NIH, USA), Avizo (VSG, France) and MicroView (General Electric, USA) software packages. Any available image analysis software can be used instead|