Stitching existing data
If your data have already been acquired and not pre-processed via
syncAndCrunch, you should handle them as follows. Each acquisition session should have its own directory set up like this. i.e. It needs:
- A meta-data file (the "recipe" file).
- A sub-directory called
rawDatathat houses the raw data directories. Move them here if they're not already here.
With the above in place you can use
help stitchAllChannels) to automate the process of conducting all the pre-stitching analyses and then stitching. e.g.
>> cd /mnt/myLocalData/SampleXYZ
>> ls -l
>> downsampleAllChannels(50) %Generate 50 micron volumes for stitchit.sampleSplitter
If you look inside
stitchAllChannelsyou'll see it really doesn't do very much. Instead of using
stitchAllChannelsyou can run the required functions yourself one at a time like this:
>> cd /mnt/myLocalData/mySample
>> generateTileIndex %Indexes tiles, determining where each fits in the final volume
>> preProcessTiles(0,1:2,1:2) %Calculate coefficients for correcting artifacts
>> collateAverageImages %Calculate grand average tiles
>> stitchSection(,1) %Stitch all data from channel 1
That's it: those are the core commands in StitchIt. So what was going on there?
generateTileIndexindexes the tiles to map their names on to their position in the final volume. Then the
preProcessTilescommand calculates coefficients for the comb correction and average tiles for illumination correction. The first input argument tells it to only work on directories that already haven’t been processed, the second and third arguments tell it to calculate bidirectional scanning coefficients and illumination correction on channels 1 and 2. Average image data for illumination correction are stored for each physical section within that section’s raw data directory. If interupted,
preProcessTilescan resume where it left off.
collateAverageImagesproduces the grand average images used for illumination correction (data from the whole specimen contribute to the illumination correction). The above steps is most of what
In case you're wondering what these various corrections are good for, here's an example of the same section before and after illumination correction by dividing out the average tile.