How to correct for chromatic shift using Volocity

Chromatic shift is a linear mis-registration that occurs between different fluorescence channels and is caused by such things as chromatic aberration in optics and dichroic mirror misalignments. It might be necessary to correct for chromatic shift in images and stacks if you are acquiring data for colocalisation analysis - especially at high resolution. Volocity software can be used to generate Registration Corrections that can be applied to 3D data to shift pixels in all channels back to their correct positions.

Creating and Using Registration Corrections

Use a suitable specimen to create a registration correction; I use a slide of 0.5 micron TetraSpeck beads from Molecular Probes because they are small, bright and are labelled with blue, green, red and far red fluorophores. It makes sense to acquire the beads during the same session as the specimens you will want to correct. Capture 3D stacks of these beads at the same XY and Z resolution you will be acquiring your data at so that the pixel shift is correct. You don't need to worry about whether the laser power, gain and offset are the same as for the specimen - just so long as you avoid saturation in the bead images.

The procedures for creating and using Registration Corrections are in the Volocity manual. Please see the following pages.

• Volocity page 95 – Creating Registration Corrections
• Volocity page 98 – Correcting Registration

Use the automatic correction option; the manual correction is a bit buggy but the automatic correction always works well.

If you have any more questions about chromatic shift correction then please email the Light Microscopy Facility: lmcb-lm-help@ucl.ac.uk