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Micro-CT of the Human Lung: Imaging of Alveoli and Virtual Endoscopy of an Alveolar Duct in a Normal Lung and in a Lung with Centrilobular Emphysema—Initial Observations¹

¹ From the Department of Pneumology, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany (H.W.); Department of Diagnostic Radiology, University of Giessen, Germany (H.W., A.B., W.S.R.); and Coriell Institute for Medical Research, Camden, NJ (A.K.). Received June 30, 2004; revision requested September 3; revision received October 15; accepted November 4. Address correspondence to H.W. (e-mail: watz.henrik{at}web.de).

	ABSTRACT

TOP ABSTRACT INTRODUCTION Materials and Methods Results Discussion References

 
The appearance of human lung parenchyma at the structural level of alveoli was investigated by the use of micro–computed tomography (CT). Approval for use of autopsy lungs was given by the head of the pathology institute of the university, in accordance with the requirements of the State Ministry of Science and Arts and without the need for institutional review board approval. Two human lungs (one normal lung and one lung with centrilobular emphysema of a mild to moderate degree) were inflated and fixed with hot formalin vapor. Lung specimens excised from the superior segment of the left lower lobe (B6) were stained with silver nitrate in a vacuum and investigated at a volume of interest of 4 mm for each side with a voxel size of 14 µm. Normal-size and enlarged alveoli became visible. A three-dimensional reconstruction of the terminal airspaces made virtual endoscopy of the alveolar ducts possible.

© RSNA, 2005

	INTRODUCTION

TOP ABSTRACT INTRODUCTION Materials and Methods Results Discussion References

 
Micro–computed tomography (CT) is emerging as an imaging technique because of the need for three-dimensional analysis of small specimens (1). Depending on the x-ray source, focus size, voltage, sample volume, geometric magnification of the optical system, and resolution of the charge-coupled device camera, a spatial resolution of 1 µm may be obtained (2). Micro-CT has been recognized as a valuable tool in research laboratories for use in the examination of trabecular structure in rodent bones (3,4) and human bone biopsy specimens (5–7). Another area of interest is the vasculature and microvasculature in rodent organs, especially in the heart and kidneys (8–11). Besides bone biopsy specimens, only the coronary arteries have been depicted in humans thus far (12).

To our knowledge, human lung parenchyma has not been investigated with micro-CT, however, because of the limited contrast of lung parenchyma, preparative difficulties, and limitations in computer-assisted analysis. In particular, the fine structure of the soft-tissue–like terminal airspaces does not provide the same level of contrast as the trabeculae or contrast medium–filled microvessels. Additionally, examination accuracy of lung specimens requires carefully inflated terminal airspaces. Inflation with hot formalin vapor seems to be suitable to fix the lungs in nearly end-inspiratory volume (13,14). The problem of contrast enhancement of the alveolar walls still must be solved. Thus, the purpose of our investigation was to evaluate the appearance of human lung parenchyma with micro-CT.

	Materials and Methods

TOP ABSTRACT INTRODUCTION Materials and Methods Results Discussion References

 
Autopsy Lungs
Two left human lungs (one normal lung and one with centrilobular emphysema) were removed at autopsy (H.W.) at the Institute of Pathology at the Justus Liebig University, Giessen, Germany. Informed consent was given by next of kin. The use of autopsy lungs was approved by the head of the pathology institute of the university, in accordance with the requirements of the State Ministry of Science and Arts. Institutional review board approval was not needed. History and cause of death in both cases excluded pneumonia or severe lung edema as the main conditions affecting the appearance of the original lung parenchyma. The patient with the normal lung died of a sudden cardiac event at 46 years of age and had no history of smoking. The patient with centrilobular emphysema died of an acute stroke at 88 years of age. He had a history of smoking of at least 40 pack-years. No erect chest radiographs were obtained before death in either case.

