The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.
Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors’ lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth.
In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.
1. Specimen preparation: Golgi-Cox
2. Specimen preparation: Nissl
3. Specimen preparation: India ink
4. Specimen preparation: Generic species, generic organs
5. KESM setup and imaging
6. Image processing and data preparation
7. Data visualization and analysis
8. Representative Results:
Here, we present whole-brain data and details. Figs. 12-15 show whole-brain India Ink, Golgi, and Nissl data sets 1, 4, 3.
Figure 1. Fixed, stained, and embedded mouse brain
Figure 2. Embedded mouse brain specimen mounted on the specimen ring.
Figure 3. The Knife Edge Scanning Microscope (KESM). A photo of the KESM is shown with its major components marked: (1) high-speed line-scan camera, (2) microscope objective, (3) diamond knife assembly and light collimator, (4) specimen tank (for water immersion imaging), (5) three-axis precision air-bearing stage, (6) white-light microscope illuminator, (7) water pump (in the back) for the removal of sectioned tissue, (8) PC server for stage control and image acquisition, (9) granite base, and (10) granite bridge.
Figure 4. Imaging principles of the KESM. The principal of operation of KESM is illustrated. The objective and the knife is held in place, while the specimen affixed on the positioning stage moves (arrow with solid line) at the resolution of 20 nm and travel speed of 1-5, and gets scraped against the diamond knife (5 mm wide for 10X objective), generating a thin section flowing over the knife (arrow with solid line). Line-scan imaging is done near the very tip of the knife.
Figure 5. KESM camera and objective focusing controls.
Figure 6. KESM knife assembly and controls.
Figure 7. Initial focusing through the observation port.
Figure 8. KESM Stage Controller 2 application (screenshot).
Figure 9. KESM stack processor application (screenshot).
Figure 10. MeVisLab application (screenshot).
Figure 11. KESM Brain Atlas: Web-interface (screenshot).
Figure 12. KESM whole-brain India ink data. Volume visualizations of KESM data stacks are shown for the vascular data set. (a) Close-up of the vascular data. Width ~100 um. (b-d) Three standard views of the whole mouse brain vasculature (subsampled from high-resolution data). Width ~10mm.
Figure 13. KESM whole-brain mouse Golgi data.
Figure 14. KESM Golgi data details.
Figure 15. KESM whole-brain Nissl data. (a) Close-up of the Nissl data. ~300 um3. (b-d) Three standard views of the whole mouse brain Nissl data (subsampled from high-resolution data). Cross section shown for a clear view of the internal structures.
The KESM allows for a submicrometer-level survey of large volumes of biological specimen (~1 cm3). This kind of volume is enough to hold whole small animal organs, such as brains, lungs, hearts, kidneys, etc. Scanned images from such organs can provide unprecedented quantitative information about the structural organization of these organs, and enable computational modeling of various functional aspects of these organs, including circuit and network dynamics, electrical properties, fluid and air flow dynamics, and muscular dynamics.
The specimen preparation protocol and post-analysis protocol detailed in this article are expected to help outside research groups to have easier access to the KESM and the resulting data. The KESM operation protocol will help these external researchers to appreciate the benefits and limitations of this unique imaging modality.
The authors have nothing to disclose.
This project was funded by NIH/NINDS (#1R01-NS54252), NSF (#0905041,#0079874), the Texas Advanced Technology Program (#ATP-000512-0146-2001, #ATP-000512-0261-2001), Texas A&M Research Foundation, Department of Computer Science and Engineering at Texas A&M University, and 3Scan. We would like to thank Bernard Mesa (Micro Star Technologies) for technical consultation and support for KESM instrumentation. Major parts of the design and implementation of the KESM was done by Bruce H. McCormick, who died in 2007.