Slice selective images demonstrating SQUARE MRI contrast (Fig. 3A–D) and the resulting T1 map (Fig. 3E) were acquired using a single animal. Images were processed and reconstructed in Prospa (v. 3.06, Magritek, Wellington, New Zealand)

by applying a sine-bell squared window function to the raw data before two-dimensional Fourier transformation. The two dimensional image data were exported for further analysis using IGOR Pro (v. 6.01; Wavemetrics, Lake Oswego, OR, USA). To construct the T1 map shown in Fig. 3E the image data were combined into a three dimensional matrix having two spatial dimensions (the slice selective images) and HDAC inhibitor one time dimension (the delay before acquisition). Linear regression analysis of the natural logarithm of the signal intensity as a function of delay time was used to obtain spatially resolved T1 values in Fig. 3E. Representative data from four selected volume elements in Fig. 3E are shown in Fig. 4. T1 values calculated outside the lung region were composed solely of background noise and were not displayed in Fig. 3E. The final T1 map was overlaid onto the lung image at delay time td = 0 s for clarity of presentation. Male Sprague–Dawley

rats (350–400 g, Charles River UK Ltd, Margate, UK) were euthanized by overdose of pentobarbital (Sigma-Aldrich Ltd, Gillingham, UK) in accordance with local animal welfare guidelines and the Animals (Scientific Procedures) Act (1986). Immediately after confirmation of death, a catheter was inserted into the caudal vena cava to allow flushing of the pulmonary circulation with BTK inhibitor 20–30 cm3 heparin 100 IU/cm3

(Wockhardt UK Ltd, Wrexham, UK) in 0.9% saline solution (Baxter Healthcare Ltd, Thetford, UK) followed with phosphate buffer solution (PBS, Sigma-Aldrich Ltd, Gillingham, UK) in order to remove residual blood from the pulmonary circulation. The heart and lungs were removed en masse. A polytetrafluorethylene (PTFE) adapter tube was inserted 5–10 mm above the carina and sutured into place. The heart and lungs were suspended in 5% glucose solution (weight/volume) with the trachea Methane monooxygenase pointing downwards in a custom-built acrylic ventilation chamber, as detailed in Fig. 1. The ex vivo lungs were repeatedly inflated with 8–10 cm3 of room air to check for leakage either from the suture around the trachea or the lungs themselves. For the presented work the lung harvesting procedure was completed with 100% success of removing the lungs intact. Normally with a skilled operator the ex vivo technique results in over 90% of lungs being suitable for imaging. The lungs were chilled to 278 K for transportation to the imaging facility. The pure gas phase relaxation time of 83Kr is sufficiently long with T1 times of several minutes at ambient pressure [16] to permit hp gas extraction and transfer.