Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

互联网2013-11-13

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实验步骤

1) This protocol closely follows the protocol of Mayerich et al.⁷ .

2) The C57BL/6J mouse is deeply anesthetized using isoflurane inhalant anesthesia and then decapitated.

3) The brain is removed and placed into a Golgi-Cox fixation solution (1% potassium chromate, 1% potassium dichromate, and 1% mercuric chloride in deionized water).

4) The brain is left in the Golgi-Cox solution in the dark, at room temperature, for 10 to 16 weeks.

5) The brains are rinsed in deionized water overnight in the dark.

6) The rinsed brain is immersed in 5% ammonium hydroxide solution in deionized water for 7 to 10 days in the dark at room temperature. This lengthy preparation time was to ensure infiltration of the entire brain, so that the black precipitate had a chance to completely form in the tissue.

7) The brain is rinsed again in deionized water at room temperature for 4 hours and then dehydrated through a graded series of ethyl alcohols: 50% and 70% in the refrigerator (4 °C), and 85%, 95% (3 changes), and 100% (4 to 5 changes) in room temperature. The brain is left in each solution for 24 hours.

8) The dehydrated brain is then put in acetone (3 to 4 changes, each for one day), followed by an araldite-acetone mixture with araldite-to-acetone ratio of 1:2, 1:1, 2:1, and finally in 100% araldite (3 changes, each time overnight), in the refrigerator (4 ° C).

9) Finally, the treated brain is embedded in 100% araldite and heated to 60 °C for 3 days (cf. embedding protocol in Abbott and Sotelo² ).
If specimens are embedded in LR White then the specimens are transferred from the last 100% ethanol solution through three changes of LR White solution that are each kept in the refrigerator overnight, which is a standard procedure prior to polymerization. Three changes are done to ensure full infiltration. The specimen is then transferred to fresh LR White that is polymerized in a closed container at 60 °C for 24 hours, which is the required amount of time and temperature for proper polymerization.

10) Fig. 1 shows a Golgi-Cox stained brain embedded in Araldite.

11) The cured specimen block is then mounted on the metal specimen ring using epoxy, and the sides of the block trimmed as necessary (see Fig. 2).

2. Specimen preparation: Nissl

1) The mouse is deeply anaesthetized using ketamine and xylazine injected intraperitoneally and then perfused transcardially using 50 mL of room temperature phosphate-buffered saline (pH 7.4), followed by 250 mL of room temperature 10% neutral buffered formalin (pH 7.4). and finally with 3.0 cc of undiluted India ink.

2) Whole body perfusion with saline and fixative is necessary to clear the blood from the cardiovascular system and to fix the tissues. Perfusion with India ink is necessary to completely fill the vasculature of the cardiovascular system.

3) The resulting brain is then dehydrated through a series of graded ethyl alcohols (25%{}-100%) and then embedded in araldite plastic following the protocol in 1.8-1.9 above. If LR white is the embedding medium then the protocol in 1.10 above is followed.

4) The mouse is deeply anaesthetized using ketamine and xylazine injected intraperitoneally and then perfused transcardially using 50 mL of room temperature phosphate-buffered saline (pH 7.4), followed by 250 mL of room temperature 10% neutral buffered formalin (pH 7.4). and finally with 3.0 cc of undiluted India ink.

5) Whole body perfusion with saline and fixative is necessary to clear the blood from the cardiovascular system and to fix the tissues. Perfusion with India ink is necessary to completely fill the vasculature of the cardiovascular system.

6) The resulting brain is then dehydrated through a series of graded ethyl alcohols (25%{}-100%) and then embedded in araldite plastic following the protocol in 1.8-1.9 above. If LR white is the embedding medium then the protocol in 1.10 above is followed.

7) The mouse is deeply anaesthetized using ketamine and xylazine injected intraperitoneally and then perfused transcardially using 50 mL of room temperature phosphate-buffered saline (pH 7.4), followed by 250 mL of room temperature 10% neutral buffered formalin (pH 7.4). and finally with 3.0 cc of undiluted India ink.

8) Whole body perfusion with saline and fixative is necessary to clear the blood from the cardiovascular system and to fix the tissues. Perfusion with India ink is necessary to completely fill the vasculature of the cardiovascular system.

9) The resulting brain is then dehydrated through a series of graded ethyl alcohols (25%{}-100%) and then embedded in araldite plastic following the protocol in 1.8-1.9 above. If LR white is the embedding medium then the protocol in 1.10 above is followed.

3. Specimen preparation: India ink

1) The mouse is deeply anaesthetized using ketamine and xylazine (1.7 mg ketamine / 0.26 mg xylazine per 20 grams body weight) injected intraperitoneally and then perfused transcardially using 50 mL of room temperature phosphate-buffered saline (pH 7.4), followed by 250 mL of room temperature 10% neutral buffered formalin (pH 7.4). and finally with 3.0 cc of undiluted India ink.

3) The resulting brain is then dehydrated through a series of graded ethyl alcohols (25%-100%) and then embedded in araldite plastic following the protocol in 1.8-1.9 above. If LR white is the embedding medium then the protocol in 1.10 above is followed.

4. Specimen preparation: Generic species, generic organs

1) For the generic case, fixation can be done with either 10% neutral buffered formalin or 4% paraformaldehyde.

2) For small tissue volumes, diffusion instead of perfusion is recommended for fixation and staining. In the case of small organisms or organs, staining also can be done during the dehydration process.

3) The embedding protocol is the same as above, although the times for solution infiltration can be reduced for organs or organisms that are much smaller than whole mouse brain.

5. KESM setup and imaging

1) Turn off pump, turn on stage, turn on camera, turn on illuminator.

