NOTE Microscopic Diffusion Anisotropy in Formalin Fixed Prostate Tissue: Preliminary Findings Roger M. Bourne, 1 * Nyoman Kurniawan, 2 Gary Cowin, 2 Paul Sved, 3 and Geoffrey Watson 1 Diffusion tensor microimaging at 16.4 T with 40 mm isotropic voxels was used to investigate anisotropic water diffusion in prostate tissue at spatial resolution approaching the cellular scale. Nine normal glandular tissue samples were collected from the peripheral zone of six formalin fixed radical prosta- tectomy specimens. Fibromuscular stromal tissue exhibited microscopic diffusion anisotropy (mean fractional anisotropy range 0.47–0.66) significantly higher (P < 0.01, Student’s t-test) than in epithelium-containing voxels (mean fractional anisot- ropy range 0.31–0.54) in six of the seven normal tissue sam- ples in which both compartments could be measured. Fiber tracking demonstrated principle stromal fiber directions con- sistent with myocyte orientation seen on light microscopy of the same sample. Diffusion tensor microimaging may be valu- able for investigation of variable results from attempts to measure diffusion anisotropy in the prostate in vivo. Magn Reson Med 000:000–000, 2012. V C 2012 Wiley Periodicals, Inc. Key words: diffusion; microimaging; prostate; anisotropy; stroma; tractography An imaging method that generates contrast based on mi- croscopic tissue structural properties would be expected to provide both sensitive and specific cancer detection if the image contrast can be made to reflect the structures that define cancer. Diffusion-weighted water imaging (DWI) is an obvious candidate for this purpose because the free diffusion of water in tissue is known to be con- strained by intra- and extracellular structures and cell walls. DWI can reveal both the scale and orientation of tissue structure because contrast depends on the net dis- placement of water over a specific time period in a spe- cific direction. Two parameters are commonly used to describe the rate and relative directional freedom of water diffusion. These are the apparent self diffusion coefficient (ADC) or diffusivity, and the fractional aniso- tropy (FA) (1), respectively. DWI studies of prostate tissue in vivo have demon- strated a decrease in the measured ADC in cancer tissue that correlates with Gleason grade (2,3). The decrease in ADC has been posited to be consistent with both the loss of high ADC lumenal and ductal spaces and increased cell density characteristic of prostatic adenocarcinoma (4), however, recent evidence from diffusion microimag- ing studies of formalin fixed prostate tissue suggest that distinct diffusivity differences between the epithelial cells, the stromal matrix, and the acinar lumena are likely to contribute to changes in ADC measured at low spatial resolution in vivo (5–7). Measurements of diffusion anisotropy in the prostate in vivo have produced equivocal results with widely differing FA values for similar tissue and no consistent correlation between pathology and FA (8–12). However, a study of for- malin fixed radical prostatectomy specimens, performed at 4.7 T with spatial resolution 0.5  0.5  0.5 mm 3 , obtained diffusion anisotropy data consistent with gross tissue architecture (13). High FA was observed in regions of primarily fibromuscular stromal tissue with the primary diffusion axis parallel to the assumed main fiber axis. The study reported here seeks to investigate the poten- tial of diffusion microimaging of fixed tissue samples to clarify the biophysical basis of diverse findings from ani- sotropy measurements of the prostate in vivo. METHODS Tissue Collection All tissue samples were collected from radical prostatec- tomy specimens with institutional ethics approval and written informed consent from tissue donors. Nine sam- ples of normal tissue were collected from the left and/or right lateral peripheral zone of the prostates of six patients. Whole organs, immersed 72 h in 10% neutral buffered formalin post surgery, were sectioned for routine histopathology. Four-mm thick transverse slices were examined by a specialist urologic pathologist and full thickness tissue samples obtained with a 3 mm core punch (sample volume 28 mm 3 ). The selection of regions for sampling was based on visual assessment of the likely tissue type. In this preliminary study we focused on samples of normal glandular tissue. Cores were placed in vials of neutral buffered formalin and stored 1–2 weeks at room temperature before MR imaging. MR Microimaging Tissue cores were transferred from neutral buffered for- malin to phosphate buffered saline containing 0.2% v/v gadolinium contrast agent (Magnevist, Schering AG, Ger- many. Dimeglumine gadopentetate 0.5 mg/mL.) giving 1 Discipline of Medical Radiation Sciences, Faculty of Health Sciences, University of Sydney, Lidcombe, Australia. 2 Center for Advanced Imaging, University of Queensland, Brisbane, Australia. 3 Department of Urology, Royal Prince Alfred Hospital, Sydney, Australia. 4 Department of Anatomical Pathology, Royal Prince Alfred Hospital, Sydney, Australia. *Correspondence to: Roger M. Bourne, Ph.D., Discipline of Medical Radiation Sciences, Faculty of Health Sciences, University of Sydney, PO Box 170, Lidcombe, 1825 Australia. E-mail: roger.bourne@sydney.edu.au Received 9 August 2011; revised 29 December 2011; accepted 30 December 2011. DOI 10.1002/mrm.24179 Published online in Wiley Online Library (wileyonlinelibrary.com). Magnetic Resonance in Medicine 000:000–000 (2012) V C 2012 Wiley Periodicals, Inc. 1