...
Your reconstruction should already be in the MRC format if it was calculated using IMOD or TxBR. If your reconstruction is from a 4k x 4k camera, you should be able to load the full volume into memory on jane without any performance issues. However, if it is from the 8k x 8k camera or is a montage, you will need to bin it so Amira's performance will not suffer.
| Note |
|---|
All files MUST have a .mrc extension. If they do not, they will not be read into Amira properly once you exit and restart the program! |
1. If your volume needs to be binned, use the following command to bin by a factor of 2:
| Code Block |
|---|
$ newstack -bin 2 basename.recmrc basename_2Dbin2.recmrc |
2. When you perform the maximum intensity projection (MIP) calculations, you need to make sure your volume is devoid of all gold particles, as they will dramatically skew the calculation and result in unwanted artifacts. Open your original reconstruction file in imod. Find the minimum and maximum Z-slices at which the volume is free of gold particles. Make note of these two values, and subtract one from each of them (we'll call the final values Zmin and Zmax). We will create a second MRC file in which these Z-slices are trimmed out in the next step.
...
| Code Block |
|---|
$ newstack -scale 255,0 -secs Zmin-Zmax basename_2Dbin2.recmrc basename_2Dbin2_inverted_trimZ.recmrc |
If you haven't already binned your volume because it is from a 4k x 4k camera, run the following command to perform the binning and contrast inversion operations simultaneously:
| Code Block |
|---|
$ newstack -bin 2 -scale 255,0 -secs Zmin-Zmax basename.recmrc basename_2Dbin2_inverted_trimZ.recmrc |
(C) Loading the Volumes into Amira
...
1. In the Pool frame, click the green 'Open Data' button. Select your current directory from the upper pull-down menu (it should be the top choice), and select the first volume (basename.rec mrc OR basename_2Dbin2.recmrc). Click the 'Load' button and select 'MRC Volume' in the 'Dialog' pop-up window. Click the 'OK' button.
...
3. Repeat step 1 for your volume with inverted contrast (basename_2Dbin2_inverted.rec)mrc. You should see a second green button appear in the Pool frame.
...