Scanning Brain Networks
Table 1 : X-ray tomographic microscopy data collection conditions. Beamline
BL20XU X-ray optics
X-ray energy (keV) Pixel size a
Viewing field size (pixels) a Viewing field size (µm) a Rotation/frame (degrees) Exposure/frame (ms) Frame/dataset
Dataset collection time (min) Spatial resolution
a Width × height
tissue was soaked in 10 mL of 100% EtOH for a few hours or overnight at room temperature (20–25°C). T is process was repeated three times in total. T e tissue was then transferred to 10 mL of n -buthyl glycigylether and leſt for several hours or overnight at room temperature. T is process was repeated twice in total. Subsequently, the tissue was soaked in a 2 mL aliquot of Petropoxy 154 (Burnham Petrographics, ID) epoxy resin overnight at 4°C. T e epoxy resin was degassed prior to use. T is process was repeated twice in total. If the tissue fl oated to the surface of the resin even aſt er two cycles of resin soaking, an additional soaking using another resin aliquot was performed.
Human brain tissues soaked in the resin were cut into rod shapes with widths of 0.3–0.5 mm under a stereomicroscope and transferred to a borosilicate glass capillary (W. Müller, Germany) fi lled with the resin. Larger tissue samples with widths of 1.5–3 mm were also prepared and embedded in resin pellets. T e fl y brain was embedded in a tiny resin drop using a nylon loop for protein crystallography (Hampton Research, CA). T e samples were then kept at 90°C for 40–90 hr for curing the resin.
Sample mounting . T e resin-embedded samples were mounted on the sample stage using brass fi ttings ( Figure 1a ). Example samples are shown in Figure 1b . Pellet samples were attached to the fl at surface of the brass fi tting using epoxy glue or double-stick tape. Epoxy glue is preferable for high-resolution analysis. Capillary samples with outer diameters greater than 0.5 mm were inserted in clay stuff ed in a hole made at the end of the brass fi tting. Because the capillary insertion compresses the residual air in the hole and its re-expansion can cause sample driſt during data collection, a minuscule side opening was made in the lateral face of the fi tting ( Figure 1c ) to vent the air and clay. Capillaries with diameters less than 0.5 mm were sleeved with a brass tube using epoxy glue and secured with a setscrew. Sample mounting was fi nished at least 30 minutes before the data collection to stabilize the samples. For tomographic microscopy with resolutions higher than 200 nm, the mounted samples should be placed near the sample stage as soon as possible in order to equilibrate their temperature with that of the apparatus. Tomographic microscopy . Simple-projection tomographic microscopy was performed at the BL20XU beamline of the SPring-8 synchrotron radiation facility, as reported previously [ 1 ].
Simple projection 12
0.50 × 0.50 µm 2,048 × 2,048 1,024 × 1,024 0.10 150
1,800 6
1.0–1.2 µm
BL37XU
Fresnel zone plate optics 8
60 × 60 nm
2,048 × 2,048 123 × 123 0.20 700 900 20
160–200 nm
BL47XU
Fresnel zone plate optics 8
262 × 262 nm 2,000 × 700 524 × 183 0.10 300
1,800 16
600–800 nm
Figure 2 : (a) Three-dimensional rendering of the network of the fruit fl y brain. The structures including the midline component called the central complex are illustrated. Scale bar = 10 µm. (b) Skeletonized model of the left hemisphere is superposed on the entire brain structure. The front side of the rendering is cut off to show inner structures. Scale bar = 20 µm. CB, central brain; OL, optic lobe.
2015 September •
www.microscopy-today.com 13
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68