Lung Fixation
The lungs were fixed by using hot formalin vapor with a respirator modified for postmortem formalin insufflation via the main bronchus (H.W. and A.B.). This technique, previously described as a volume-controlled fixation (13,14), was slightly modified for pressure-controlled inflation. An 8-hour ventilation cycle with inflation pressures of up to 40 cm of water was applied to fix the lungs in nearly end-inspiratory volume and avoid collapse.

High-Resolution CT Imaging and Interpretation
Before specimens for histologic and micro-CT analysis were excised from each lung, we performed high-resolution CT scanning of each complete intact lung (1-mm section thickness, 120-kV tube voltage, 330-mA tube current, 2-second imaging time) (Somatom Plus; Siemens, Erlangen Germany) by using the smallest available field of view (ie, 3 cm) (H.W.). Scanning was performed with a distance of 2–3 cm to the pleural surface, and scans were focused on imaging the lung parenchyma without any segmental or subsegmental bronchi or vessels.

Lung Specimen Preparation, Analysis, and Imaging
Six lung specimens (2 x 2 x 4 cm) excised subpleurally from six different regions of each lung (three from the upper lobe and three from the lower lobe, with one specimen obtained from the superior segment of the lower lobe) were obtained for further investigation of the lung parenchyma (H.W.). First, all of the specimens were analyzed for regularly inflated terminal airspaces by inspecting their freshly cut surface. A photograph of the surface was obtained at a magnification of x20 (H.W.). Afterward, a tissue block (approximately 5 mm on each side) was obtained from the medial part of these specimens and used for paraffin embedding and histologic investigation (H.W.). Histologic sections with a thickness of 5 µm were obtained and stained with hematoxylin-eosin. One histologic section from each block was photographed and printed at a magnification of x25. These six photographs were analyzed for the presence and severity of emphysema with section assessment (H.W., A.B., and W.S.R., in consensus), which is a method for assessing and grading emphysema by using reference images (15,16). The reference images were obtained in a series of postmortem inflated lungs and aligned with the image scores reported by Nagai et al (15). Nagai et al (15) devised a scoring system that ranges from grade 0 (no emphysema present) to grade 9 (complete destruction and disappearance of lung parenchyma). Lungs 1 and 2 were assessed (H.W., A.B., and W.S.R.) in consensus. Lung 1 had a normal parenchymal pattern, without any destruction (grade 0, according to the scale of Nagai et al). Lung 2 showed destruction, mainly in the central portion of the acinus, with a preserved regular pattern of the alveoli in the distal portions that represented centrilobular emphysema of a mild to moderate degree (grade 3–4, according to the scale of Nagai et al).

The lung specimens (8 x 8 x 10 mm) used for micro-CT scanning originated from the middle part of the specimen excised from the superior segment of the lower lobe (B6). Following the concept of the unequal dichotomy of the bronchial system with a nearly perpendicular position of B6 to the pleural surface, we assumed to have a complete acinus or at least most of its alveolar ducts within the specimen.

The lung specimens (8 x 8 x 10 mm) were stained with 0.8 mol/L silver nitrate in a vacuum by using a routine water stream–driven device, so that all terminal airspaces were evacuated and could be equally stained with silver nitrate. After a period of 72 hours, the remaining silver solution was removed with centrifugation (100 rpm), and the specimens were gently dried (H.W.).

The silver-stained lung specimens were examined with micro-CT at the Institute for Biomedical Engineering, University of Zurich, Zurich, Switzerland. This micro-CT system is based on a Kevex PXS5 (Thermo Electron, Scotts Valley, Calif) microfocus x-ray source, and it has been described previously (17,18). This system is commercially available (µCT 20; Scanco Medical, Bassersdorf, Switzerland) and has been slightly modified to our experimental setting. It is a fan-beam radiation-based system with a focal spot of the x-ray tube of 10 µm. The 40-kVp x-ray spectrum is filtered with 0.3 mm aluminum, which leads to an energy spectrum that peaks at 25 keV. The object is mounted on a turntable that is rotating and shifting automatically in the transverse direction. The linear charge-coupled device array detector consists of 1024 photodiode elements, with a pitch of 25 µm. A 50-µm-thick amorphous scintillator transforms the x-ray into light. A fiber plate guides the scintillation light to the charge-coupled device array, where it passes amplification and digitization. Via small computer system interface, the projections are transferred to the computer. A total of 600 projections are obtained over 216° (180° plus half the fan angle on either side). A standard convolution back-projection procedure with a Shepp-Logan filter is used to reconstruct the CT images. The sampling time of the instrument is limited by the readout time of the charge-coupled device array, which is 16 msec and results in a minimal measuring time for the 600 projections of nearly 10 seconds per section. Section thickness of each image is 28 µm, with an intersection distance of 14 µm in the transverse direction. Thus, the resulting effective section thickness of overlapping sections is 14 µm.

