We have received many inquiries regarding our methods during and after the cutting of the brain of patient H.M. that occurred from December 2nd through December 4th. We have addressed the most frequently asked questions and we will continue explaining our protocols in the future. We are very pleased and honored to be able to share our research efforts with the public as well as the scientific community.
Q: We saw you picking up the tissue with a painter’s brush while you were cutting. What happens to the tissue sections after they are cut from the block, and can they be damaged when they are collected from the blade?
A: The first thing to keep in mind is that the whole brain was ‘fixed’ in a solution of formaldehyde (4%) that makes the tissue very durable and mechanically resilient. Secondly, we ran the whole brain through a process of ‘cryo-protection’ in which thick solutions of sucrose infuse the tissue and prevent the formation of ice crystals when the block is frozen. When the blade cuts through the top of the frozen block, the tissue melts onto it, but does not break apart because of the hardening effect of formaldehyde. Having said this, the speed and the temperature at which the brain is cut are crucial. Even with formalin fixation and cryo-protection, if the temperature is too low and/or the speed of cutting is too high, the section can crack or even shatter.
(The fact that we froze the brain before cutting should not lead to confuse our technique with that which is adopted more widely in neuropathology for much smaller blocks. The latter are cut within refrigerated cabinets (cryostats) and placed on the glass still frozen).
Q: Are the slices still usable?
A: Of course; the sectioning procedure was not only meant to acquire anatomical images to reconstruct a 3-D model of the brain (and the exact boundaries of the 1953 surgical lesion). Most importantly, once flattened on large-format glass slides and stained they will provide the microscopic detail that is necessary to understand the neurological processes triggered by the surgery and aging in the brain of patient H.M.
Q: We noticed that the slices come off the microtome crumpled. We are curious about what happens after the slices are swept away. How are they flattened out?
A: The sections are collected with a brush and placed into wells that have been serially numbered and filled with saline solution containing a combination of salts in order to keep the pH in check. When we are ready to run specific staining protocols, we select batches of sections and we use smaller painter’s brushes to unfold each tissue slice and to position them correctly onto the glass, just like it is shown in this video.
Q: A question about whether there is a risk of contamination – contamination to the slides relative to the environment. Seems like a lot of people close to the subject matter.
A: It is impractical to maintain a completely sterile environment, but all surfaces and equipment in the wet lab are kept clean and disinfected at all times. To avoid contamination of the tissue sections after the procedure, and to prolong their ‘shelf life’ we use small amounts of chemicals that slow down the growth of bacteria or mold. In addition, the sections that are not mounted and stained in the very short term are stored long-term at -30°C in special sealed vials filled with cryogenic solution.
Q: How did you keep the entire mass of the brain and gelatin from melting over the long span of time required to collect the entire series of sections?
A: A prerequisite for the success of the procedure was the ability to keep the whole brain frozen within a narrow range of temperatures around -35°C. To accomplish this, we engineered a refrigeration system that was built as a stack of blocks containing the freezing fluid. Initially, the gelatin/brain block was 20 centimeters tall. As we cut through the brain from top to bottom, the hollow tower of frozen blocks was disassembled layer by layer until the frozen microtome stage was enough to keep the last thick slab of tissue cold enough for cutting.
Q: Can the public get access to the video and images that the project is creating?
A: The Brain Observatory will continue to provide access to the scientific results that the project will generates. We are now working to reconstruct a 3-dimensional model from the images that were acquired while we were cutting. The data set is massive in terms of file size because it is produced by stacking 2401 high-resolution images obtained with the 16 Megapixel camera mounted right above the brain. However, we will convert the 3-D image into a format that can be viewed via the web.
Q: What next?
A: The best is still to come. In January, we will begin staining the first series of sections using the Nissl method which uses a dye to differentiate the main body of neurons, thereby revealing the cellular architecture and microscopic boundaries of different brain structures. We have engineered special microscope scanners to digitize our large-format slides and to deliver the first images of the whole brain at cellular resolution.