2P Stage Control
Python scripts to automate two-photon stage positioning, image acquisition, and montage assembly using ImageJ and TrackEM

Stuff related to software to control the stage on the 2P microscope, acquire images, and recreate them as mosaics. Also covers deconvolution code.

  • SWHStage.py seems to be the main thing I use currently. It saves XYZ coords in the filename.

  • SWHstage_to_trackEM_xml.py is how I turn it into an XML file ready to load into TrackEM (natively installed in FIJI).

Steps

  • run SWHstage.py on the 2p microscope and note where the images were saved. 1 montage series must be alone in a single folder.
  • copy this folder from the X-drive to your local computer. Calling individual images from the X drive is too slow to proceed otherwise.
  • run SWHstage_to_trackEM_xml.py on the folder that images were saved to locally. It will generate auto.xml in that folder
  • drag/drop auto.xml onto ImageJ (FIJI) and TrackEM will launch.
  • right-click, adjust images, set min and max layer-wise and go 0-4092 (I think?) or whatever looks good
  • alternatively do layer-wise enhance contrast, do NOT normalize, and do click use full stack, 0.01% saturated pixels
  • right-click, align, all images in this layer
  • I like the phase correlation method
  • right-click, adjust images, blend layer-wise (this will take a little while to run)
  • export flat image (you can adjust scale here, I like 25%)

Background Subtraction

  • image an empty field (single frame)
  • gaussian blur 50 px
  • math subtract the minimum value in the frame
  • analyze, set measurements, check min and max gray value
  • analyze, measure (for me it shows 320)
  • process, math, subtract (stack - background frame)
  • file, save as, image sequence, slice labels as filenames
  • process, image calculator (subtract original stack from new BG)

Screenshots

immediately on loading

After subtracting DIC background

After blending

Source Code