The experimental setting evaluated for the nondestructive evaluation of bone biopsy specimens (17,18) was directly transferred to our imaging of lung specimens. Working with the same sample size and the same 4-mm region of interest, a 4-mm volume of interest for each side resulted, represented by a cubic voxel size of 14 µm. The proved in-plane spatial resolution of bone biopsy specimens is 28 µm, measured at 10% modulation transfer function (18).

The cubes represent the 4-mm volume of interest of each side. They are reconstructed with the volume-rendering technique from 286 micro-CT sections. Reconstruction time, including measurement time, is nearly 2 hours.

Image pre- and postprocessing was necessary to depict the surface of these spongelike specimens. A software package (3D Top; Vimana, Naastricht, the Netherlands) for topologic investigation (19) was used (A.K.) in conjunction with a rendering technique capable of generating endoscopic views (Analyze; Mayo Foundation, Rochester, Minn). Outlining and coloring of the terminal airspaces was performed (W.S.R. and A.K. together) with computer software (Photo-Paint, version 9; Corel, Ottawa, Ontario, Canada).

	Results

TOP ABSTRACT INTRODUCTION Materials and Methods Results Discussion References

 
After 8 hours of ventilation at a maximum ventilation pressure of 40 cm H₂O, the lung was fixed in nearly end-inspiratory volume. The lung tissue remains elastic, and the terminal airspaces are inflated fully. Histologic analysis, micro-CT, photography, and high-resolution CT of the parenchyma reveal the structural differences between a normal lung (Fig 1) and a centrilobular emphysema of a mild to moderate degree (Fig 2). In both cases, alveoli can be identified in photomicrographs, micro-CT images, and photographs of a freshly cut surface (Figs 1a–1c, 2a–2c). The terminal airspaces cannot be detected with high-resolution CT (Figs 1d, 2d). In the normal specimen, the high-resolution CT scan shows a regular texture pattern of the parenchyma with interlobular septa and lobular arteries in between (Fig 1d). In the emphysematous sample, the parenchymal destruction around the centrilobular arteries is visible and demonstrates the centrilobular character of the emphysema (Fig 2d).

View larger version (177K): [in this window] [in a new window] [Download PPT slide]   Figure 1a. (a) Photomicrograph of a normal lung parenchyma. The alveoli are of normal size. Note the respiratory bronchiole (arrow) dividing two alveolar ducts. (Hematoxylin-eosin stain; original magnification, x25.) (b) Micro-CT section shows the clearly demarcated alveoli of a normal lung parenchyma. A respiratory bronchiole is dividing two alveolar ducts. The respiratory bronchiole (arrow) is the starting point for the virtual endoscopy of the alveolar duct (field of view, 4 mm). (c) Photograph of a freshly cut surface of the normal specimen. Single alveoli can be identified. Two alveolar ducts can be detected (arrow). (Original magnification, x20.) (d) High-resolution CT scan of the normal lung parenchyma (field of view, 3 cm). A regular texture pattern of the lung parenchyma is visible between the interlobular septa and the centrilobular arteries of the secondary lobuli. Alveoli cannot be identified because of the lack of resolution.

