Post hoc Correction of Chromatic Aberrations in Large-Scale Volumetric Images in Confocal Microscopy

Over the last decade, tissue-clearing techniques have expanded the scale of volumetric fluorescence imaging of the brain, allowing for the comprehensive analysis of neuronal circuits at a millimeter scale. Multicolor imaging is particularly powerful for circuit tracing with fluorescence microscopy. However, multicolor imaging of large samples often suffers from chromatic aberration, where different excitation wavelengths of light have different focal points. In this study, we evaluated chromatic aberrations for representative objective lenses and a clearing agent with confocal microscopy and found that axial aberration is particularly problematic. Moreover, the axial chromatic aberrations were often depth-dependent. Therefore, we developed a program that is able to align depths for different fluorescence channels based on reference samples with fluorescent beads or data from guide stars within biological samples. We showed that this correction program can successfully correct chromatic aberrations found in confocal images of multicolor-labeled brain tissues. Our simple post hoc correction strategy is useful to obtain large-scale multicolor images of cleared tissues with minimal chromatic aberrations.

Automated summary

In this study, chromatic aberrations in multicolor imaging of large samples were evaluated and found to be particularly problematic in axial aberrations, often dependent on depth. A program was developed to align depths for different fluorescence channels based on reference samples, which successfully corrected chromatic aberrations found in confocal images of multicolor-labeled brain tissues. The simple post hoc correction strategy proved useful for obtaining large-scale multicolor images of cleared tissues with minimal chromatic aberrations.