Two-Photon Linescans
How to acquire fast imaging data using two-photon linescans

This page describes how to acquire two-photon LineScan recordings commanded by ClampEx. Acquisition settings are stored in the XML files generated by PrairieView. Pair linescan folders with ABF files using the time codes stored in the XML file and ABF header.

Green Red

Imaging Settings

  • Dwell time: Amount of time the laser spends at each pixel. Increasing this reduces noise but increases laser exposure.

  • Resolution: 256 is recommended. Higher resolutions don’t yield much benefit, are slower to scan, and increase exposure all else being equal.

  • Zoom: Linescans on small structures are best performed using 4x or 8x optical zoom. Somatic linescans could be performed at 1x.

Linescan Settings

  • LineType: In order to average linescan data together across cells it is critical that this is set to Straight Line, the width of the line is always the same (typically 60 pixels), and all the following settings are identical across experiments.

  • Number of Lines: Size of the linescan along the time axis. Do not exceed 1000 or individual linescans will be broken into multiple tif files.

  • Lines to Skip: Increase this to achieve longer linescans for a given number of lines.

  • Repetitions: Number of replicates for each linescan. This number should be 3 for linescans triggered by 3-sweep ABFs.

  • Trigger: Set to No Trigger for a linescan to occur when you press the “Start Linescan” button, or Trigger Each Rep. to trigger a linescan from an ABF (starting on the rising edge of the trigger line).

  • Synchronization: This can be used to synchronize laser targeted stimulation with linescan imaging. See the two-photon uncaging page for details.

Considerations

  • Resting G should be near or lower than resting R
  • Routinely inspect the R curve to ensure photo-bleaching is not occurring
  • Laser exposure kills cells
    • Minimize laser power
    • Minimize linescan duration
    • Minimize dwell time
    • Minimize repetitions
    • Minimize resolution
    • Minimize number of lines

Linescan Analysis