View larger version (141K): [in this window] [in a new window] [Download PPT slide]   Figure 1b. (a) Photomicrograph of a normal lung parenchyma. The alveoli are of normal size. Note the respiratory bronchiole (arrow) dividing two alveolar ducts. (Hematoxylin-eosin stain; original magnification, x25.) (b) Micro-CT section shows the clearly demarcated alveoli of a normal lung parenchyma. A respiratory bronchiole is dividing two alveolar ducts. The respiratory bronchiole (arrow) is the starting point for the virtual endoscopy of the alveolar duct (field of view, 4 mm). (c) Photograph of a freshly cut surface of the normal specimen. Single alveoli can be identified. Two alveolar ducts can be detected (arrow). (Original magnification, x20.) (d) High-resolution CT scan of the normal lung parenchyma (field of view, 3 cm). A regular texture pattern of the lung parenchyma is visible between the interlobular septa and the centrilobular arteries of the secondary lobuli. Alveoli cannot be identified because of the lack of resolution.

View larger version (181K): [in this window] [in a new window] [Download PPT slide]   Figure 1c. (a) Photomicrograph of a normal lung parenchyma. The alveoli are of normal size. Note the respiratory bronchiole (arrow) dividing two alveolar ducts. (Hematoxylin-eosin stain; original magnification, x25.) (b) Micro-CT section shows the clearly demarcated alveoli of a normal lung parenchyma. A respiratory bronchiole is dividing two alveolar ducts. The respiratory bronchiole (arrow) is the starting point for the virtual endoscopy of the alveolar duct (field of view, 4 mm). (c) Photograph of a freshly cut surface of the normal specimen. Single alveoli can be identified. Two alveolar ducts can be detected (arrow). (Original magnification, x20.) (d) High-resolution CT scan of the normal lung parenchyma (field of view, 3 cm). A regular texture pattern of the lung parenchyma is visible between the interlobular septa and the centrilobular arteries of the secondary lobuli. Alveoli cannot be identified because of the lack of resolution.

View larger version (162K): [in this window] [in a new window] [Download PPT slide]   Figure 1d. (a) Photomicrograph of a normal lung parenchyma. The alveoli are of normal size. Note the respiratory bronchiole (arrow) dividing two alveolar ducts. (Hematoxylin-eosin stain; original magnification, x25.) (b) Micro-CT section shows the clearly demarcated alveoli of a normal lung parenchyma. A respiratory bronchiole is dividing two alveolar ducts. The respiratory bronchiole (arrow) is the starting point for the virtual endoscopy of the alveolar duct (field of view, 4 mm). (c) Photograph of a freshly cut surface of the normal specimen. Single alveoli can be identified. Two alveolar ducts can be detected (arrow). (Original magnification, x20.) (d) High-resolution CT scan of the normal lung parenchyma (field of view, 3 cm). A regular texture pattern of the lung parenchyma is visible between the interlobular septa and the centrilobular arteries of the secondary lobuli. Alveoli cannot be identified because of the lack of resolution.

View larger version (155K): [in this window] [in a new window] [Download PPT slide]   Figure 2a. (a) Photomicrograph of the centrilobular emphysema. The centrilobular portions of the acinus are involved mainly in the destruction of the lung parenchyma. (Hematoxylin-eosin stain; original magnification, x25.) (b) Micro-CT section of the centrilobular emphysema. The alveoli are enlarged, and the alveolar septa are missing. Some parts of the acinus are more heavily damaged than others (field of view, 4 mm). (c) Photograph of a freshly cut surface in the emphysematous specimen. The lung parenchyma is destroyed. Some parts of the terminal airspaces are more involved than others. (Original magnification, x20.) (d) High-resolution CT scan of the emphysematous lung parenchyma (field of view, 3 cm). The destruction around the centrilobular arteries is shown between radiologically intact lung parenchyma (arrow).

