Accurate and fiducial-marker-free correction for three-dimensional chromatic shift in biological fluorescence microscopy

Atsushi Matsuda,Lothar Schermelleh,Tokuko Haraguchi,Yasuhiro Hirano,Yasushi Hiraoka

doi:10.1038/s41598-018-25922-7

Abstract

Correction of chromatic shift is necessary for precise registration of multicolor fluorescence images of biological specimens. New emerging technologies in fluorescence microscopy with increasing spatial resolution and penetration depth have prompted the need for more accurate methods to correct chromatic aberration. However, the amount of chromatic shift of the region of interest in biological samples often deviates from the theoretical prediction because of unknown dispersion in the biological samples. To measure and correct chromatic shift in biological samples, we developed a quadrisection phase correlation approach to computationally calculate translation, rotation, and magnification from reference images. Furthermore, to account for local chromatic shifts, images are split into smaller elements, for which the phase correlation between channels is measured individually and corrected accordingly. We implemented this method in an easy-to-use open-source software package, called Chromagnon, that is able to correct shifts with a 3D accuracy of approximately 15 nm. Applying this software, we quantified the level of uncertainty in chromatic shift correction, depending on the imaging modality used, and for different existing calibration methods, along with the proposed one. Finally, we provide guidelines to choose the optimal chromatic shift registration method for any given situation.

Highlights

Multicolor imaging is a key modality in biological fluorescence microscopy to determine the relationships between different targets within a specimen
Log-polar transformation and optimization-based methods could be used for biological images, but, from our experience, these were not sufficiently accurate for use in super-resolution microscopy
Our method does not set the highest benchmark in the literature (e.g., Gahlmann et al attained a mean 3D fiducial registration error of 7.84 nm10), it should be noted that our registration method is able to measure chromatic shift from biological images

Summary

Introduction

Multicolor imaging is a key modality in biological fluorescence microscopy to determine the relationships between different targets within a specimen. Various registration methods have been presented that can correct the chromatic shift at or around the surface of a coverslip[4,5,6,7,8,9,10] These typically rely on fiducial markers analyzed separately from the sample of interest (e.g., a 2D layer of multispectral fluorescent beads attached to the coverslip). We devised a new method to calculate registration parameters from any type of biological image, allowing for fluorescent-bead-free measurements of chromatic shift. We implemented this method in an easy-to-use stand-alone software, named “Chromagnon”. We present a simple yet accurate method to correct chromatic shift

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Conclusion