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.

Rapid Directed Molecular Evolution of Fluorescent Proteins in Mammalian Cells

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2x107 independent random genes of fluorescent proteins expressed in HEK cells completing one iteration directed evolution in a course of ~8 days. We employed this approach to develop a set of green and near-infrared fluorescent proteins with enhanced intracellular brightness. The developed near-infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as C.elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near-infrared fluorescent proteins enabled crosstalk-free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual-color near-infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multicolor imaging of small model organisms.