It might be necessary to correct for chromatic shift in images and stacks if you are acquiring data for colocalisation analysis. Volocity can 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 multicolour beads because they are small, bright and are labelled with blue, green, red and far red fluorophores. 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.
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
The LMCB Light Microscopy Facility will be creating and storing bead data and registration corrections acquired using a range of objective lenses on different microscopes. These will routinely be captured at the highest resolutions possible for the objectives so if you want to use these previously prepared corrections you should capture your data at the same resolutions.