ASC Z-Resolution

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  • Leica Z-resolution mirror (coverslip on top of mirror)


  • use immersion for the respective objectives
  • use ~488 nm laser for illumination
  • set main beamsplitter to reflection (i.e. 30/70 or 80/20 splitter, AOBS in reflection mode)
  • set detection to the range of the laser wavelength (i.e. LP420, LP470, spectral detection 480-500 nm)
  • close pinhole to minimum
  • set acquisition to good quality (laser power high enough, line average, low scan speed, no saturated pixels)
  • acquire line-stack (XZ-scan) over the reflective side
    • if XZ-scan is not available: acquire XYZ-stack and reslize it afterwards in Fiji


  • open the image in Fiji
    • make sure the image is calibrated (use Plugins > LOCI > Bioformats Importer, if File > Open ignores the metadata)
  • draw a vertical line over the peak intensity (select line tool from the toolbar, left-click for start point, hold shift, left-click for end point)
  • Analyze > Plot Profile for a rough estimation of the FWHM
  • precise analysis: save the list (button List in the profile window) and measure the FWHM in i.e. Excel


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