View larger version (142K): [in this window] [in a new window] [Download PPT slide]   Figure 2b. (a) Photomicrograph of the centrilobular emphysema. The centrilobular portions of the acinus are involved mainly in the destruction of the lung parenchyma. (Hematoxylin-eosin stain; original magnification, x25.) (b) Micro-CT section of the centrilobular emphysema. The alveoli are enlarged, and the alveolar septa are missing. Some parts of the acinus are more heavily damaged than others (field of view, 4 mm). (c) Photograph of a freshly cut surface in the emphysematous specimen. The lung parenchyma is destroyed. Some parts of the terminal airspaces are more involved than others. (Original magnification, x20.) (d) High-resolution CT scan of the emphysematous lung parenchyma (field of view, 3 cm). The destruction around the centrilobular arteries is shown between radiologically intact lung parenchyma (arrow).

View larger version (198K): [in this window] [in a new window] [Download PPT slide]   Figure 2c. (a) Photomicrograph of the centrilobular emphysema. The centrilobular portions of the acinus are involved mainly in the destruction of the lung parenchyma. (Hematoxylin-eosin stain; original magnification, x25.) (b) Micro-CT section of the centrilobular emphysema. The alveoli are enlarged, and the alveolar septa are missing. Some parts of the acinus are more heavily damaged than others (field of view, 4 mm). (c) Photograph of a freshly cut surface in the emphysematous specimen. The lung parenchyma is destroyed. Some parts of the terminal airspaces are more involved than others. (Original magnification, x20.) (d) High-resolution CT scan of the emphysematous lung parenchyma (field of view, 3 cm). The destruction around the centrilobular arteries is shown between radiologically intact lung parenchyma (arrow).

View larger version (180K): [in this window] [in a new window] [Download PPT slide]   Figure 2d. (a) Photomicrograph of the centrilobular emphysema. The centrilobular portions of the acinus are involved mainly in the destruction of the lung parenchyma. (Hematoxylin-eosin stain; original magnification, x25.) (b) Micro-CT section of the centrilobular emphysema. The alveoli are enlarged, and the alveolar septa are missing. Some parts of the acinus are more heavily damaged than others (field of view, 4 mm). (c) Photograph of a freshly cut surface in the emphysematous specimen. The lung parenchyma is destroyed. Some parts of the terminal airspaces are more involved than others. (Original magnification, x20.) (d) High-resolution CT scan of the emphysematous lung parenchyma (field of view, 3 cm). The destruction around the centrilobular arteries is shown between radiologically intact lung parenchyma (arrow).

An alveolar duct of the normal specimen can be identified on the photomicrograph, micro-CT image, and photograph (Fig 1a–1c). A dichotomous division of a respiratory bronchiole in two alveolar ducts is visible on the photomicrograph and micro-CT image (Fig 1a, 1b). On the photograph, only two alveolar ducts can be identified; their origin, a dichotomous dividing respiratory bronchiole, is not represented in this plane (Fig 1c). The respiratory bronchiole in Figure 1b is the starting point for virtual endoscopy. In the emphysematous lung specimen, a regular dichotomous division of the terminal airspaces cannot be detected between the destroyed parenchyma.

Both cubes (Fig 3a, 3c) represent the 4-mm volume of interest of each side. Alveoli can be identified on the surface of both cubes. The top views of the surfaces (Fig 3b, 3d) emphasize the normal size of alveoli in Figure 3a and the enlarged size of alveoli in Figure 3d.

View larger version (187K): [in this window] [in a new window] [Download PPT slide]   Figure 3a. Images generated with Photo-Paint software (Corel). a) Cube of the normal lung specimen, reconstructed in three-dimensions from 286 micro-CT sections (volume of interest, 4 mm per side). (b) Top view of the surface of the normal cube. Single alveoli of normal size can be identified (arrow). (c) Three-dimensional reconstructed cube of the emphysematous specimen (volume of interest, 4 mm per side). (d) Top view of the surface of the emphysematous cube. Note the enlarged size of the alveoli (arrow) when compared with b.

View larger version (174K): [in this window] [in a new window] [Download PPT slide]   Figure 3c. Images generated with Photo-Paint software (Corel). a) Cube of the normal lung specimen, reconstructed in three-dimensions from 286 micro-CT sections (volume of interest, 4 mm per side). (b) Top view of the surface of the normal cube. Single alveoli of normal size can be identified (arrow). (c) Three-dimensional reconstructed cube of the emphysematous specimen (volume of interest, 4 mm per side). (d) Top view of the surface of the emphysematous cube. Note the enlarged size of the alveoli (arrow) when compared with b.

