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

Marcus N Leiwe,Takeshi Imai,Satoshi Fujimoto

doi:10.3389/fnana.2021.760063

Abstract

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.

Highlights

Combined with genetically encoded fluorescent markers, fluorescence imaging is a powerful strategy for mesoscopic mapping of neuronal circuits
Chromatic aberration is a common problem in multicolor imaging of cleared samples
Our analyses of fluorescent beads revealed that lateral chromatic aberration is almost negligible, while axial aberration is often a significant issue

Summary

Introduction

Combined with genetically encoded fluorescent markers, fluorescence imaging is a powerful strategy for mesoscopic mapping of neuronal circuits. Various types of tissue clearing agents compatible with fluorescent proteins have been developed (Richardson and Lichtman, 2015). This expanded the imaging scale to a millimeter scale. Chromatic aberration occurs as different wavelengths of light refract at different angles depending on the substance it is passing through. This can produce two different types of chromatic aberrations, namely, lateral (displacement along the x- and y-axis) and axial (displacement along the z-axis, Figure 1A). When tracing fine structures such as neurites and synapses with multiple fluorophores, this distortion can lead to a difference in the position

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