Chromatic aberration measurement and correction

One main issue in multi-color imaging is the precise overlay of the single channels. This becomes especially important for pixel-precise colocalization analysis. Therefore this test does make most sense for high resolution objectives. Several points are to consider for a perfect overlay of colors:

• coupling of the lasers: often "UV" and "VIS" lines have separate couplings due to the necessity for different fibers; the coupling precision influences the registration of "UV" and "VIS" lines
• collimator lenses: various systems allow to further influence the overlay of "UV" and "VIS" via collimator lenses
• objective corrections: the level of correction for chromatic abberations varies between objectives
• objective damages: the correction of objectives can be ruined by objective damages
• pinhole registration: on systems with multiple pinholes such as the Zeiss LSM 510, one might run into the additional trouble of pinhole alignment; if the pinholes are not well registered to each other, signals get displaced

Now it's clear that several combinations of acquisition settings should be tested. A good start is a stack of 3 colors (RGB, i.e. EX 405, 488, 561 nm) with open pinhole(s).

requirements

• 200 or 500 nm fluorescent beads sample
  • no need to use sub-resolution beads here, since color shifts can be corrected in the range

acquisition

• open pinhole(s)
• use high resolution apochromatic objective (NA 1.2 or above)
• set up beam path for Z-stack RGB fluorescence (i.e. EX 405, 488, 561 nm)
  • take care of pixel size and Z-step size (optimal sampling, i.e. pixel size 100 nm, step size 200 nm)
  • do not oversaturate pixels, rather go for 12/16 bit
  • use sequential line-by-line scanning, to reduce both bleed-through and influence of stage/system vibrations
  • set the margins of the stack to cover the beads and the majority of the point spread function
  • stay in the center of the field of view (optimal performance of scanner and objectives)
• acquire stack of images

analysis

• load the acquired stack into Fiji
• X-shift
  • select the main focal plane
  • draw a horizontal line with the line tool across a single bead close to the center
    • get a straight line by holding down the shift key while drawing
    • the line can be moved around with the arrow keys
  • go to Analyze > Plot Profile
  • go to the next channel w/o changing the line and plot another profile
  • compare the peak of the profiles; or click on List and compare the peak values
• Y-shift:
  • repeat the line drawing and profile plotting for the vertical direction
• Z-shift:
  • remove the line
  • generate XZ-stack: Image > Stacks > Reslice...
  • select the plane of interest, with a clear cut through the PSF of a bead
  • draw a straight vertical line across the center of the bead

images

• 

  coregistration analysis in Fiji, 200 nm beads, 2 line profiles (X-direction) for "blue" and "green"

• 

  coregistration analysis in Fiji, 200 nm beads, 2 line profiles (Z-direction) for "green" and "red", Z-shift of ~500 nm visible