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).

• overlay UV/V and VIS (confocal systems with separate UV/V laser coupling)
  • purpose: check the coupling precision of the UV/V laser
  • requirements: 0.2/0.5µm fluorescent beads sample
  • work flow: open pinhole(s), use high resolution apochromatic lens (NA 1.2 or above), acquire two images (UV/V plus VIS channel) with good sampling (pixel size ~100nm)

images

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