View larger version (146K): [in this window] [in a new window] [Download PPT slide]   Figure 3b. Images generated with Photo-Paint software (Corel). a) Cube of the normal lung specimen, reconstructed in three-dimensions from 286 micro-CT sections (volume of interest, 4 mm per side). (b) Top view of the surface of the normal cube. Single alveoli of normal size can be identified (arrow). (c) Three-dimensional reconstructed cube of the emphysematous specimen (volume of interest, 4 mm per side). (d) Top view of the surface of the emphysematous cube. Note the enlarged size of the alveoli (arrow) when compared with b.

View larger version (140K): [in this window] [in a new window] [Download PPT slide]   Figure 3d. Images generated with Photo-Paint software (Corel). a) Cube of the normal lung specimen, reconstructed in three-dimensions from 286 micro-CT sections (volume of interest, 4 mm per side). (b) Top view of the surface of the normal cube. Single alveoli of normal size can be identified (arrow). (c) Three-dimensional reconstructed cube of the emphysematous specimen (volume of interest, 4 mm per side). (d) Top view of the surface of the emphysematous cube. Note the enlarged size of the alveoli (arrow) when compared with b.

View larger version (154K): [in this window] [in a new window] [Download PPT slide]   Figure 4a. (a) Virtual endoscopic image of the terminal airspaces of the normal specimen. View from a respiratory bronchiole into two alveolar ducts (arrows). (b) Virtual endoscopic image of the terminal airspaces of the normal specimen. Image shows the entrance of an alveolar duct (arrow). (c) Virtual endoscopic image of the terminal airspaces of the normal specimen. View into the whole alveolar duct. Alveoli can be identified by their regular standing walls (arrow). (d) Virtual endoscopic image of the terminal airspaces of the normal specimen. The virtual endoscopic view ends in front of an alveolus (arrow).

View larger version (150K): [in this window] [in a new window] [Download PPT slide]   Figure 4b. (a) Virtual endoscopic image of the terminal airspaces of the normal specimen. View from a respiratory bronchiole into two alveolar ducts (arrows). (b) Virtual endoscopic image of the terminal airspaces of the normal specimen. Image shows the entrance of an alveolar duct (arrow). (c) Virtual endoscopic image of the terminal airspaces of the normal specimen. View into the whole alveolar duct. Alveoli can be identified by their regular standing walls (arrow). (d) Virtual endoscopic image of the terminal airspaces of the normal specimen. The virtual endoscopic view ends in front of an alveolus (arrow).

View larger version (167K): [in this window] [in a new window] [Download PPT slide]   Figure 4c. (a) Virtual endoscopic image of the terminal airspaces of the normal specimen. View from a respiratory bronchiole into two alveolar ducts (arrows). (b) Virtual endoscopic image of the terminal airspaces of the normal specimen. Image shows the entrance of an alveolar duct (arrow). (c) Virtual endoscopic image of the terminal airspaces of the normal specimen. View into the whole alveolar duct. Alveoli can be identified by their regular standing walls (arrow). (d) Virtual endoscopic image of the terminal airspaces of the normal specimen. The virtual endoscopic view ends in front of an alveolus (arrow).

View larger version (165K): [in this window] [in a new window] [Download PPT slide]   Figure 4d. (a) Virtual endoscopic image of the terminal airspaces of the normal specimen. View from a respiratory bronchiole into two alveolar ducts (arrows). (b) Virtual endoscopic image of the terminal airspaces of the normal specimen. Image shows the entrance of an alveolar duct (arrow). (c) Virtual endoscopic image of the terminal airspaces of the normal specimen. View into the whole alveolar duct. Alveoli can be identified by their regular standing walls (arrow). (d) Virtual endoscopic image of the terminal airspaces of the normal specimen. The virtual endoscopic view ends in front of an alveolus (arrow).

