These are some of the multiscale mouse brain imaging protocols used by the members of the mouse BIRN.
MR Microscopy
Submitted by G. Allan Johnson, 4/10/02
The Center for In Vivo Microscopy (CIVM) at Duke University
All scans will be performed at 9.4 T using a 1 cm solenoid rf coil.
All scans will be acquired in a single setting so the data sets will be registered.
All scans will be acquired with a fixed field of view of 11x11x22 mm with a matrix of 256x256x512 yielding voxels of 0.043x0.043x0.043 mm i.e. 8 x 10-5 mm3.
We will acquire three registered data sets using standard 3DFT spin warp encoding. The TR and NEX have been adjusted so that each set will require 14.56 hrs:
- Data set A: T1 weighted @ TR=100 ms, TE=5.5 ms, Bandwidth ±31.25 kHz, NEX=8.
- Data set B: T2 weighted @ TR=200 ms, TE- 20 ms, Bandwidth ±15.62 kHz,NEX=4.
- Data set C: Spin density @ T2 weighted @ TR=200 ms, TE- 5.5 ms, Bandwidth ±15.62 kHz,NEX=4.
Diffusion Tensor Imaging
Submitted by Russ Jacobs, 10/25/05
The MRI Center in the Biological Imaging Center (BIC) at the California Institute of Technology
All images will be acquired using a vertical bore 11.7 Tesla Bruker Avance DRX500 system (Bruker Biospin, Germany) equipped with a Micro2.5 imaging gradient set capable of a peak gradient strength of 1T/m and a maximum slew rate of 12.5kT/m/s.
The specimen will be secured in a Teflon® holder and submerged in a perfluoropolyether (Fomblin®, Solvay Solexis, Inc, Thorofare, NJ) within a 50ml vial and imaged using a 35mm birdcage transmit/receive volume resonator. Specimens will be equilibrated at room temperature prior to imaging. The ambient bore temperature is maintained at 20°C by thermostatically controlled airflow. Optimized second order shimming is achieved across the whole sample using the Bruker implementation of Fastmap.
Diffusion weighted images will be acquired using a conventional pulsed-gradient spin echo (PGSE) sequence (TR/TE = 150ms/11.6ms, 256 x 150 x 130 matrix, 19.2mm x 15mm x 12mm FOV, 80µm isotropic voxel size, 1 average, = 3ms, ∆ = 5ms, Gd = 750mT/m, nominal b-factor = 1450 s/mm2). An optimized six point icosahedral encoding scheme (Hasan et al. 2001) is used for diffusion weighted acquisitions with a single un-weighted reference.
The apparent diffusion tensor is calculated conventionally by inversion of the encoding b-matrix. Small shifts and rotations of the diffusion weighted images are often observed and ascribed to residual eddy currents from the high-amplitude diffusion encoding pulses persisting through the echo acquisition. Diffusion weighted images are consequently registered to the diffusion un-weighted image using a rigid-body transform under the assumption that the original diffusion encoding directions were exact and that un-weighted im age best represents the true orientation of the mouse brain relative to the imaging gradient axes. The b-matrix for each diffusion encoding is determined by numerical simulation of the pulse sequence k-space trajectory in order to account for gradient cross-terms. Eigenvalues, eigenvectors, tensor trace and fractional anisotropy are calculated conventionally using built-in and custom Matlab functions.
Histology
Submitted by Allan MacKenzie-Graham, 10/31/05
The Laboratory of Neuro Imaging (LONI) at the University of California at Los Angeles
After removal from the skull, the brains are dipped in 5% gelatin (Sigma) to maintain the integrity of the tissue after sectioning. The brains are then embedded in OCT compound (Sakura) at 4° C with dry ice.
Once the samples are prepared, the brains are attached to a chuck with OCT compound (Sakura), and sections are cut serially in 50µm thick sagittal sections on a modified CM3050S cryostat (Leica). A DMCIe digital camera (Polaroid) captures images of the blockface prior to each section at a resolution of 1600 x 1200 (approximately 6.7µm/pixel) in 24-bit color. Sections 200µm apart are Nissl-stained (Thionin) and alternating sections 200µm apart are myelin-stained using a modified myelin impregnation stain. Stained preparations are digitized using a 0.5X objective on an AX70 microscope (Olympus) with a DMCIe digital camera (Polaroid) at a resolution of 1600 x 1200 (approximately 6.1µm/pixel) in 24-bit color. The images are acquired using a macro imaging system that provides undistorted high-resolution images with even illumination across the entire field of view.
