Fast virtual histology of unstained mouse brains using in-line X-ray phase tomography
Résumé
IntroductionWhole-brain imaging with cell resolution is one of the most important challenges in neuroscience. Conventional histology techniques are laborious and require tissue staining and sectioning. We have previously proposed an in-line phase contrast tomography set-up that identified mouse brain anatomy as clearly as histology in 44 min (1). The current work presents an optimized pipeline and demonstrates its potential for fast virtual histology in a mouse model of acute cerebral injury.MethodsSix mice (healthy: N=1, acute cerebral ischemia: N=5) were imaged in-vivo with MRI. Imaging of brains fixed with PFA4% was performed on beamline ID19 at ESRF at 19 keV selected from undulator radiation. An indirect detection-based detector with a LuAg scintillator, standard microscope optics and a 2048x2048 pixel CCD camera was positioned 1 m from the sample to have phase contrast. The pixel size was set to 7.5 µm. Acquisition time was 14 minutes per brain.ResultsFigure 1 shows 3 different virtual histologic slices of the healthy brain (Fig 1A;E;G). Minimum intensity projection (MinIP) resulted in angiography with sufficient resolution to visualize pial vessels (Fig 1B-C). T2-weighted MRI revealed abnormal hyperintense signals (Fig 1D, arrow), corresponding to a choroid plexus (CP) cyst as clearly depicted on phase contrast images (Fig 1E). CP layers were well resolved (Fig 1F). In the cerebellum, there was an excellent contrast between grey and white matter (Fig 1G-H), with detection of individual Purkinje cells (arrow in Fig 1I).In mice with ischemic injury, MRI showed an extensive lesion across the frontoparietal cortex (Fig 2A, plain line) with a well-delineated core (dotted line) and vasogenic edema along the ipsilateral external capsule (arrow). Native phase contrast images also discriminated the core/periphery of the lesion (Fig 2B). MinIP allowed further characterizing the lesion, with a disorganization of vessels in the core and avascularity in the periphery (Fig 1C). In addition, maximum intensity projection (Fig 2D) showed (i) enhanced cellularity on the ipsilateral side (Fig 2E, arrow), (ii) vessels rich in collagen such as carotids (Fig 2F) and (iii) upregulation of collagen in the ischemic cortex (Fig 2G-H) (2).ConclusionsThe proposed set-up of in-line phase contrast tomography allows fast virtual histology of the whole unstained brain, which is promising for phenotyping transgenic mouse and characterizing models of neurovascular diseases, with the potential to scan 30 brains in an 8-hours shift. Because it is non-destructive, this approach will be used for guiding further immunohistologic analysis of the brain.References1. Marinescu M and others. Mol Imaging Biol 2013;15(5):552-559.2. Hawkes CA and others. Exp Neurol 2013;250:270-281.AcknowledgementThis work was performed within the framework of the LABEX PRIMES (ANR-11-LABX-0063) of Université de Lyon and was supported by the European Synchrotron Research Facility (ESRF, project LS-2292) by allocation of beam time.