View larger version (153K): [in this window] [in a new window] [Download PPT slide]   Figure 5a. (a) Virtual endoscopic image of the terminal airspaces of the emphysematous specimen. View into two dividing respiratory bronchioles (arrows). (b) Virtual endoscopic image of the terminal airspaces of the emphysematous specimen. Two alveolar ducts are divided and widened. The virtual endoscopic image proceeds in the direction of the right-hand duct (arrow). (c) Virtual endoscopic image of the terminal airspaces of the emphysematous specimen. View into the whole alveolar duct. The terminal airspaces are enlarged, and the alveolar walls are destroyed. (d) Virtual endoscopic image of the terminal airspaces of the emphysematous specimen. Single alveoli cannot be identified.

View larger version (172K): [in this window] [in a new window] [Download PPT slide]   Figure 5b. (a) Virtual endoscopic image of the terminal airspaces of the emphysematous specimen. View into two dividing respiratory bronchioles (arrows). (b) Virtual endoscopic image of the terminal airspaces of the emphysematous specimen. Two alveolar ducts are divided and widened. The virtual endoscopic image proceeds in the direction of the right-hand duct (arrow). (c) Virtual endoscopic image of the terminal airspaces of the emphysematous specimen. View into the whole alveolar duct. The terminal airspaces are enlarged, and the alveolar walls are destroyed. (d) Virtual endoscopic image of the terminal airspaces of the emphysematous specimen. Single alveoli cannot be identified.

View larger version (192K): [in this window] [in a new window] [Download PPT slide]   Figure 5c. (a) Virtual endoscopic image of the terminal airspaces of the emphysematous specimen. View into two dividing respiratory bronchioles (arrows). (b) Virtual endoscopic image of the terminal airspaces of the emphysematous specimen. Two alveolar ducts are divided and widened. The virtual endoscopic image proceeds in the direction of the right-hand duct (arrow). (c) Virtual endoscopic image of the terminal airspaces of the emphysematous specimen. View into the whole alveolar duct. The terminal airspaces are enlarged, and the alveolar walls are destroyed. (d) Virtual endoscopic image of the terminal airspaces of the emphysematous specimen. Single alveoli cannot be identified.

View larger version (189K): [in this window] [in a new window] [Download PPT slide]   Figure 5d. (a) Virtual endoscopic image of the terminal airspaces of the emphysematous specimen. View into two dividing respiratory bronchioles (arrows). (b) Virtual endoscopic image of the terminal airspaces of the emphysematous specimen. Two alveolar ducts are divided and widened. The virtual endoscopic image proceeds in the direction of the right-hand duct (arrow). (c) Virtual endoscopic image of the terminal airspaces of the emphysematous specimen. View into the whole alveolar duct. The terminal airspaces are enlarged, and the alveolar walls are destroyed. (d) Virtual endoscopic image of the terminal airspaces of the emphysematous specimen. Single alveoli cannot be identified.

	Discussion

TOP ABSTRACT INTRODUCTION Materials and Methods Results Discussion References

Installing formalin in different liquid fixatives via the main bronchus is a method often used to fix lungs in an inflated state, and many studies for correlations between findings at CT and at pathologic analysis have been performed with this method (20,21). In high-resolution CT images, the secondary lobuli, their appearance at pathologic analysis, the lobular artery, and the interlobular septa within the inflated lungs are visible (22).

We decided to inflate the lungs with hot formalin vapor to ensure that no fluid remains in the terminal airspaces after the fixation process, which would be a handicap for subsequent homogeneous staining with silver nitrate. Our method, previously described as volume-controlled fixation (13,14), provides inflated dry, but still elastic, lung tissue, without artificial condensation of water within the specimen. The inflated terminal airspaces can be examined with any morphologic method, as demonstrated by the radiologic appearance of the acini and lobuli on high-spatial-resolution radiographic images (23).