Image Processing
Submitted by Allan MacKenzie-Graham, 10/31/05
The Laboratory of Neuro Imaging (LONI) at the University of California at Los Angeles
The method we use to extract mouse brain tissue from the embedding medium used during cryosectioning and the slide background for histologically stained sections takes advantage of the rich textural information of the images, for example, the texture of the frozen OCT compound is quite different from that of the brain tissue.
A previously trained neural net classifies a region within the image as either tissue or background based on the textural descriptors and output a segmented image. Combined with color information, it provides sufficient discriminating power to perform the segmentation.
The two dimensional digital images of the stained sections are brought roughly into register with their corresponding blockface images acquired during sectioning using automated software tools produced at LONI. The preregistration program produces an initialization file for Automated Image Registration (AIR) 4.0.
Once the registered images are aligned, they are reconstructed into a three-dimensional volume. The resulting volume is brought into register with an inherently three-dimensional MRM in a defined and common coordinate system. All image processing is done on a 64-processor Origin 3000 supercomputer (SGI).
Ultra-wide Field Photon Microscopy
Submitted by Maryann Martone, 11/2005
The National Center for Microscopy and Imaging Research (NCMIR) at the University of California at San Diego
Higher resolution data on normal and abnormal tissue and cellular architecture are obtained on relatively large expanses of mouse brain through the creation of wide-field high resolution fluorescent brain maps using multiphoton microscopy. This approach involves the use of a motorized computer-controlled precision stage coupled to a multiphoton microscope fitted with high quality optics. Using this set up, fluorescently labeled brain sections are scanned at close to the resolution limit of the light microscope across wide extents. Image stacks are then knit together to create high-resolution 3-D large-scale brain maps (LSBM’s) that are integrated into an informatics environment for viewing and analysis. These large scale maps are currently upwards ot 10 Gb in size and require robust resources like BIRN for strage and access. The resulting image mosaics provide detailed views of cellular and subcellular structure and macromolecular distributions in a larger tissue context. Details of this work will appear in the January 2006 issue of Neuroinformatics (Price et al., in press). Examples of these brain maps can be viewed through the Cell Centered Database (
http://ccdb.ucsd.edu). To browse through the image maps using a web-based virtual microscopy environment, log into the CCDB as guest and select project P1187 and record number 102103a. Because of the requirements for good morphological preservation, this technique is generally not compatible with other histological techniques in the mouse BIRN because tissue cannot be frozen or subjected to anoxia. As with most higher resolution techniques, the sample size is limited compared to lower resolution imaging. However, when used in conjunction with the histological and magnetic resonance imaging techniques employed in mouse BIRN, ultrawide field microscopy provides a powerful tool to delve deeper into pathological processes occurring on the sub-micron level.
Electron Tomography Imaging
Submitted by Maryann Martone, 11/2005
The National Center for Microscopy and Imaging Research (NCMIR) at the University of California at San Diego
Electron tomography involves the derivation of 3D ultrastructure contained within an electron microscopic specimen from a series of 2D electron micrographs taken as the specimen is rotated through a wide angular range. It is analogous in concept to computerized axial tomography. Within the Mouse BIRN, researchers are using electron tomography in conjunction with high voltage electron microscopes to create 3D reconstructions of cellular and subcellular processes in mouse models of human disease. High voltage electron microscopy allows for the use of thicker sections than is possible with conventional transmission electron microscopy, on the order of 5-10 times, depending on the microscope. Researchers at NCMIR, in collaboration with mouse BIRN partners, have been sending samples of filled spiny neurons to Osaka, Japan, for tomographic imaging on the world’s most powerful electron microscope, the 3MeV Hitachi microscope at the University of Osaka. Three-dimensional volumes have been obtained of selectively stained spiny dendrites at electron microscopic resolution. These preparations have been used to document changes in spine ultrastructure in several mouse models of human disease. This work is currently in progress and will be submitted for publication within the next year. Examples of tomographic reconstructions of spiny dendrites can be viewed in the Cell Centered Database (
http://ccdb.ucsd.edu) by searching for “spiny dendrite”.
Mouse Brain Library (MBL) Tissue Preparation
Submitted by Robert W. Williams, 10/28/05
The University of Tennessee Health Science Center in Memphis
This
online manual explains the detailed protocol of tissue embedding and coverslipping brain tissue currently used by the MBL. For more information on the purpose of the MBL, visit
http://www.mbl.org/.