2) Raise knife and objective.

3) Install specimen ring.

4) Measure specimen dimension and create a configuration file for the KESM Stage Controller2 application.

5) Start KESM Stage Controller2 and initialize stage.

6) Lower knife/objective, turn on pump.

7) Focus objective and camera, by turning the focusing knob on the optical train and adjusting the camera's field of view relative to the knife edge.

8) Press on [Go] to initiate imaging.

9) See Figs. 3-8. See ^{9, 7} for technical details.

6. Image processing and data preparation

1) Copy the KESM Stack Processor executable into the data folder under the configuration file folder (00000, 00001, etc.).

2) Run the KESM Stack Processor to remove lighting artifact and normalize the background intensity in all images. Repeat for all data subfolders.

3) Prepare multiscale tiles from the data set and upload and set up the files in the KESM Brain Atlas web server.

4) See Fig. 9.

7. Data visualization and analysis

1) Visualization using the KESM Brain Atlas. Go to http://kesm.cs.tamu.edu and select the appropriate atlas.

i. Navigate as in Google maps.

ii. Zoom in and out as in Google maps.

iii. To go to a different depth, select how deep to go at each step from the pull-down menu, and then click on either [ ] or [-] to increase or decrease z depth.

iv. Adjust the overlay number by using the pull-down menu.

v. Adjust the overlay interval by using the pull-down menu.

vi. See Fig. 11.

2) Visualization using MeVisLab.

i. Launch MeVisLab.

ii. Create a new project.

iii. In the following, new modules can be creating by typing in the module name in the command box.

iv. Create module [Compose3Dfrom2DFiles], and go to the desired folder. Enter file string and click in [GetFileList]. Make sure the selected images can fit in memory. Click [Create3D] to create volume.

v. Create module [SetWorldMatrix], and set the "Scale" under "Elementary Transforms" x, y, z to the appropriate voxel size (0.6, 0.7, 1.0 for a 10X 0.45NA objective). Link [Compose3Dfrom2DFiles] to [SetWorldMatrix].

vi. Set Matrix and Transforms under "Matrix Composition" to "Ignore", "Ignore", "Forward".

vii. Create module [Arithmetic1] and set the "Function" to "Invert(Max-Img)". Link [SetWorldMatrix] to [Arithmetic1].

viii. Create module [View3D] and connect [Arithmetic1].

ix. In View3D, while right mouse button is pressed, move the mouse around to adjust the threshold. You can crop regions, change orientation (move mouse while left mouse button is pressed), change illumination (volume rendering or MIP), turn on/off background, etc.

x. See Fig. 10.

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} .

References:

1. Abbott, L.C. High-throughput imaging of whole small animal brains with the knife-edge scanning microscope. In Neuroscience Meeting Planner, Washington, DC: Society for Neuroscience Program No. 504.2 (2008).

2. Abbott, L.C. & Sotelo, C. Ultrastructural analysis of catecholaminergic innervation in weaver and normal mouse cerebellar cortices. Journal of Comparative Neurology , 426, 316-329, (2000).

3. Choe, Y., Abbott, L.C., Miller, D.E., Han, D., Yang, H.-F., Chung, J.R., Sung, C., Mayerich, D., Kwon, J., Micheva, K., & Smith, S.J. Multiscale imaging, analysis, and integration of mouse brain networks. In Neuroscience Meeting Planner, San Diego, CA: Society for Neuroscience . Program No. 516.3. Online (2010).

4. Choe, Y., Han, D., Huang, P.-S., Keyser, J., Kwon, J., Mayerich, D., & Abbott, L.C. Complete submicrometer scans of mouse brain microstructure: Neurons and vasculatures. In Neuroscience Meeting Planner, Chicago, IL: Society for Neuroscience Program No. 389.10. Online (2009).

5. Choe, Y., Abbott, L.C., Han, D., Huang, P.S., Keyser, J. Kwon, J., Mayerich, D., Melek, Z., & McCormick, B.H. Knife-edge scanning microscopy: High-throughput imaging and analysis of massive volumes of biological microstructures. In A. Ravi Rao and Guillermo Cecchi, editors, High-Throughput Image Reconstruction and Analysis: Intelligent Microscopy Applications , Artech House, Boston, MA, 11-37 (2008).

6. Choe, Y., Abbott, L.C., Ponte, G., Keyser, J., Kwon, J., Mayerich, D., Miller, D., Han, D., Grimaldi, A.M., Fiorito, F., Edelman, D.B., & McKinstry, J.L. Charting out the octopus connectome at submicron resolution using the knife-edge scanning microscope. Nineteenth Annual Computational Neuroscience Meeting: CNundefined2010, BMC Neuroscience. , 11 (Suppl 1), 136, (2010).

7. Mayerich, D., Abbott, L.C., & McCormick, B.H. Knife-edge scanning microscopy for imaging and reconstruction of three-dimensional anatomical structures of the mouse brain. Journal of Microscopy , 231, 134-143, (2008).

8. Mayerich, D., Kwon, J., Sung, C., Abbott, L.C., Keyser, J., & Choe, Y. Fast macro-scale Transmission Imaging of Microvascular Networks Using KESM. Biomedical Optics Express , 2, 2888-2896, (2008).

9. McCormick, B.H. Development of the Brain Tissue Scanner, Technical Report, Department of Computer Science, Texas A & M University, College Station, TX, March 18, 2002 [http://research.cs.tamu.edu/bnl/static/pubs/McC02.pdf], (2002).

10. Sporns, O., Tononi, G., & Kötter, R. The human connectome: A structural description of the human brain. PLoS Computational Biology. , 1, e42 (2005).

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