Structure-function studies of blood and air capillaries in chicken lung using 3D electron microscopy.

2.1.

Preparation of specimens

The animal protocols for these experiments were approved by the Animal Subject Committees of the University of California San Diego. White Leghorn chickens (G allus gallus domesticus ) were anesthetized with intravenous pentobarbital sodium (40 mg/kg) and a cannula was inserted into the trachea. The lungs were then fixed by intratracheal installation of 3% glutaraldehyde using a pressure of 25 cm H 2 O for 15 min. Lung samples were taken from the paleopulmo portion of lung from each animal at about one-third distance from the most caudal aspect of the lung. A section of tissue 0.5 cm thick was excised from the entire width of each lung transverse to the cranial-caudal axis. This section was then cut into approximately 10 vertical slices and trimmed into blocks. 2.2.

Transmission electron microscopy

Blocks were rinsed overnight in 0.1 M phosphate buffer (350 mOsm, pH 7.4) and postfixed for two hours in osmium tetroxide (1% osmium tetroxide in 0.125 sodium cacodylate buffer; 400 mOsm, pH 7.4). The samples were then passed through stepwise dehydration in increasing concentrations of ethanol (50–100 percent), rinsed with propylene oxide and embedded in Araldite. Blocks were then cut into ultra thin sections (50–70 nm) and contrast stained with saturated uranyl acetate and bismuth subnitrate. Sections were examined at an accelerating voltage of 60 kV using a Zeiss EM 10C transmission electron microscope. Micrographs of a carbon grating replica were taken for calibration. 2.3.

Electron tomography

Electron tomography was carried out on sections of 0.5 and 1.0 µm thickness. Images were acquired using a JEM4000EX IVEM (400 kV) microscope equipped with a NCMIR custom design 4K lens coupled CCD camera ( Fan et al., 2000 ). The sections were successively tilted through 2 degree increments from −64 to +64 degrees. Part of an epithelial bridge was collected as a single tilt series and a central portion of the epithelial bridges was collected as a double tilt series. The backprojected volume images were reconstructed using both the Boulder Laboratory’s IMOD Tomography package suite and NCMIR’s Transform Based Reconstruction (TxBR) software. Visualizations of the backprojected volumes was completed using Visage Imaging’s Amira software suite. General information about electron tomography is available in Frank (1992) . 2.4.

Serial block-face scanning electron microscopy

Blocks of lung tissue were specially prepared for serial block-face scanning EM as follows. After primary aldehyde fixation the tissue was rinsed in 0.1 sodium cacodylate and post-fixed in 0.1% potassium ferrocyanide-reduced 2% osmium tetroxide in cacodylate buffer for 1 hour. Tissue was then rinsed in distilled water and treated with 0.1% aqueous thiocarbohydrazide for 20 minutes. After further rinsing in distilled water the tissue was again treated with 2% osmium tetroxide for 30 minutes, rinsed in distilled water , dehydrated in an ethanol series and infiltrated with Durcupan ACM resin. The tissue blocks were mounted on an aluminum pin and trimmed to 1 mm × 0.5 mm in size. The specimen was placed in a scanning electron microscope (FEI Quanta FEG) equipped with a serial block-face sectioning unit (Gatan 3 View) and a backscatter electron image of the face obtained. An automatic microtome then removed a 60 nm thick slice from the face and another image was recorded. This procedure was then repeated 500 times to give a data set from which a complete three-dimensional reconstruction 30 µm thick could be derived. The separate images were processed using Amira (Visage Imaging) to create maximum intensity projections, slice by slice renderings and segmentations of epithelial bridges. A general description of serial block face scanning EM is given in Denk and Horstmann (2004) .

Supplementary Material 01 SM1. Electron tomograph showing the pulmonary capillary with its three bridges that make up Fig. 1A . By rotating the capillary in three dimensions, the junctions between the bridges and the capillary wall are more clearly seen. 02 SM2. Electron tomograph showing a ribbon of epithelial bridge running between two capillaries Rotation of the image shows the nature of the junctions between the bridge and the capillary walls clearly. 03 SM3. Serial block-face SEM study showing an epithelial plate surrounded by blood capillaries. The plate has been colored green while the capillaries are blue. This was done by tracing around the periphery of the plate with a video pen. In part of the clip it is possible to see structures through the plate because it is so thin. In a later part of the clip the capillaries have removed by the operator and the plate is well seen both in plane view and side view. 04 SM4. Serial block-face SEM study showing the blood and air capillaries in a block of tissue 30 µm thick. The configuration of the two sets of capillaries can be seen as the section is rotated. 05 SM5. Serial block-face SEM study in which we appear to move through a 30 µm thick tissue block in a series of small steps. As we do this and concentrate on a single capillary, its thickness changes and eventually we see the connecting epithelial bridge. For some of the capillaries there is no bridge on the far side but simply the empty air capillary.