This protocol is the first to introduce 3D scanning technology in the anatomical, specifically neuroanatomical research field, and has achieved highly accurate overlapping performance. The 3D neuro embedding overlap, or 3D neuro protocol, embeds micro-scale sterile sockets into macro-scale brain images, and bridges these different spatial scales seamlessly. We also have to apply the 3D neuro protocol to human MRIs and post-mortem brain.
The overlapping enables us to identify known MRI contrast patterns relating to disease. 3D scan systems are sensitive to water surrounding the extracted bio organism, so it is critical to wipe the water very carefully. Furthermore, the procedure must be performed as quickly as possible.
Begin this procedure with preparation of a mouse, as well as MRI acquisitions and equipment preparations as described in the text protocol. On day two, cut the scalp of a previously euthanized mouse along the sagittal suture using surgical scissors. Use surgical scissors to cut the skull in diagonal directions from the lambda point to a point a little in front of the bregma.
Then, gently peel off the skull from the brain using curved tweezers. Lift the brain with a spatula and transfer it to a Petri dish with ice-cold cutting solution. Using an external gas bomb with a controlled rotating pump speed, flow air containing 95%oxygen and 5%carbon dioxide to generate small bubbles in the ice-cold cutting solution.
After one to two minutes, put the brain on a microfiber cloth whose surface is slightly covered by a flour sieve. Gently wipe the fluid from the brain surface using the microfiber cloth. Put the brain, with its dorsal aspect up, onto the sample stand.
Then, place the sample stand at the center of the automatic turntable. Darken the room during the 3D scan and perform a 3D scan on the turntable. To test whether the 3D scan works well, click 3D Scan by selecting the angle between two shots as 22.5, the starting angle as zero, and the final angle as 360.
Move the brain to a Petri dish and bubble the brain in the cutting solution for approximately 10 seconds. Cut the brain into two blocks at the middle of the coronal plane, using a scalpel. Then, move the two brain blocks gently to the flat surface using a surgical spatula.
Attach the brain blocks of the brain block base using instant glue. Softly and carefully wipe the fluid from the brain surface within one to two minutes, using the microfiber cloth. Then, perform a 3D scan again.
Attach a blade to the blade holder of the vibratome. Attach the black tape covering the center of the brain block base to the center of the cutting stage for a vibratome. Pour cutting solution into the cutting stage and set the cutting stage on the vibratome.
Adjust the cutting speed and amplitude. Make two to five coronal slices of 300 microns thick from the two brain blocks. Keep the solution in the cutting stage bubbling if possible.
Optimize the cutting speed, frequency, and vibration amplitude of the system by setting them as 12.7 millimeters per minute for the speed, 87 to 88 hertz for the frequency, and 0.8 to 1.0 millimeters for the swing width. While cutting the brain slices, record the sliced coordinate in the format, including the anterior-posterior coordinate, hemisphere, and other conditions. Gently transfer the brain slices to a beaker filled with pre-warmed ACSF using a thick plastic pipette.
Incubate the brain slices in the beaker at approximately 34 degrees Celsius for one hour. During this time, perform a 3D scan of the remaining brain blocks on the cutting stage. Now, set a multielectrode array, or MEA chip, on a recording unit.
Connect the chip to a peristaltic pump using two tubes. Use one tube to guide the same ACSF into the MEA chip and the other tube to guide the ACSF out of the MEA chip. Attach two needles, connected at the tips of the two tubes, to the top of the wall of the MEA chip.
Fix their positions with their tips following the inner wall of the MEA chip. Set the flow rate of the ACSF to 4.1 RPM. Perform MRI data processing to extract cortical volumes as described in the text protocol.
To perform MRI image processing, download the 3D Slicer free software. Open the MR images of the extracted brains that were produced using the volume rendering and editor modules in the 3D Slicer. Change the mode from editor to volume rendering, and click a target button to make the image of the brain come to the center of the screen.
Select the MRT-2 brain mode and tune the thresholds by moving the shift bar. Then, move back from volume rendering to editor and click the threshold effect button. Apply the label 41, cerebral cortex.
Then, save the brain surface data as an STL file by changing the file format from VTK to STL on the form checklist. Perform an automatic coregistration among eight or 16 images taken from eight or 16 different angles within a sequence of a scan to correct small mismatches. To do so, click Global Registration included in the alignment option and integrate the images.
Repeat scans from different angles to obtain the whole brain surface. If the integration among images scanned from different angles was not successful, click Manual Alignment and select a pair of fixed image and a moving image. Start the manual alignment of the images by selecting three or four common points in different images.
Then, click OK.The optimization algorithm is the iterative closest-point algorithm and does not include a non-linear deformation. Make a mesh of all the aligned images by selecting all images and clicking the mesh generation button. Then, select the option, small artistic object, to get the mesh at the highest resolution.
Save the image as STL Binary or an ASCII format. Now, open the MRI surface and merge it with the 3D scan surface using the surface processing software. Perform a manual alignment process as before.
Then, save these coregistered surface images again. If necessary, clean the individual surface data by erasing small noises surrounding the brain region, especially in the case of the MRI data, and by filling any holes, especially in the case of the 3D scan data. Finally, open the surface data with data analysis software such as MATLAB.
Generate and evaluate the histograms of the minimum distances between the two surfaces. Distances were evaluated between cortical surfaces produced by stripping MRI volume and surfaces obtained from 3D scans of extracted brains. The mode values of the histogram of the distances are only 55 microns.
Additionally, when accumulating the histogram from the point where the distance equals zero, the accumulated value reaches 90%of total sample numbers at approximately 300 microns. The final histogram of the distances between two surfaces showed a typical peak around 50 microns. From a macroscopic viewpoint, this mode value corresponds to the geometrical limitation, which was 100 microns from the voxel size of MRIs.
This point indirectly suggests that the overlapping algorithm between the MRI and 3D scan worked superbly well, and that the noise levels of both the MRI and the 3D scan were suppressed as a low value. This protocol is the first to uprise through the scanning technology to bio-organism. The technology was originally utilized for purely engineering demands.
Applying this technology to medicine can answer new questions. Following 3D scanning, we can use calcium-imaging wall patch gram recordings to get complementary knowledge in terms of temporal and the spacial resolution and recordable numbers of cells. The highly accurate overlapping performance this protocol provides will make a seamless association between the anatomical spatial scale and the zero-spatial scale more realistic than before.