The silver staining method is similar to conventional staining methods used with light or electron microscopy (24,25). The high concentration (eg, 0.8 mol/L) of silver nitrate and the special vacuum procedures used to stain the evacuated terminal air spaces are the main modifications for this work. Staining of the lung tissue is essential to generate sufficient contrast between the very thin walls of the lung parenchyma and the air-filled alveoli. As the x-ray energy spectrum peaks at 25 keV and the photoelectric effect is the dominating factor of x-ray attenuation (18), the excellent contrast obtained with density differences of bone versus marrow (for which the micro-CT system is optimized) can be leveraged with the staining method presented in this article. Other studies investigating vasculature and soft tissue of organs overcame this problem by filling the vessels with a contrast agent (8–11). We found silver staining of the lung parenchyma to be practical and uncomplicated, and it provided excellent contrast. Different micro-CT scanner types exist, and they work with either cone-beam (8,12,26) or fan-beam (17) radiation. All scanners are based on a microfocus x-ray source and a high-spatial-resolution charge-coupled device array system (1). On the basis of the organ examined, sample volume, type of scanner, and geometric settings of the scanner, voxel sizes quoted in the literature differ between 2 µm in human coronary arteries (12), 6.65 µm in trabeculae (4), 21 µm in corporeal vasculature (27), and 81 µm in root canals of teeth (28). Further improvement of spatial resolution can be achieved with synchrotron radiation that produces a monochromatic x-ray beam. Salome et al (2) achieved a voxel size of around 1 µm in trabeculae with this technique.

To balance the field of view with at least a few alveolar ducts within the sample volume and the spatial resolution with a good image quality of the alveoli, we decided to investigate the lung specimens by using the same scanning parameters that are approved for bone biopsy specimens (17). A proved in-plane spatial resolution of 28 µm, obtained with a nominal isotropic voxel size of 14 µm, seems to be appropriate when compared with the size of a normal alveolus (eg, 250–300 µm).

In studies of lung tissue, the vasculature of a rat lung has been investigated with a micro-CT technique (26,29,30). Johnson et al (26) reconstructed the vascular pulmonary tree down to the level of the 130-µm diameter of the vessels. Spatial resolution in this study was 100 µm. Quantitative analysis of the pulmonary arteries was performed by measuring the diameter of the vessels over a range of experimental vascular pressures and provided the distensibility coefficient of pulmonary arteries (29).

Micro-CT has become an essential component of many structural research laboratories (1) because it fills a gap between conventional microscopic techniques and clinical imaging. The main advantage in comparison with conventional histologic analysis is the easy quantitative volumetric measurement of a three-dimensional isotropic volume (10). It is superior to microscopic serial sectioning and confocal microscopy. These methods are limited by the problems of (a) accurate alignment of the sections, (b) lost sections due to cutting of the specimens, (c) time-consuming process, and (d) small sample volume (9,31). The virtual endoscopy of the terminal airspaces presented in this work by the use of the micro-CT data set emphasizes this advantage.

The limitations of our study are the number of lung specimens evaluated and the missing lung disorders other than emphysema, such as fibrosis or lung edema. This has to be evaluated with further studies that involve imaging of the lung parenchyma with a micro-CT technique.

By combining pressure-controlled inflation of the lungs with hot formalin vapor and subsequent staining of lung specimens with silver nitrate in a vacuum, we were able to establish the preparative requirements for use of micro-CT in the investigation of human lung parenchyma and assess the potential of this technique for imaging of alveoli.

	FOOTNOTES

 
Authors stated no financial relationship to disclose.

Author contributions: Guarantors of integrity of entire study, H.W., W.S.R.; study concepts, H.W., W.S.R.; study design, all authors; literature research, H.W., A.K.; experimental studies, H.W., A.B., W.S.R.; data acquisition, H.W., A.K., W.S.R.; data analysis/interpretation, all authors; manuscript preparation and definition of intellectual content, all authors; manuscript editing, H.W., A.B., W.S.R.; manuscript revision/review, all authors; manuscript final version approval, H.W., A.K., W.S.R.

	References

TOP ABSTRACT INTRODUCTION Materials and Methods Results Discussion References

 

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