Embedded Files
save tilt series in both .tif and .dat format and transfer them to Roberts linux computer using SSH file transfer
Move to a directory you put files
tomo-setup.sh
this makes four directories, raw, clean, align, and out. All files are put in to raw directory.
cd raw
move into raw directory
./tomo-clean.sh
notes:if file not present, copy from elsewhere (usually you can find it under /home/roberts-lab/ks/)
it takes while to clean images. .img files are generated.
mv *.img ../clean
format information from save_log.txt into text table
[image name]_001 [Spe dir] [Spe ang] [x middle] [y middle]
example:
series_001 102.26 52.41 1024 1024
series_002 102.26 51.8 1024 1024
...
series_041 102.26 -49.81 1024 1024
series_042 102.26 -51.81 1024 1024
notes:
tab between columns
middle pixel is 1024 for 2048 X 2048 image
easiest to format using OpenOffice spreadsheet
save as [filename].txt in raw/
tomo-create.sh [filename].txt [comment] [filename]-01-itr.tlt
note: single word comment such as "filaments" (w/o quotes) but do not use underbar or hyphen for comment
less [filename]-01-itr.tlt
check format for errors
q to close
mv [filename]-01-itr.tlt ../align
copy a .param file to align/ from elsewhere
rename to [filename]-01.param
note: save at least one *-01.param for future use
nano [filename]-01.param
open a .param file with text editor and modify
imgref = file number of image at 0◦ tilt, e.g.37
check table in raw/[filename].txt if unsure
alismp=3
alibox_x=500 corbox_x=256 refmsk_x=480
alibox_y=500 corbox_y=256 refmsk_y=480
Control+x to exit nano, then press y and return
tomo-ali.sh [filename]-01.param [filename]-01-itr.tlt
A window showing a image pops up
Actions > Align All
Actions > Show Movie, make sure images are aligned properly, if not align them by manually
File > Save
close window
cp [filename]-01.param [filename]-02.param
cp [filename]-01-itr-ali.tlt [filename]-02-itr.tlt
optional step:
tomo-box.sh [filename]-02.param [filename]-02-itr.tlt
tests for errors using specified alignment box
if errors are present
reduce alignment box size until errors are gone
or remove images from .tlt file
tomo-refine.sh [filename]-02.param >[filename]-02.log 2>&1 &
note: to check progress, type:
more [filename]-02.log | grep max
It takes 30 minutes to an hour (depending on for this step, when it is finished
tomo-fit.sh [filename]-02.param
four .ps files showing results of alignment are generated
gimp [filename]-02-fit-cof.ps
§ Import
make sure top curve < 1.01 and bottom curve > 0.99. gimp is image editor like photoshop
cp [filename]-02.param [filename]-03.param
cp [filename]-02-fitted.tlt [filename]-03-itr.tlt
open [filename]-03.param with text editor and modify
alismp=2
alibox_x=750 corbox_x=256 refmsk_x=730
alibox_y=750 corbox_y=256 refmsk_y=730
tomo-refine.sh [filename]-03.param >[filename]-03.log 2>&1 &
It takes a few hours.
tomo-fit.sh [filename]-03.param
gimp [filename]-03-fit-cof.ps
§ Import
make sure top curve < 1.01 and bottom curve > 0.99
cp [filename]-03.param [filename]-04.param
cp [filename]-03-fitted.tlt [filename]-04-itr.tlt
open [filename]-03.param with text editor and modify
alismp=1.5
alibox_x=1000 corbox_x=256 refmsk_x=960
alibox_y=1000 corbox_y=256 refmsk_y=960
tomo-refine.sh [filename]-04.param >[filename]-03.log 2>&1 &
tomo-fit.sh [filename]-04.param
gimp [filename]-04-fit-cof.ps
§ Import
make sure top curve < 1.01 and bottom curve > 0.99
cp [filename]-04.param [filename]-05.param
cp [filename]-04-fitted.tlt [filename]-05-itr.tlt
open [filename]-04.param with text editor and modify
alismp=1.0
alibox_x=1500 corbox_x=256 refmsk_x=1460
alibox_y=1500 corbox_y= 256 refmsk_y=1460
tomo-refine.sh [filename]-05.param >[filename]-05.log 2>&1 &
tomo-fit.sh [filename]-05.param
gimp [filename]-05-fit-cof.ps
§ Import
make sure top curve < 1.01 and bottom curve > 0.99
repeat tomo-refine until two lines in [filename]-xx-fit-cof.ps get close to 1.0
cp [filename]-05.param [filename]-06.param
cp [filename]-05-fitted.tlt [filename]-06-itr.tlt
Optional: put numbers in size of map volume, if you want to specify the volume size
x=
y=
z=
tomo-map.sh [filename]-05.param [filename]-05-itr.tlt
this generate 3D map in mrc format
cd ../out
displayimage [filename]-06-bck.img
check 3D map. Press up and down arrow to go up and down stacks.
Convert 3D map for IMOD:
Open terminal
cd ~/ImageJ
Plugins->MRC reader
Open 3D map
Image->Adjust
Adjust image contrast. Press Apply to apply new setting
Image->Type->8-bit
Convert images to 8-bit
Image->Stacks->Delete Slices
Delete useless slices if there are any
File->Save As->Image Sequence
Save as tif images
move to the directory containing tif files
tif2mrc [filename]-???.tif [filename].mrc
this converts tif files to single mrc file for IMOD
When 3D map is huge use ImageJ on HPC
Open terminal
ssh -x roberts@submit.fsu.edu (or hpcx)
Password required to access to HPC@FSU
cd /ks-scrach/ImageJ
Move to ImageJ directory
./run
Start ImageJ
After adjusting contrast, converting to 8-bit, and saving as tif fles, move a directory containing the tif images
scp *.tif roberts-lab@bio334-pc2.bio.fsu.edu:/home/xxx/xxx/xxx
transfer tif files to Roberts linux
exit
logout HPC
move to the directory containing tif files
tif2mrc [filename]-???.tif [filename].mrc
this converts tif files to single mrc file for IMOD
FOR AMIRA:
sample –cnv –smp 2 2 2 –c –s [map_x/2] [map_y/2] [map_z/2] [filename].thr [filename].bin
preproc –med –m 3 3 3 [filename].bin [filename]-01.med
preproc –med –m 3 3 3 [filename]-01.med [filename]-02.med
preproc –med –m 3 3 3 [filename]-02.med [filename]-03.med
compute [filename].rev = [filename]-03.med / -1
cutimage –fmt mrc [filename].rev [filename]-pretrim.mrc
trimvol [filename]-pretrim.mrc [filename].trim
mrcbyte [filename].trim [filename].mrc
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